ORCID Profile
0000-0002-2107-2738
Current Organisation
RMIT University
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Analytical chemistry | Sensor technology (incl. chemical aspects) | Characterisation of Biological Macromolecules | Biochemistry and Cell Biology | Biomaterials | Nanotechnology | Glycobiology | Receptors and Membrane Biology | Structural Biology (incl. Macromolecular Modelling) | Nanobiotechnology |
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in Technology
Publisher: Elsevier BV
Date: 03-1999
DOI: 10.1016/S1380-2933(98)00025-6
Abstract: Malignant B cells from patients with chronic B lymphocytic leukemia (B CLL) generally express both surface IgM and the pan T cell antigen CD5, a characteristic of the B1 population of B lymphocytes. The IgM on the surface of these B CLL cells is frequently polyreactive with respect to its capacity to recognize multiple structurally dissimilar antigens (Ag). To understand the structural characteristics of the polyreactive binding sites of human IgM molecules expressed on B CLL cells by: (1) analyzing the nucleotide and protein sequences of the variable (V) domains of five IgM molecules expressed in cases of B CLL and (2) utilizing these sequences to generate three-dimensional (3D) models of Fv (VL - VH) molecules. Peripheral blood leukocytes obtained from five cases of B CLL were tested for polyreactive binding properties by assessing their capacity to bind mouse IgG by indirect immunofluorescence. The V region genes of light and heavy chains were lified using the polymerase chain reaction, subsequently cloned and their nucleotide sequences obtained. Translated amino acid sequences of the V domains were used to generate homology models of the Fv molecules. Low affinity binding of mouse IgG was demonstrated for all B CLL s les examined, confirming the polyreactive nature of the IgM expressed on these cells. There was an absence or minimal mutation within V region genes when compared to germline Ig genes. Junctional ersity was not observed for VL regions, although truncations and insertions were frequent in D minigenes of VH regions. The binding sites were predicted to form either relatively flat surfaces with occasional protrusions or cavities at the VL - VH domain interface. Aromatic side chains covered a large proportion of the potential binding surfaces in the models of B CLL Fv components. Primary DNA sequences can be categorized as germline, suggesting that the B cells involved in B CLL are germline or naive in origin. The medium to large HCDR3s provide the majority of probable contact residues for antigens. While prominent aromatic residues are likely to engage in binding patterns which are conserved (e.g. mouse Ig reactivity), the erse binding sites predicted for B CLL-derived IgMs also have properties which are conducive to polyreactive antigen binding.
Publisher: Public Library of Science (PLoS)
Date: 10-11-2009
Publisher: Elsevier BV
Date: 02-2010
DOI: 10.1016/J.MOLIMM.2009.11.011
Abstract: Bovine herpesvirus 1 (BoHV-1) causes respiratory and genital diseases in cattle for which available vaccines do not confer adequate protection. Since passive immunization with antibodies permits disease prevention, single-chain fragment variable (scFv), originating from a monoclonal bovine IgG1 antibody against BoHV-1, were constructed and expressed in Pichia pastoris in V(lambda)-V(H) orientation via a flexible seven-amino acid linker. Similar to the intact IgG, the purified recombinant scFv neutralized BoHV-1 in vitro and recognized viral antigens in BoHV-1 infected MDBK cells by immunofluorescence. Homology modeling of the Fv predicts two distinct conformations for CDR3H. Firstly, a long protruding CDR3H conformation where no disulfide linkage occurred between two "non-canonical" Cys residues resulted in a large binding cavity between V(lambda) and V(H). Secondly, a smaller potential antigen-binding cavity is predicted with a disulfide linkage between the two Cys residues of CDR3H creating a six-membered loop in the ascending polypeptide, which fitted into the space between V(lambda) and V(H). Despite such potential configurational ersity of the antigen-binding site, the electrostatic surface potentials that would interact with the BoHV-1 epitope are largely similar for both the topographies where salt-bridge type electrostatic interactions likely occur at the edges of the binding site. Given that IgG1 antibody against BoHV-1 is clonally selected, it is likely that disulfide-stabilized broader and flatter surface topography is specifically generated to accommodate the predicted carbohydrate neutralizing B-epitope on BoHV-1. The specificity and neutralizing capacity for BoHV-1 of the scFv should make this bovine antibody fragment a useful diagnostic and potential therapeutic candidate for an important viral pathogen in cattle.
Publisher: Elsevier BV
Date: 04-2000
DOI: 10.1016/S0161-5890(00)00049-3
Abstract: The crystal structure of the Fv molecule from a human monoclonal IgM cryoglobulin (Mez) was determined at 2.6 A resolution. Amino acid sequences of framework regions (FR) of the Mez light (L) and heavy (H) chain variable domains (VL and VH) are highly similar to their counterparts in another human Fv (Pot) previously subjected to X-ray analysis in our laboratory. As expected, the three-dimensional (3-D) structures of FR are quite similar in the two proteins, as are four of the six complementarity-determining regions (CDRs): CDRs 1 and 2 for both L and H chains. Absence of Pro 95L from the LCDR3 loop in Mez VL (relative to Pot LCDR3) results in compression of this loop and creates more space in the VL-VH interface. In the two IgMs, HCDR3 conformations differ significantly from all previously defined conformations for these loops. Pot has a 12-residue HCDR3 that collapses to fill all available space in the VL-VH domain interface, resulting in the formation of a relatively flat platform for antigen binding. In Mez, the HCDR3 is two residues longer and is comprehensively different. A semi-rigid ascending segment dominated by a Pro-Pro-Tyr sequence protrudes out into solvent. The descending portion has the sequence Gly-Trp-Gly-Gly-Gly, which promotes high local flexibility. This segment folds across the VL-VH domain interface to interact with residues in LCDR3. These features partition the Mez active site into two compartments, a large cavity between VL and VH and a smaller cavity lined entirely by constituents of the three heavy chain CDRs. Such an unusual topographical feature indicates why the Mez IgM does not bind to the Fc portion of intact human IgG antibodies in immunoassays yet interacts with high avidity with many Fc-derived octapeptides. The cavities are expected to be the repositories for the Fc-derived peptides, while the semi-rigid protrusion of the Mez HCDR3 prevents the close approach of another macromolecule (e.g. intact IgG) to the active site.
Publisher: American Chemical Society (ACS)
Date: 03-08-2018
Abstract: Bacterial adhesion to human epithelia via lectins constitutes a therapeutic opportunity to prevent infection. Specifically, BambL (the lectin from Burkholderia ambifaria) is implicated in cystic fibrosis, where lectin-mediated bacterial adhesion to fucosylated lung epithelia is suspected to play an important role. We employed structure-based virtual screening to identify inhibitors of BambL-saccharide interaction with potential therapeutic value. To enable such discovery, a virtual screening protocol was iteratively developed via 194 retrospective screening protocols against 4 bacterial lectins (BambL, BC2L-A, FimH, and LecA) with known ligands. Specific attention was given to the rigorous evaluation of retrospective screening, including calculation of analytical errors for enrichment metrics. The developed virtual screening workflow used crystallographic constraints, pharmacophore filters, and a final manual selection step. The protocol was applied to BambL, predicting 15 active compounds from virtual libraries of approximately 7 million compounds. Experimental validation using fluorescence polarization confirmed micromolar inhibitory activity for two compounds, which were further characterized by isothermal titration calorimetry and surface plasmon resonance. Subsequent testing against LecB from Pseudomonas aeruginosa demonstrated binding specificity of one of the hit compounds. This report demonstrates the utility of virtual screening protocols, integrating ligand-based pharmacophore filtering and structure-based constraints, in the search for bacterial lectin inhibitors.
Publisher: Elsevier BV
Date: 1999
DOI: 10.1016/S0145-305X(98)00049-4
Abstract: We have employed a COS cell expression cloning procedure to isolate a full length cDNA clone encoding a hagfish leukocyte-associated membrane protein (HLMP1). The protein, which is identified by a monoclonal antibody (JB3) generated in our laboratory, is present on the majority of hagfish leukocytes and is also expressed on erythrocytes. The cDNA clone contained an open reading frame encoding a 120 residue polypeptide which exhibits 33% amino acid sequence identity with the precursor protein of human CD59, a leukocyte-associated membrane protein which regulates the action of the complement membrane attack complex on homologous cells. CD59 belongs to a family of structurally related glycoproteins which includes the Ly-6 proteins expressed on mouse lymphocytes. In addition to significant overall sequence homology HLMP1 shows conservation of 8 key cysteine residues with members of the CD59/Ly-6 family. Comparison of the hagfish sequence with that of the mature human CD59 protein suggested a processed protein consisting of 74 amino acids associated with the cell membrane via a GPI anchor. The latter was confirmed by immuno-flow cytometry following treatment of transfected COS cells with phospholipase. Phylogenetic analysis and tissue distribution of this protein in the hagfish are consistent with HLMP1 being a homologue of CD59. A three-dimensional model of HLMP1, constructed using the NMR-determined structure for human CD59 as a template, indicated conservation of a core structure of five strands of beta-sheet and a short helix stabilised by four disulfide bonds. These findings, when taken together with our previous identification of C5a-like chemotactic activity in LPS-activated serum, provide indirect evidence for the existence of the terminal lytic complement pathway (C5 to C9) in these primitive vertebrates.
Publisher: Wiley
Date: 14-12-2006
DOI: 10.1111/J.1365-3083.2006.01865.X
Abstract: Analysis of the heavy-chain gene (pTGHC9907) encoding a bovine IgG1 antibody against bovine herpes virus type 1 (BHV-1) isolated from a Holstein cow has led to the identification of a new IgG1 sequence allele. A comparison of nucleotide sequence of pTGHC9907 with the IgG1(a) (clone 2) and IgG1(b) (clone 8.10) sequence variants and unclassified IgG1 cDNA sequence (clone 8.75) has revealed significant differences in the hinge region spanning codons 216-230. The Thr224 and Thr226 of IgG1(a) were replaced with Arg224 and Pro226, while both Thr218 and Pro224 of IgG1(b) were substituted with Arg with deletion of Ser225 in HB9907 antibody. Additional amino acid substitutions were noted in the CH1 (positions 190, 192), CH2 (position 281) and CH3 (position 402) exons. Thus, the polymorphic sites occurred in all constant domains, but were clustered in the hinge region of IgG1. Examination of a three-dimensional model of the HB9907 heavy chain revealed that all sequence variations were on the surface of the IgG and are possible targets for recognition by antisera and effector molecules such as cellular adhesion molecules. The presence in the CH1 domain of a repeating motif of Pro-Ala-Ser-Ser indicated a potential structure-enhancing function and a role in cellular adhesion and migration. Replacement of Thr with Arg residues within the hinge was predicted to have a dual effect of reducing the number of O-linked glycosylation sites and increasing the susceptibility to degradation by protease-secreting bacteria of the hinge region. As unclassified IgG1 cDNA sequence (clone 8.75) is structurally distinct from other variants, it is also classified as IgG1(d). Collectively, these observations support the identification of a new allotypic variant of bovine IgG1, designated as IgG1(c) that is distinct in both sequence and structure from the known sequence variants.
Publisher: Elsevier BV
Date: 08-2008
DOI: 10.1016/J.JNEUROIM.2008.06.013
Abstract: A number of treatment options are available to multiple sclerosis patients, however this needs to be improved. Herein, we designed and synthesized a number of peptides by mutating principal TCR contact residues based on MBP(83-99) peptide epitope. Immunization of SJL/J mice with MBP(83-99) and mutant [A(91)]MBP(83-99), [E(91)]MBP(83-99), [F(91)]MBP(83-99), [Y(91)]MBP(83-99), and [R(91), A(96)]MBP(83-99) peptides, induced IFN-gamma, and only [R(91), A(96)]MBP(83-99) mutant peptide was able to induce IL-4 secretion by T cells. T cells against the native MBP(83-99) peptide cross-reacted with all peptides except [Y(91)]MBP(83-99) and [R(91),A(96)]MBP(83-99). The double mutant [R(91), A(96)]MBP(83-99) was able to antagonize IFN-gamma production in vitro by T cells against the native MBP(83-99) peptide. Antibodies generated to [R(91), A(96)]MBP(83-99) did not cross-react with whole MBP protein. Molecular modeling between peptide analogs and H2 I-A(s) demonstrated novel interactions. The [R(91), A(96)]MBP(83-99) double mutant peptide analog is the most promising for further therapeutic studies.
Publisher: Wiley
Date: 2000
DOI: 10.1002/1099-1352(200007/08)13:4<198::AID-JMR499>3.0.CO;2-D
Publisher: Springer Vienna
Date: 11-10-2011
Publisher: Wiley
Date: 07-1999
DOI: 10.1002/(SICI)1099-1352(199907/08)12:4<249::AID-JMR463>3.0.CO;2-B
Publisher: Oxford University Press (OUP)
Date: 2013
DOI: 10.1189/JLB.0612308
Abstract: Along with an enhanced interaction with CD4, highly M-tropic HIV-1 Envs have an altered mechanism of engagement with CCR5. BR-derived HIV-1 strains have an exceptional ability to enter macrophages via mechanisms involving their gp120 Env that remain incompletely understood. Here, we used cell-based affinity-profiling methods and mathematical modeling to generate quantitative VERSA metrics that simultaneously measure Env-CD4 and Env-CCR5 interactions. These metrics were analyzed to distinguish the phenotypes of M-tropic and non-M-tropic CCR5-using HIV-1 variants derived from autopsy BRs and LNs, respectively. We show that highly M-tropic Env variants derived from brain can be defined by two distinct and simultaneously occurring phenotypes. First, BR-derived Envs demonstrated an enhanced ability to interact with CD4 compared with LN-derived Envs, permitting entry into cells expressing scant levels of CD4. Second, BR-derived Envs displayed an altered mechanism of engagement between CD4-bound gp120 and CCR5 occurring in tandem. With the use of epitope mapping, mutagenesis, and structural studies, we show that this altered mechanism is characterized by increased exposure of CD4-induced epitopes in gp120 and by a more critical interaction between BR-derived Envs and the CCR5 N-terminus, which was associated with the predicted presence of additional atomic contacts formed at the gp120-CCR5 N-terminus interface. Our results suggest that BR-derived HIV-1 variants with highly efficient macrophage entry adopt conformations in gp120 that simultaneously alter the way in which the Env interacts with CD4 and CCR5.
Publisher: Wiley
Date: 2001
DOI: 10.1002/JMR.537
Abstract: Using X-ray crystallography, a human monoclonal IgM cryoglobulin (Mez) was found to have an unusual combining site topography. Analysis of the unliganded Fv at 2.6 A resolution revealed that the HCDR3 had partitioned the active site into two compartments [Ramsland PA et al. 2000. Mol. Immunol. 37: 295-310]. The two cavities had dimensions and chemical properties that were compatible with the binding of peptides. In this study, libraries of peptides were prepared using solid-phase synthesis. Binding of the intact Mez IgM to these peptides was tested by enzyme-linked immunoassays. Screening of 400 dipeptides revealed that binding was markedly skewed toward amino acids with aromatic side-chains (Phe and Trp), especially when located in the second position. Preferential recognition of aromatic side-chains by Mez IgM was confirmed with larger peptides of three to five residues, but C-terminal positioning was not favored in these peptides. Mez IgM also showed binding propensities for acidic residues (Asp and Glu) as well as several other side-chains with different chemical properties, including His, Pro, Asn and Gln. Mez IgM recognized sets of overlapping octapeptides representing the sequences of the constant domains of human IgG1 heavy chains. These peptides represented similar stretches of polypeptide on the three-dimensional structures of all three constant domains (CH1, CH2 and CH3). Thus, Mez IgM may recognize structurally homologous regions of immunoglobulin domains, which were conserved during the evolution of the immune system.
Publisher: Informa UK Limited
Date: 11-07-2009
DOI: 10.1517/14712590903066703
Abstract: Transplantation of organs across species (xenotransplantation) is being considered to overcome the shortage of human donor organs. However, unmodified pig organs undergo an antibody-mediated hyperacute rejection that is brought about by the presence of natural antibodies to Galalpha(1,3)Gal, which is the major carbohydrate xenoantigen. Genetic modification of pig organs to remove most of the Galalpha(1,3)Gal epitopes has been achieved, but the human immune system may still recognize residual lipid-linked Galalpha(1,3)Gal carbohydrates, new (cryptic) carbohydrates or additional non-Galalpha(1,3)Gal carbohydrate xenoantigens. The structural basis for lectin and antibody recognition of Galalpha(1,3)Gal carbohydrates is starting to be understood and is discussed in this review. Antibody binding to Galalpha(1,3)Gal carbohydrates is predicted to primarily involve end-on insertion of the terminal alphaGal residue, but it is possible that groove-type binding can occur, as for some lectins. It is likely that similar antibody and lectin recognition will occur with other non-Galalpha(1,3)Gal xenoantigens, which potentially represent new barriers for pig-to-human xenotransplantation.
Publisher: Elsevier BV
Date: 10-2003
DOI: 10.1016/S0161-5890(03)00167-6
Abstract: This study explores the structural features of murine monoclonal IgG2a anti-dinitrophenyl (DNP) antibodies that were previously shown to form immune complexes (IC) differing in their capacity to bind complement, their clearance from the circulation and their deposition in the kidney. Interestingly, the sequence of one of these antibodies has a missing stretch of 14 amino acids within FR3. Molecular modeling suggests that this sequence deletion corresponds to the loss of beta-pleated sheet structure for two beta-strands (designated 4-3 and 4-4) on the external surface of the V(H) domain. Despite this sequence and conformational abnormality, the antibody retains affinity for DNP comparable to other IgG2a antibodies. Data presented here identify monoclonal IgG2a antibodies that form IC with varying propensity for both complement binding and renal deposition and yet have similar V(H) domain sequences. In fact, in the case of two IgG2a antibodies that form IC with very different renal tropisms and complement binding capacity, sequence variation within V(H) was observed only at three clustered residues within FR2, a single residue within FR3 and nine clustered residues spanning CDR3 and FR4. Sequence and modeling analysis also yielded the paradoxical finding that an antibody forming IC with a relatively high capacity to serve as a target for complement binding displays a relatively low number of solvent exposed acceptor residues for C4b and C3b. These data underscore the complex relationship between V domain structure, complement activation and renal deposition of model IC.
Publisher: Elsevier BV
Date: 03-2013
DOI: 10.1016/J.JMGM.2013.01.001
Abstract: Prediction of the protein residues most likely to be involved in ligand recognition is of substantial value in structure-based drug design. Considering multiple ligand binding modes is of potential relevance to studying ligand recognition, but is generally ignored by currently available techniques. We have previously presented the site mapping technique, which considers multiple ligand binding modes in its analysis of protein-ligand recognition. AutoMap is a partially automated implementation of our previously developed site mapping procedure. It consists of a series of Perl scripts that utilize the output of molecular docking to generate "site maps" of a protein binding site. AutoMap determines the hydrogen bonding and van der Waals interactions taking place between a target protein and each pose of a ligand ensemble. It tallies these interactions according to the protein residues with which they occur, then normalizes the tallies and maps these to the surface of the protein. The residues involved in interactions are selected according to specific cutoffs. The procedure has been demonstrated to perform well in studying carbohydrate-protein and peptide-antibody recognition. An automated procedure to optimize cutoff selection is demonstrated to rapidly identify the appropriate cutoffs for these previously studied systems. The prediction of key ligand binding residues is compared between AutoMap using automatically optimized cutoffs, AutoMap using a previously selected cutoff, the top ranked pose from docking and the predictions supplied by FTMap. AutoMap using automatically optimized cutoffs is demonstrated to provide improved predictions, compared to other methods, in a set of immunologically relevant test cases. The automated implementation of the site mapping technique provides the opportunity for rapid optimization and deployment of the technique for investigating a broad range of protein-ligand systems.
Publisher: Wiley
Date: 23-01-2007
DOI: 10.1002/JMR.814
Abstract: Cryoglobulinemia is associated with a range of diseases including rheumatoid arthritis, B-cell malignancies, and chronic viral infections. This "cold-sensitivity" condition is caused by cryoglobulins that precipitate, gel, or occasionally crystallize in the cold. Clinical manifestations vary widely in severity, depending on many factors, including the type of cryoglobulin (monoclonal or mixed immunoglobulins) and the physical nature of the aggregates (precipitate, gel, or crystal). Dynamic light scattering (DLS) was used to examine the cold-induced precipitation or gelation of two human cryoglobulins, namely, Pot IgM and Yvo IgM. The DLS assay was highly reproducible, sensitive, and had low intra-assay variations for both IgM cryoglobulins. Distinct processes were revealed to contribute to precipitation and gelation of cryoglobulins. The precipitation of Pot IgM displayed a rapid transition from solution to solid phases, with a wide distribution of aggregate sizes. In contrast, the gelation of Yvo IgM progressed gradually across a broad temperature range to produce a relatively uniform gel matrix. Initial cryoglobulin concentrations determined the kinetics and critical temperatures for both precipitation and gelation. Moreover, the Yvo IgM was observed to have a distinct relationship between concentrations and mean hydrodynamic diameters or particle sizes. Concentration-dependent effects on particle sizes were present, but not as pronounced for the Pot IgM. Precipitation and gelation of cryoglobulins were also found to be differentially responsive to changes in the aqueous environment. Our results indicate that DLS is a rapid, reliable, and sensitive method for characterizing the nature of disease-associated cryoglobulins.
Publisher: Frontiers Media SA
Date: 21-06-2017
Publisher: Elsevier BV
Date: 02-2008
Publisher: Public Library of Science (PLoS)
Date: 20-04-2012
Publisher: Elsevier BV
Date: 03-2009
DOI: 10.1016/J.MOLIMM.2008.11.007
Abstract: The major histocompatibility complex (MHC) on the surface of antigen presenting cells functions to display peptides to the T cell receptor (TCR). Recognition of peptide-MHC by T cells initiates a cascade of signals, which results in the initiation of a T cell dependent immune response. An understanding of how peptides bind to MHC molecules is important for determining the structural basis for T cell dependent immune responses and facilitates the structure-based design of peptides as candidate vaccines to elicit a specific immune response. To date, crystal structures, immunogenicity and in vivo biological relevance have mainly been characterized for high affinity peptide-MHC interactions. From the crystal structures of numerous peptide-MHC complexes it became apparent what canonical sequence features were required for high affinity binding, which led to the ability to predict in most instances peptides with high affinity for MHC. We previously identified the crystal structures of non-canonical peptides in complex with MHC class I (one bound with low affinity and the other with high affinity, but utilizing novel peptide anchors and MHC pockets). It is becoming increasingly evident that other non-canonical peptides can also bind, such as long-, short- and glyco-peptides. However, the in vivo role of non-canonical peptides is not clear and we present here the immunogenicity of two non-canonical peptides and their affinity when bound to MHC class I, H2K(b). Comparison of the three-dimensional structures in complex with MHC suggests major differences in hydrogen bonding patterns with H2K(b), despite sharing similar binding modes, which may account for the differences in affinity and immunogenicity. These studies provide further evidence for the erse range of peptide ligands that can bind to MHC and be recognized by the TCR, which will facilitate approaches to peptide-based vaccine design.
Publisher: Elsevier BV
Date: 12-2009
DOI: 10.1016/J.MOLIMM.2009.09.031
Abstract: Antibody-carbohydrate interactions play central roles in stimulating adverse immune reactions. The most familiar ex le of such a process is the reaction observed in ABO-incompatible blood transfusion and organ transplantation. The ABO blood groups are defined by the presence of specific carbohydrates expressed on the surface of red blood cells. Preformed antibodies in the incompatible recipient (i.e., different blood groups) recognize cells exhibiting host-incompatible ABO system antigens and proceed to initiate lysis of the incompatible cells. Pig-to-human xenotransplantation presents a similar immunological barrier. Antibodies present in humans recognize carbohydrate antigens on the surface of pig organs as foreign and proceed to initiate hyperacute xenograft rejection. The major carbohydrate xenoantigens all bear terminal Gal alpha(1,3)Gal epitopes (or alphaGal). In this study, we have developed and validated a site mapping technique to investigate protein-ligand recognition and applied it to antibody-carbohydrate systems. This site mapping technique involves the use of molecular docking to generate a series of antibody-carbohydrate complexes, followed by analysis of the hydrogen bonding and van der Waals interactions occurring in each complex. The technique was validated by application to a series of antibody-carbohydrate crystal structures. In each case, the majority of interactions made in the crystal structure complex were able to be reproduced. The technique was then applied to investigate xenoantigen recognition by a panel of monoclonal anti-alphaGal antibodies. The results indicate that there is a significant overlap of the antibody regions engaging the xenoantigens across the panel. Likewise, similar regions of the xenoantigens interact with the antibodies.
Publisher: The American Association of Immunologists
Date: 10-2010
Abstract: Free κ L chains (FκLCs) are expressed on the surface of myeloma cells and are being assessed as a therapeutic target for the treatment of multiple myeloma. Despite its clinical potential, the mechanism by which FκLCs interact with membranes remains unresolved. In this study, we show that FκLCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound FκLCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of FκLCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated FκLCs exposed on the plasma membrane after exocytosis. Although it is well known that protein aggregates bind membranes, this is only the second ex le of an aggregate being found on the surface of cells that also secrete the protein in its native form. We postulate that many other aggregation-prone proteins may associate with cell membranes by similar mechanisms.
Publisher: Wiley
Date: 2001
DOI: 10.1002/JMR.533
Abstract: A monoclonal IgM cryoglobulin with erse binding behavior was isolated from a patient (Mez) with Waldenström's macroglobulinemia. It gave very high titers in the binding of combinatorially synthesized libraries of peptides ranging in size from two to eight residues. The crystal structure of Mez Fv revealed that the binding site was ided into two cavities of unequal volumes with dimensions and chemical properties that were compatible with the binding of peptides. Access to this unique combination of structural information and peptide binding data led us to carry out Mez-peptide docking simulations to gain insight into the Mez binding propensities. In the present article, the results for docking of five peptide libraries are combined with discussions of the methods and approximations involved in the docking process. We analyze the origins of peptide binding affinity for Mez IgM in terms of its cross-reactivity and its structural preferences.
Publisher: Elsevier BV
Date: 09-2000
Publisher: Oxford University Press (OUP)
Date: 10-09-2015
Abstract: Euonymus europaeus lectin (EEL) is a carbohydrate-binding protein derived from the fruit of the European spindle tree. EEL was first identified for its erythrocyte agglutinating properties and specificity for B and H blood groups. However, a detailed molecular picture of the structural basis of carbohydrate recognition by EEL remains to be developed. In this study, we performed fluorescence titrations of a range of carbohydrates against EEL. Binding of EEL to a wide range of carbohydrates was observed, including a series of blood group-related carbohydrates, mannosides, chitotriose and sialic acid. Affinity was strongest for carbohydrates with H-related structures and the B trisaccharide. A homology model of EEL was produced from templates identified using the HHPred server, which employs hidden Markov models (HMMs) to identify templates. The HMM approach identified that the best templates for EEL were proteins featuring a ricin B-like (R-type) fold. Separate templates were used to model the core and binding site regions of the lectin. Through the use of constrained docking and spatial comparison with a template ligand, binding modes for the carbohydrate ligands were predicted. A relationship between the experimental binding energies and the computed binding energies of the selected docked poses was determined and optimized. Collectively, our results suggest that EEL utilizes a single site for recognition of carbohydrates terminating in a variety of monosaccharides.
Publisher: Elsevier BV
Date: 11-2001
Publisher: Elsevier BV
Date: 09-2011
Publisher: American Society for Microbiology
Date: 05-2011
DOI: 10.1128/JVI.00106-11
Abstract: Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro -generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop–CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.
Publisher: Elsevier BV
Date: 08-1999
Publisher: Public Library of Science (PLoS)
Date: 14-10-2022
DOI: 10.1371/JOURNAL.PONE.0276287
Abstract: The blood fluke Cardicola forsteri (Trematoda: Aporocotylidae) is a pathogen of ranched bluefin tuna in Japan and Australia. Genomics of Cardicola spp. have thus far been limited to molecular phylogenetics of select gene sequences. In this study, sequencing of the C . forsteri genome was performed using Illumina short-read and Oxford Nanopore long-read technologies. The sequences were assembled de novo using a hybrid of short and long reads, which produced a high-quality contig-level assembly (N50 430 kb and L50 = 138). The assembly was also relatively complete and unfragmented, comprising 66% and 7.2% complete and fragmented metazoan Benchmarking Universal Single-Copy Orthologs (BUSCOs), respectively. A large portion ( 55%) of the genome was made up of intergenic repetitive elements, primarily long interspersed nuclear elements (LINEs), while protein-coding regions cover 6%. Gene prediction identified 8,564 hypothetical polypeptides, 77% of which are homologous to published sequences of other species. The identification of select putative proteins, including cathepsins, calpains, tetraspanins, and glycosyltransferases is discussed. This is the first genome assembly of any aporocotylid, a major step toward understanding of the biology of this family of fish blood flukes and their interactions within hosts.
Publisher: International Union of Crystallography (IUCr)
Date: 26-04-2002
DOI: 10.1107/S0907444902004183
Abstract: The X-ray structure of an immunoglobulin light-chain dimer isolated from the urine as a "Bence-Jones protein" from a patient with multiple myeloma and amyloidosis (Sea) was determined at 1.94 A resolution and refined to R and R(free) factors of 0.22 and 0.25, respectively. This "amyloidogenic" protein crystallized in the orthorhombic P2(1)2(1)2(1) space group with unit-cell parameters a = 48.28, b = 83.32, c = 112.59 A as determined at 100 K. In the vital organs (heart and kidneys), the equivalent of the urinary protein produced fibrillar amyloid deposits which were fatal to the patient. Compared with the amyloidogenic Mcg light-chain dimer, the Sea protein was highly soluble in aqueous solutions and only crystallized at concentrations approaching 100 mg ml(-1). Both the Sea and Mcg proteins packed into crystals in highly ordered arrangements typical of strongly diffracting crystals of immunoglobulin fragments. Overall similarities and significant differences in the three-dimensional structures and crystalline properties are discussed for the Sea and Mcg Bence-Jones proteins, which together provide a generalized model of abnormalities present in lambda chains, facilitating a better understanding of amyloidosis of light-chain origin (AL).
Publisher: Frontiers Media SA
Date: 10-06-2015
Publisher: Wiley
Date: 09-1999
DOI: 10.1002/(SICI)1099-1352(199909/10)12:5<335::AID-JMR487>3.0.CO;2-3
Publisher: Humana Press
Date: 2012
DOI: 10.1007/978-1-61779-845-0_4
Abstract: Naturally occurring and elicited anti-carbohydrate antibodies play a major role in immune responses to xenografts. The original obstacles associated with the Gal antigen have been largely resolved by the generation of knockout pigs. In contrast, much less is known about the nature and role of non-Gal carbohydrate antigens and the antibodies recognizing these. These antibodies can be identified and characterized by enzyme-linked immunosorbent assay. Furthermore, the biological significance of the non-Gal antigen(s) can be determined by expression of the relevant glycosyltransferase(s) by transfection and analyzed by antibody and/or lectin binding.
Publisher: The American Association of Immunologists
Date: 15-03-2010
Abstract: Reactive oxygen species (ROS) have been implicated in various physiological activities. However, their role in dendritic cell (DC) activation and generation has not been investigated. Using the bone marrow-derived GM-CSF–induced ex vivo DC model, we characterize how induction of ROS correlates with inflammatory DC functionality and expansion. We describe that the functionality of GM-CSF–induced DCs is distinct in two developmental stages. Whereas ision of DC-committed hematopoietic progenitor cells (HPCs) neared completion by day 6, the level of ROS soared after day 4. Day 3 ROSlo DCs were highly responsive to TLR stimuli such as LPS and zymosan by rapid upregulation of CD80, CD86, and MHC class II, in contrast to the low response of day 6 ROShi DCs. ROShi DCs could not initiate and sustain a significant level of NF-κB phosphorylation in response to LPS and zymosan, although demonstrating hyperactivation of p38 MAPK by LPS, in a fashion disparate to ROSlo DCs. ROSlo DCs stimulated a higher level of allogeneic and OVA-specific T cell proliferative responses, although ROShi DCs were much more proficient in processing OVA. In response to pathogenic stimuli, ROShi DCs also demonstrated rapid cellular adhesion and H2O2 release, indicating their role in immediate microbial targeting. Moreover, HPC expansion and DC generation were dependent on the surge of ROS in an NADPH oxidase-independent manner. These findings point to the potential role of cellular ROS in mediating functionality and development of DCs from HPCs during inflammation.
Publisher: Springer Science and Business Media LLC
Date: 02-06-2011
Abstract: CD4-binding site (CD4bs) alterations in gp120 contribute to HIV-1 envelope (Env) mediated fusogenicity and the ability of gp120 to utilize low levels of cell-surface CD4. In a recent study, we constructed three-dimensional models of gp120 to illustrate CD4bs conformations associated with enhanced fusogenicity and enhanced CD4-usage of a modestly-sized panel of blood-derived HIV-1 Envs (n = 16). These conformations were characterized by a wider aperture of the CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop. Here, we sought to provide further validation of the utility of these models for understanding mechanisms that influence Env function, by characterizing the structure-function relationships of a larger panel of Envs derived from brain and other tissues (n = 81). Three-dimensional models of gp120 were generated by our recently validated homology modelling protocol. Analysis of predicted CD4bs structures showed correlations between the aperture width of the CD4bs cavity and ability of the Envs to mediate cell-cell fusion, scavenge low-levels of cell-surface CD4, bind directly to soluble CD4, and bind to the Env mAb IgG1b12 whose epitope overlaps the gp120 CD4bs. These structural alterations in the CD4bs cavity were associated with repositioning of the V5 loop. Using a large, independent panel of Envs, we can confirm the utility of three-dimensional gp120 structural models for illustrating CD4bs alterations that can affect Env function. Furthermore, we now provide new evidence that these CD4bs alterations augment the ability of gp120 to interact with CD4 by increasing the exposure of the CD4bs.
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.VIROL.2012.06.024
Abstract: Structure-guided approaches to HIV-1 vaccine design depend on knowledge of the presentation of neutralizing epitopes on gp120, such as the epitope for the broadly neutralizing mAb b12. Here, we characterized predicted three-dimensional structures of functionally erse gp120 proteins in their b12-bound conformation, to better understand the gp120 determinants that expose or occlude the b12 epitope. Mapping the gp120-b12 binding interface identified amino acid polymorphisms within the C2, C3, C4 and V5 regions of gp120 associated with augmented b12 binding, and importantly, identified residues in the b12-exclusive binding domain of gp120 that are important for b12 neutralization resistance. Structural studies suggest that these b12 resistance variants promote reduced conformational flexibility in the b12 recognition site, which we show involves structural alterations within the gp120 CD4 binding loop and the V4 loop. Together, our studies provide new mechanistic insights into the gp120 determinants influencing sensitivity and resistance to HIV-1 neutralization by b12.
Publisher: Public Library of Science (PLoS)
Date: 08-04-2015
Publisher: Springer Science and Business Media LLC
Date: 14-06-2014
Publisher: Wiley
Date: 23-10-2010
DOI: 10.1002/JMR.1077
Abstract: Docking is a computational technique that places a small molecule (ligand) in the binding site of its macromolecular target (receptor) and estimates its binding affinity. This review addresses methodological developments that have occurred in the docking field in 2009, with a particular focus on the more difficult, and sometimes controversial, aspects of this promising computational discipline. These developments aim to address the main challenges of docking: receptor representation (such aspects as structural waters, side chain protonation, and, most of all, flexibility (from side chain rotation to domain movement)), ligand representation (protonation, tautomerism and stereoisomerism, and the effect of input conformation), as well as accounting for solvation and entropy of binding. This review is strongly focused on docking advances in the context of drug design, specifically in virtual screening and fragment-based drug design.
Publisher: Elsevier BV
Date: 12-2011
Publisher: Springer New York
Date: 23-09-2011
DOI: 10.1007/978-1-4614-0106-3_6
Abstract: A common site in the constant region (Fc) of immunoglobulins is recognized by host receptors and is a frequent target of proteins expressed by pathogens. This site is located at the junction of two constant domains in the antibody heavy chains and produces a large shallow cavity formed by loops of the CH2 and CH3 domains in IgG and IgA (CH3 and CH4 domains in IgM). Crystal structures have been determined for complexes of IgG-Fc and IgA-Fc with a structurally erse set of host, pathogen and in vitro selected ligands. While pathogen proteins may directly block interactions with the immunoglobulins thereby evading host immunity, it is likely that the same pathogen molecules also interact with other host factors to carry out their primary biological function. Herein we review the structural and functional aspects of host and pathogen molecular recognition of the common site on the Fc of immunoglobulins. We also propose that some pathogen proteins may promote virulence by affecting the bridging between innate and adaptive immunity.
Publisher: Bentham Science Publishers Ltd.
Date: 10-2009
DOI: 10.2174/138161209789105090
Abstract: Central to the initiation of a T cell dependent immune response is the recognition of major histocompatibility complex (MHC) class I or class II molecules (in humans termed HLA and in mice termed H-2) bound to antigenic peptide. T cell receptors (TCR) have programmed specificity for particular peptide/MHC complexes, which ensures focused immune responses are generated against the antigen source. To design effective peptide based vaccines a comprehensive understanding of the specific interactions between MHC molecules and peptide, and of TCR recognition of MHC eptide is valuable. We place particular emphasis on non-canonical bound peptides and their use in immunotherapy studies.
Publisher: Portland Press Ltd.
Date: 29-08-2013
DOI: 10.1042/BJ20130575
Abstract: We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby in idual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.
Publisher: Elsevier BV
Date: 05-2011
DOI: 10.1016/J.MOLIMM.2011.03.003
Abstract: The murine mAb, K-1-21, recognizes a conformational epitope expressed on free Ig kappa light chains (FκLCs) and also on cell membrane-associated FκLCs found on kappa myeloma cells. This has led to the development of a chimeric version of K-1-21, MDX-1097, which is being assessed in a Phase II clinical trial for the treatment of multiple myeloma. The epitope recognized by K-1-21 is of particular interest, especially in the context that it is not expressed on heavy chain-associated light chains such as in an intact Ig molecule. Using epitope excision techniques we have localized the K-1-21 epitope to a region spanning residues 104-110 of FκLC. This short strand of residues links the variable and constant domains, and is a flexible region that adopts different conformations in FκLC and heavy chain-associated light chain. We tested this region using site-directed mutations and found that the reactivity of K-1-21 for FκLC was markedly reduced. Finally, we applied in silico molecular docking to generate a model that satisfied the experimental data. Given the clinical potential of the Ag, this study may aid the development of next generation compounds that target the membrane form of FκLC expressed on the surface of myeloma plasma cells.
Publisher: Elsevier BV
Date: 06-2011
Publisher: Elsevier BV
Date: 06-2004
Publisher: Wiley
Date: 22-07-2016
DOI: 10.1002/JMR.2555
Abstract: The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc-protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc-binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc-protein complexes are available, numerous others have not yet been determined. Protein-protein docking could be used to investigate Fc-protein complexes however, improved approaches are necessary to efficiently model such cases. In this study, a docking-based structural bioinformatics approach is developed for predicting the structures of Fc-protein complexes. Based on the available set of X-ray structures of Fc-protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc-protein complexes, as well as in the design of peptides and proteins that target Fc.
Publisher: Springer Science and Business Media LLC
Date: 02-05-2018
Publisher: Informa UK Limited
Date: 10-01-2011
DOI: 10.1517/14712598.2011.542140
Abstract: The existence of specific carbohydrates on the surface of a wide range of cells provides the opportunity for the development of highly targeted therapeutic agents. The potential applications of such agents are erse, and include vaccines against pathogenic microorganisms, cancer and HIV, and anti-rejection agents for organ transplantation. However, the use of carbohydrates as either therapeutic agents or immunogens is frequently problematic, as they are often rapidly metabolized and poorly immunogenic. Therefore, the search for carbohydrate-mimetic agents is of considerable therapeutic value, for the potential of such agents to both interfere with carbohydrate-protein interactions and to generate carbohydrate-specific immune responses. The review discusses recent ex les of carbohydrate-mimetic peptides with regard to the structural and functional aspects of mimicry and the implications of peptide mimicry for application in therapeutics. The reader will gain knowledge of the various mechanisms of peptide carbohydrate mimicry, and the potential importance of these mechanisms in targeted therapeutic design. Peptide carbohydrate mimicry is manifested by distinct mechanisms, any one of which may be relevant to specific protein targets. As structural information becomes available for a wider variety of systems, the questions about mimicry will be more effectively addressed.
Publisher: The American Association of Immunologists
Date: 15-09-2011
Abstract: The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag–Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.
Publisher: Wiley
Date: 06-12-2003
DOI: 10.1002/PROT.10278
Abstract: Aldose reductase, a monomeric NADPH-dependent oxidoreductase, catalyzes the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The X-ray structure of human aldose reductase holoenzyme in complex with statil was determined at a resolution of 2.1 A. The carboxylate group of statil interacted with the conserved anion binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Statil's hydrophobic phthalazinyl ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group penetrating the "specificity" pocket. The interactions between the inhibitor's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding to residues Leu300 and Thr113. Based on the model of the ternary complex, the program GRID was used in an attempt to design novel potential inhibitors of human aldose reductase with enhanced binding energies of the complex. Molecular modeling calculations suggested that the replacement of the fluorine atom of statil with a carboxylate functional group may enhance the binding energies of the complex by 33%.
Publisher: Public Library of Science (PLoS)
Date: 31-10-2013
Publisher: Elsevier BV
Date: 08-2008
DOI: 10.1016/J.MOLIMM.2008.04.024
Abstract: Multiple sclerosis (MS) is an autoimmune demyelinating disease mediated primarily by CD4+ T cells. The design of peptide mutants of disease-associated myelin epitopes to alter immune responses offers a promising avenue for the treatment of MS. We designed and synthesized a number of peptide analogs by mutating the principal TCR contact residue based on MBP83-99 epitope and these peptides were conjugated to reduced mannan. Immune responses were erted from Th1 to Th2 in SJL/J mice and generated antibodies which did not cross-react with native MBP protein. Peptide [Y91]MBP83-99 gave the best cytokine and antibody profile and constitutes a promising candidate peptide for immunotherapy of MS. Structural alignment of existing crystal structures revealed the peptide binding motif of I-As. Molecular modeling was used to identify H-bonding and van der Waals interactions between peptides and MHC (I-A(s)).
Publisher: Springer Science and Business Media LLC
Date: 12-03-2018
DOI: 10.1038/S41598-018-22592-3
Abstract: Humoral immune responses against the malaria parasite are an important component of a protective immune response. Antibodies are often directed towards conformational epitopes, and the native structure of the antigenic region is usually critical for antibody recognition. We examined the structural features of various Plasmodium antigens that may impact on epitope location, by performing a comprehensive analysis of known and modelled structures from P. falciparum . Examining the location of known polymorphisms over all available structures, we observed a strong propensity for polymorphic residues to be exposed on the surface and to occur in particular secondary structure segments such as hydrogen-bonded turns. We also utilised established prediction algorithms for B-cell epitopes and MHC class II binding peptides, examining predicted epitopes in relation to known polymorphic sites within structured regions. Finally, we used the available structures to examine polymorphic hotspots and Tajima’s D values using a spatial averaging approach. We identified a region of Pf AMA1 involving both domains II and III under a high degree of balancing selection relative to the rest of the protein. In summary, we developed general methods for examining how sequence-based features relate to one another in three-dimensional space and applied these methods to key P. falciparum antigens.
Publisher: Portland Press Ltd.
Date: 10-09-2020
DOI: 10.1042/BCJ20200454
Abstract: Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea–Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.
Publisher: International Union of Crystallography (IUCr)
Date: 21-12-2001
DOI: 10.1107/S090744490101811X
Abstract: Dihydrodiol dehydrogenase catalyzes the NADP(+)-linked oxidation of trans-dihydrodiols of aromatic hydrocarbons to corresponding catechols and exists in multiple forms in mammalian tissues. The dimeric form of mammalian dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and may constitute a novel protein family with the prokaryotic proteins. Monkey kidney dimeric dihydrodiol dehydrogenase was crystallized from buffered ammonium phosphate solution using the hanging-drop vapour-diffusion method. The crystals diffract to 2.65 A resolution in the laboratory and belong to the hexagonal P6(1)22 or P6(5)22 space group, with unit-cell parameters a = b = 122.8, c = 121.3 A, alpha = beta = 90, gamma = 120 degrees.
Publisher: Elsevier BV
Date: 06-2019
DOI: 10.1016/J.JCIS.2019.03.022
Abstract: Hepatitis C virus-like particles (VLPs) are being developed as a quadrivalent vaccine candidate, eliciting both humoral and cellular immune responses in animal trials. Biophysical, biomechanical and biochemical properties are important for virus and VLP interactions with host cells and recognition by the immune system. Atomic force microscopy (AFM) is a powerful tool for visualizing surface topographies of cells, bionanoparticles and biomolecules, and for determining biophysical and biomechanical attributes such as size and elasticity. In this work, AFM was used to define morphological and nanomechanical properties of VLPs representing four common genotypes of hepatitis C virus. Significant differences in size of the VLPs were observed, and particles demonstrated a wide range of elasticity. Ordered packing of the core and potentially envelope glycoproteins was observed on the surfaces of the VLPs, but detailed structural characterization was hindered due to intrinsic dynamic fluctuations or AFM probe-induced damage of the VLPs. All VLPs were shown to be glycosylated in a manner similar to native viral particles. Together, the results presented in this study further our understanding of the nanostructure of hepatitis C VLPs, and should influence their uptake as viable vaccine candidates.
Publisher: Wiley
Date: 04-1999
DOI: 10.1002/(SICI)1097-0134(19990401)35:1<80::AID-PROT8>3.0.CO;2-3
Abstract: The phospholipase A2 enzyme, acanthoxin, found in the venom of the common death adder (Acanthophis antarcticus) as with other snake PLA2 enzymes displays neurotoxic activity. It is unclear whether this neurotoxic activity particular to some snake PLA2 enzymes is a result of structural differences solely within the catalytic sites or at a distant location upon the molecules. We have predicted the three-dimensional structure of one of the two predominant isoforms of acanthoxin (A1) using comparative protein modeling techniques. Given the high degree of homology and the availability of a high quality crystallographic structure, notexin was used as a molecular template to construct an all atom model of acanthoxin. The model was made using the program MODELLER3 and then refined with X-PLOR. Comparison between the predicted structure of acanthoxin and several X-ray structures of toxic and nontoxic PLA2 enzymes has led to a testable two-step proposal of neurotoxic PLA2 activity involving the favorable binding to acceptor molecules followed by enzymatic intrusion upon the target membrane. The electrostatic potentials across the molecular surfaces of toxic and nontoxic PLA2 enzymes were calculated (GRASP) and it was found that the toxic PLA2 enzymes possessed a charge distribution on the noncatalytic surface not identified in the nontoxic PLA2 enzymes. Thus we have identified residues potentially involved in the interaction of the PLA2 enzymes with their acceptor molecules. Furthermore, the proposed acceptor molecule recognition site is distant from the catalytic site which upon binding of the PLA2 to the acceptor molecule may enhance the enzymatic ability of the toxic PLA2 enzymes on particular cell types.
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.DCI.2005.06.011
Abstract: All jawed vertebrates contain the genetic elements essential for the function of the adaptive/combinatorial immune response, have erse sets of natural antibodies resulting from segmental gene recombination, express comparable functional repertoires and can produce specific antibodies following appropriate immunization. Profound variability occurs in the third hypervariable (CDR3) segments of light and heavy chains even within antibodies of the same ostensible specificity. Germline VH and VL elements, as well as the joining (J) segments are highly conserved among the distinct vertebrate species. Conservation is particularly noted among the VH3-like sequences of all jawed vertebrates in the FR2 and FR3 segments, as well as in the FGXGT(R or K)L J-segment characteristic of light chains and TCRs and the WGXGT(uncharged)VT JH segments. Human VH3-53 and Vlambda6 family orthologs may be present over the entire range of vertebrates. Models of the three-dimensional structures of shark VH/VL combining sites indicate similarity in framework structure and comparable CDR usage to those of man. Although carcharhine shark VH regions show greater than 50% identity to the human VH germline prototype, searches of lower deuterostome and invertebrate databases fail to detect molecules with significant relatedness. Overall, antibodies of jawed vertebrates show tremendous in idual ersity, but are constructed incorporating design features that arose with the evolutionary emergence of the jawed vertebrates and have been conserved through at least 450 million years of evolutionary time.
Publisher: Elsevier BV
Date: 04-2018
Publisher: Wiley
Date: 25-03-2013
DOI: 10.1002/JMR.2266
Abstract: The aim of docking is to accurately predict the structure of a ligand within the constraints of a receptor binding site and to correctly estimate the strength of binding. We discuss, in detail, methodological developments that occurred in the docking field in 2010 and 2011, with a particular focus on the more difficult, and sometimes controversial, aspects of this promising computational discipline. The main developments in docking in this period, covered in this review, are receptor flexibility, solvation, fragment docking, postprocessing, docking into homology models, and docking comparisons. Several new, or at least newly invigorated, advances occurred in areas such as nonlinear scoring functions, using machine-learning approaches. This review is strongly focused on docking advances in the context of drug design, specifically in virtual screening and fragment-based drug design. Where appropriate, we refer readers to exemplar case studies.
Publisher: Springer Science and Business Media LLC
Date: 20-04-2013
Abstract: The CCR5 antagonist maraviroc (MVC) inhibits human immunodeficiency virus type 1 (HIV-1) entry by altering the CCR5 extracellular loops (ECL), such that the gp120 envelope glycoproteins (Env) no longer recognize CCR5. The mechanisms of HIV-1 resistance to MVC, the only CCR5 antagonist licensed for clinical use are poorly understood, with insights into MVC resistance almost exclusively limited to knowledge obtained from in vitro studies or from studies of resistance to other CCR5 antagonists. To more precisely understand mechanisms of resistance to MVC in vivo , we characterized Envs isolated from 2 subjects who experienced virologic failure on MVC. Envs were cloned from subjects 17 and 24 before commencement of MVC (17-Sens and 24-Sens) and after virologic failure (17-Res and 24-Res). The Envs cloned during virologic failure showed broad ergence in resistance levels, with 17-Res Env exhibiting a relatively high maximal percent inhibition (MPI) of ~90% in NP2-CD4/CCR5 cells and peripheral blood mononuclear cells (PBMC), and 24-Res Env exhibiting a very low MPI of ~0 to 12% in both cell types, indicating relatively “weak” and “strong” resistance, respectively. Resistance mutations were strain-specific and mapped to the gp120 V3 loop. Affinity profiling by the 293-Affinofile assay and mathematical modeling using VERSA (Viral Entry Receptor Sensitivity Analysis) metrics revealed that 17-Res and 24-Res Envs engaged MVC-bound CCR5 inefficiently or very efficiently, respectively. Despite highly ergent phenotypes, and a lack of common gp120 resistance mutations, both resistant Envs exhibited an almost superimposable pattern of dramatically increased reliance on sulfated tyrosine residues in the CCR5 N-terminus, and on histidine residues in the CCR5 ECLs. This altered mechanism of CCR5 engagement rendered both the resistant Envs susceptible to neutralization by a sulfated peptide fragment of the CCR5 N-terminus. Clinical resistance to MVC may involve ergent Env phenotypes and different genetic alterations in gp120, but the molecular mechanism of resistance of the Envs studied here appears to be related. The increased reliance on sulfated CCR5 N-terminus residues suggests a new avenue to block HIV-1 entry by CCR5 N-terminus sulfopeptidomimetic drugs.
Publisher: American Society for Microbiology
Date: 15-10-2011
DOI: 10.1128/JVI.05510-11
Abstract: Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 ( n = 14) or CXCR4 ( n = 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of ergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.
Publisher: Wiley
Date: 03-2002
DOI: 10.1046/J.0300-9475.2002.01032.X
Abstract: A monoclonal immunoglobulin (Ig)M cryoglobulin (Mez) with interesting binding behaviour was isolated from a Waldenström's macroglobulinemia (WM) patient. It demonstrated very strong binding to peptides derived from the sequences of human IgG. However, when tested for binding to intact IgG, this antibody (Ab) did not show any rheumatoid factor (RF) activity. We propose several nonexclusive structural interpretations of the Mez-binding propensities, based on the orientations and solvent accessibilities of ligand residues and the nature of the Ab-binding site. To further characterize the structural features of Mez-peptide binding, IgG-derived octapeptides were docked into the Mez fragment variable (Fv)-binding site, revealing additional reasons for Mez-binding selectivity based on the interactions of the docked peptides with the Mez Fv. The problem was also approached from an immunological perspective. Comparisons of Mez variable region of the light chain (VL)/variable region of the heavy chain (VH) sequences with those of human germlines and known IgM RFs allowed us to provide a possible outline tracing the structural and functional origins of the Mez IgM. Coupled with examinations of interactions in docked complexes, this analysis led us to propose that the potential for RF activity, demonstrated through Mez binding to IgG-derived peptides, was owing to the inherent sequence and structure of the Mez IgM, rather than to somatic mutations. Thus, Mez IgM may occupy an intermediate niche between IgMs with and without RF activity.
Publisher: IEEE
Date: 2012
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.MOLIMM.2015.02.028
Abstract: Monoclonal antibodies represent the most successful class of biopharmaceuticals for the treatment of cancer. Mechanisms of action of therapeutic antibodies are very erse and reflect their ability to engage in antibody-dependent effector mechanisms, internalize to deliver cytotoxic payloads, and display direct effects on cells by lysis or by modulating the biological pathways of their target antigens. Importantly, one of the universal changes in cancer is glycosylation and carbohydrate-binding antibodies can be produced to selectively recognize tumor cells over normal tissues. A promising group of cell surface antibody targets consists of carbohydrates presented as glycolipids or glycoproteins. In this review, we outline the basic principles of antibody-based targeting of carbohydrate antigens in cancer. We also present a detailed structural view of antibody recognition and the conformational properties of a series of related tissue-blood group (Lewis) carbohydrates that are being pursued as potential targets of cancer immunotherapy.
Publisher: Informa UK Limited
Date: 04-2008
Publisher: Springer New York
Date: 2015
DOI: 10.1007/978-1-4939-2874-3_4
Abstract: Carbohydrate-protein recognition is vital to many processes in health and disease. In particular, elucidation of the structural basis of carbohydrate binding is important to the development of oligosaccharides and oligosaccharide mimetics as vaccines for infectious diseases and cancer. Computational structural techniques are valuable for the study of carbohydrate-protein recognition due to the challenges associated with experimental determination of carbohydrate-protein complexes. AutoMap is a computer program that we have developed to study protein-ligand recognition. AutoMap determines the interactions taking place in a set of highly ranked poses obtained from molecular docking and processes these to identify the protein residues most likely to be involved in interactions. In this protocol, we describe the use of AutoMap and illustrate its suitability for studying antibody recognition of the Lewis Y tetrasaccharide, which is a potential cancer vaccine antigen.
Publisher: S. Karger AG
Date: 2001
DOI: 10.1159/000049197
Abstract: Available data suggest that ‘primitive’ antibody-combining sites often include longer than average HCDR3s. Long HCDR3 sequences have been reported in erse vertebrates, including humans, cattle, camels and sharks. These long HCDR3 segments contain unusual sequence features such as stretches of Gly or Pro residues and multiple Cys residues. We examined how longer than average HCDR3s were accommodated in the V domains of human, murine and camel antibodies with known three-dimensional structures. The main conclusions were that (1) HCDR3s longer than 12 residues should protrude outward from the V domains (2) descending HCDR3 polypeptides may utilize VL (including LCDR3) constituents as a platform, supporting the protruding segments (3) intra- and inter-HCDR disulfides are frequently formed to rigidify the structure of HCDR3 or the combining site, and (4) V and C domains were possibly more similar in primordial antibodies than they are in their present day counterparts.
Publisher: Oxford University Press (OUP)
Date: 22-02-2010
Abstract: Carbohydrates are notoriously flexible molecules. However, they have an important role in many biochemical processes as specific ligands. Understanding how carbohydrates are recognized by other biological macromolecules (usually proteins) is therefore of considerable scientific value. Interfering with carbohydrate-protein interactions is a potentially useful strategy in combating a range of disease states, as well as being of critical importance in facilitating allo- and xenotransplantation. We have devised an in silico protocol for analyzing carbohydrate-protein interactions. In this study, we have applied the protocol to determine the structures of alphaGal-terminating carbohydrate antigens in complex with a panel of xenoreactive antibodies. The most important feature of the binding modes is the fixed conformation of the Galbeta(1,4)Glc/GlcNAc linkage across all of the binding modes. The preferred conformation of the terminal Galalpha(1,3)Gal linkage varies depending on the antibody binding site topography, although it is possible that some of the antibodies studied recognize more than one Galalpha(1,3)Gal conformation. The binding modes obtained indicate that each antibody uses distinct mechanisms in recognizing the target antigens.
Publisher: Elsevier BV
Date: 06-2017
DOI: 10.1016/J.SBI.2016.10.009
Abstract: Carbohydrate-binding antibodies and carbohydrate-based vaccines are being actively pursued as targeted immunotherapies for a broad range of cancers. Recognition of tumor-associated carbohydrates (glycans) by antibodies is predominantly towards terminal epitopes on glycoproteins and glycolipids on the surface of cancer cells. Crystallography along with complementary experimental and computational methods have been extensively used to dissect antibody recognition of glycan epitopes commonly found in cancer. We provide an overview of the structural biology of antibody recognition of tumor-associated glycans and propose potential rearrangements of these targets in the membrane that could dictate the complex biological activities of these antibodies against cancer cells.
Publisher: Frontiers Media SA
Date: 2011
Publisher: Oxford University Press (OUP)
Date: 22-11-2018
DOI: 10.1093/JB/MVY099
Abstract: In order to facilitate a number of processes including nutrient acquisition and immune evasion, malaria parasites extensively remodel their host erythrocyte. This remodelling is to a large extent accomplished through protein export, a crucial process mediated by the Plasmodium translocon for exported proteins (PTEX) translocon which is comprised of three core components, HSP101, PTEX150 and EXP2. EXP2 has been structurally and electrophysiologically shown to form the pore that spans the vacuole membrane enveloping the parasite. Here, we biochemically investigate the structure and function of EXP2. By differential alkylation we provide direct evidence that cysteines C113 and C140 form an intramolecular disulphide bond, while C201 is predominantly in a reduced state. We demonstrate that EXP2 possesses a protease resistant, membrane-associated, N-terminal region of ∼20 kDa that does not project into the infected erythrocyte cytosol however, its C-terminus does project into the vacuole space. We show that a putative transmembrane peptide derived from the N-terminal region of EXP2 is haemolytic and in a polymer-based osmotic protection assay, we demonstrate that this peptide forms a discrete haemolytic pore. This work provides further biochemical insight into the role, function and cellular arrangement of EXP2 as the pore-forming component for protein translocation.
Publisher: Oxford University Press (OUP)
Date: 07-2003
Abstract: Naturally occurring antibody repertoires of cattle (Bos taurus) include a group of IgMlambda antibodies with exceptionally long complementarity-determining region 3 of the heavy chain (CDR3H) segments, containing multiple Cys residues. These massive CDR3H segments will greatly influence the tertiary and quaternary structures of the bovine IgM combining sites. As an antibody's combining site is formed by both heavy and light chains, we have analyzed the nucleotide sequences and structural properties of the lambda-light chains that pair with micro -heavy chains containing exceptionally long CDR3H. There appears to be an exquisite selective pressure for the use of three V(lambda)1 genes (V(lambda)1x and two new V(lambda)1d and V(lambda)1e genes) in IgM with unusually long CDR3H. The V(lambda)1d and V(lambda)1e genes are similar to each other, but erge from the other V(lambda)1 genes into two closely related subfamilies. The available bovine V(lambda) genes are classified into three V(lambda) gene families: V(lambda)1, V(lambda)2 and V(lambda)3 based on nucleotide similarity >/=80%. Further, analysis of total Ser content and positions of Ser residues in the sequences was found to be sufficient to classify the cattle V(lambda)1 subfamilies. Patterns of Ser residues differ for V(lambda) domains from ruminant species (e.g. cattle, sheep and goats) and other mammals (e.g. humans and mice). These 'Ser signatures' can be used to track ergent evolution in lambda-light chains. Interestingly, Ser90L in complementarity-determining region 3 of the light chain (CDR3L) occurred in all V(lambda) domains that pair with V(H) regions containing exceptionally long CDR3H. A structural role for Ser90L was revealed in homology models of V(lambda) domains, i.e. to hold the ascending polypeptide of CDR3L in a relatively tight space between the N-terminal segment and residues from CDR1L. The CDR3L of V(lambda) domains also occupied smaller volumes if paired to V(H) domains with extremely long CDR3H (>/=48 residues), and were more variable in their conformation and filled larger volumes if CDR3Hs were </=22 residues. Thus, the role of the lambda-light chains in these unusual cattle antibodies is probably to act as a relatively featureless supporting platform for the extremely long CDR3H regions, which undoubtedly are dominantly involved in binding to an antigen.
Publisher: Portland Press Ltd.
Date: 11-04-2006
DOI: 10.1042/BJ20051739
Abstract: The 2.6 Å (1 Å=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenström's macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611–39619 Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024–14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds.
Publisher: Wiley
Date: 2003
DOI: 10.1002/JMR.610
Abstract: Crystals of a human (Sea) Bence-Jones dimer were produced in a capillary by vapor diffusion under microgravity conditions in the 9 day US Space Shuttle Mission STS-95. In comparison to ground-based experiments, nucleation was facile and spontaneous in space. Appearance of a very large (8 x 1.6 x 1.0 mm) crystal in a short time period is a strong endorsement for the use of microgravity to produce crystals sufficiently large for neutron diffraction studies. The Sea dimer crystallized in the orthorhombic space group P2(1)2(1)2(1), with a = 48.9 A, b = 85.2 A, and c = 114.0 A. The crystals grown in microgravity exhibited significantly lower mosaicities than those of ground-based crystals and the X-ray diffraction data had a lower overall B factor. Three-dimensional structures determined by X-ray analysis at two temperatures (100 and 293 K) were indistinguishable from those obtained from ground-based crystals. However, both the crystallographic R factor and the free R factor were slightly lower in the models derived from crystals produced in microgravity. The major difference between the two crystal growth systems is a lack of convection and sedimentation in a microgravity environment. This environment resulted in the growth of much larger, higher-quality crystals of the Sea Bence-Jones protein. Structurally, heretofore unrecognized grooves on the external surfaces of the Sea and other immunoglobulin-derived fragments are regular features and may offer supplementary binding regions for super antigens and other elongated ligands in the bloodstream and perivascular tissues.
Publisher: Springer Science and Business Media LLC
Date: 17-03-2016
Publisher: Oxford University Press (OUP)
Date: 21-06-2018
DOI: 10.1093/BIOINFORMATICS/BTY474
Abstract: A sliding window analysis over a protein or genomic sequence is commonly performed, and we present a Python tool, BioStructMap, that extends this concept to three-dimensional (3D) space, allowing the application of a 3D sliding window analysis over a protein structure. BioStructMap is easily extensible, allowing the user to apply custom functions to spatially aggregated data. BioStructMap also allows mapping of underlying genomic sequences to protein structures, allowing the user to perform genetic-based analysis over spatially linked codons—this has applications when selection pressures arise at the level of protein structure. The Python BioStructMap package is available at ndrewguy/biostructmap and released under the MIT License. An online server implementing standard functionality is available at biostructmap.burnet.edu.au. Supplementary data are available at Bioinformatics online.
Publisher: Public Library of Science (PLoS)
Date: 14-10-2014
Publisher: Wiley
Date: 06-11-2009
Publisher: The American Association of Immunologists
Date: 15-01-2014
Abstract: Little is known of the impact of Fc receptor (FcR) polymorphism in macaques on the binding of human (hu)IgG, and nothing is known of this interaction in the pig-tailed macaque (Macaca nemestrina), which is used in preclinical evaluation of vaccines and therapeutic Abs. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcγRIIa (mnFcγRIIa) and inhibitory FcγRIIb (mnFcγRIIb) and predicted their structures using the huIgGFc/huFcγRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcγRIIa and mnFcγRIIb compared with their human FcR counterparts. MnFcγRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcγRIIa (His131). In contrast, mnFcγRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcγRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcγRIIa showed that Pro159 and Tyr160 impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcγRIIb was shown to be dependent on His131 and Met132. Significantly, both His131 and Met132 are conserved across FcγRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcγRIIa wherein proline at position 131, also an important polymorphic site in huFcγRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcγRIIa His131. These marked interspecies differences in IgG binding between human and macaque FcRs and polymorphisms within species have implications for preclinical evaluation of Abs and vaccines in macaques.
Publisher: Elsevier BV
Date: 06-2006
Publisher: Elsevier BV
Date: 06-2021
Publisher: Wiley
Date: 18-06-2020
DOI: 10.1111/ALL.14410
Publisher: American Chemical Society (ACS)
Date: 08-12-2009
DOI: 10.1021/CI900388A
Abstract: Cell surface glycoproteins play vital roles in cellular homeostasis and disease. Antibody recognition of glycosylation on different cells and pathogens is critically important for immune surveillance. Conversely, adverse immune reactions resulting from antibody-carbohydrate interactions have been implicated in the development of autoimmune diseases and impact areas such as xenotransplantation and cancer treatment. Understanding the nature of antibody-carbohydrate interactions and the method by which saccharides fit into antibody binding sites is important in understanding the recognition process. In silico techniques offer attractive alternatives to experimental methods (X-ray crystallography and NMR) for the study of antibody-carbohydrate complexes. In particular, molecular docking provides information about protein-ligand interactions in systems that are difficult to study with experimental techniques. Before molecular docking can be used to investigate antibody-carbohydrate complexes, validation of an appropriate docking method is required. In this study, four popular docking programs, Glide, AutoDock, GOLD, and FlexX, were assessed for their ability to accurately dock carbohydrates to antibodies. Comparison of top ranking poses with crystal structures highlighted the strengths and weaknesses of these programs. Rigid docking, in which the protein conformation remains static, and flexible docking, where both the protein and ligand are treated as flexible, were compared. This study has revealed that generally molecular docking of carbohydrates to antibodies has been performed best by Glide.
Publisher: Elsevier BV
Date: 09-2016
DOI: 10.1016/J.MOLIMM.2016.07.014
Abstract: We discovered that some bovine antibodies are amongst the largest known to exist due to the presence of an exceptionally long CDR3H (≥49 amino acids) with multiple cysteines that provide a unique knob and stalk structure to the antigen binding site. The large CDR3H size, unlike mouse and human, provides a suitable platform for antigenization with large configurational B-epitopes. Here we report the identification of a B-epitope on the gC envelope protein of bovine herpes virus type-1 (BoHV-1) recognized by a bovine IgG1 antibody. The identified 156 amino acid long gC fragment (gC156) was expressed as a recombinant protein. Subsequently, a functional scFv fragment with a 61 amino-acid long CDR3H (scFv1H12) was expressed such that gC156 was grafted into the CDR3H, replacing the "knob" region (gC156scFv1H12 or Ag-scFv). Importantly, the Ag-scFv could be recognized by a neutralizing antibody fragment (scFv3-18L), which suggests that the engraftment of gC156 into the CDR3H of 1H12 maintained the native conformation of the BoHV-1 B-epitope. A 3D model of gC156 was generated using fold-recognition approaches and this was grafted onto the CDR3H stalk of the 1H12 Fab crystal structure to predict the 3D structure of the Ag-scFv. The grafted antigen in Ag-scFv is predicted to have a compact conformation with the ability to protrude into the solvent. Upon immunization of bovine calves, the antigenized scFv (gC156scFv1H12) induced a higher antibody response as compared to free recombinant gC156. These observations suggest that antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing the next generation of vaccines against infectious agents that require induction of protective humoral immunity.
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.MOLIMM.2015.05.004
Abstract: Therapeutic antibodies have come of age with major progress being made in cancer, autoimmunity and chronic inflammation, as well as a wide range of other human diseases. Antibody engineering is further driving development of novel antibody formats and genetically modified cell-based therapies that harness the power of the immune system to progress cures in otherwise intractable human diseases. Nevertheless, there are still significant challenges ahead for basic and applied research relating to therapeutic antibodies. This special issue of the journal provides reviews and opinions that relate to the discovery, design and deployment of antibodies as therapeutics.
Publisher: Wiley
Date: 09-2002
DOI: 10.1002/JMR.582
Publisher: Springer Science and Business Media LLC
Date: 25-06-2019
DOI: 10.1038/S41598-019-45461-Z
Abstract: The introduction of directly acting antiviral agents (DAAs) has produced significant improvements in the ability to cure chronic hepatitis C infection. However, with over 2% of the world’s population infected with HCV, complications arising from the development of cirrhosis of the liver, chronic hepatitis C infection remains the leading indication for liver transplantation. Several modelling studies have indicated that DAAs alone will not be sufficient to eliminate HCV, but if combined with an effective vaccine this regimen would provide a significant advance towards achieving this critical World Health Organisation goal. We have previously generated a genotype 1a, 1b, 2a, 3a HCV virus like particle (VLP) quadrivalent vaccine. The HCV VLPs contain the core and envelope proteins (E1 and E2) of HCV and the vaccine has been shown to produce broad humoral and T cell immune responses following vaccination of mice. In this report we further advanced this work by investigating vaccine responses in a large animal model. We demonstrate that intradermal microneedle vaccination of pigs with our quadrivalent HCV VLP based vaccine produces long-lived multi-genotype specific and neutralizing antibody (NAb) responses together with strong T cell and granzyme B responses and normal Th1 and Th2 cytokine responses. These responses were achieved without the addition of adjuvant. Our study demonstrates that our vaccine is able to produce broad immune responses in a large animal that, next to primates, is the closest animal model to humans. Our results are important as they show that the vaccine can produce robust immune responses in a large animal model before progressing the vaccine to human trials.
Publisher: Wiley
Date: 09-2002
DOI: 10.1002/JMR.585
Abstract: Sequencing of all human immunoglobulin (Ig) germline gene segments has recently been completed. However, our first glimpses of the recombined products of this combinatorial gene system were in the 1970s, in landmark publications, reporting the crystal structures of two human myeloma proteins, the Mcg lambda light chain dimer and the New IgG1(lambda) Fab. Although numerous crystal structures of murine and human antibodies have now been determined, only a relatively small proportion of the human germline genes have had their corresponding protein three-dimensional structures resolved. Therefore, further structural investigations are required before the inherent ersity of the antibody repertoire can be fully appreciated. We discuss the detailed structural information available for human antibodies with regard to their immune functions. Also discussed, is how the structural information is finding application in the 'humanization' of murine antibodies as part of their development as 'biopharmaceuticals' for the treatment of human disease.
Publisher: Elsevier BV
Date: 09-2010
Publisher: Proceedings of the National Academy of Sciences
Date: 12-2003
Abstract: Peptides bind MHC class I molecules by anchoring hydrophobic side chains into pockets in the peptide binding groove. Here, we report an immunogenic ( in vitro and in vivo ) MUC1 glycopeptide (MUC1–8-5GalNAc) bound to H-2K b , fully crossreactive with the nonglycosylated variant. Molecular modeling showed that the central P5-Thr-GalNAc residue points into the C pocket and forms van der Waals and hydrogen bond interactions with the MHC class I. As predicted, GalNAc, a modified peptide carrying an additional anchor in the central C anchor pocket, increased the affinity by ≈100-fold compared with the native low-affinity peptide (MUC1–8). The findings demonstrate that glycopeptides associated with MHC class I molecules can use GalNAc to anchor the peptide in the groove and enable high-affinity binding.
Publisher: Public Library of Science (PLoS)
Date: 04-04-2013
Publisher: Wiley
Date: 2005
DOI: 10.1002/JMR.742
Abstract: As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.
Publisher: Portland Press Ltd.
Date: 05-07-2018
DOI: 10.1042/BCJ20180139
Abstract: Antibody engineering is important for many diagnostic and clinical applications of monoclonal antibodies. We recently reported a series of fragment crystallizable (Fc) mutations targeting the neonatal Fc receptor (FcRn) site on a Lewis Y (Ley) binding IgG1, hu3S193. The hu3S193 variants displayed shortened in vivo half-lives and may have potential for radioimaging or radiotherapy of Ley-positive tumors. Here, we report Fc crystal structures of wild-type hu3S193, seven FcRn-binding site variants, and a variant lacking C1q binding or complement-dependent cytotoxicity (CDC) activity. The Fc conformation of the FcRn-binding sites was similar for wild-type and all mutants of hu3S193 Fc, which suggests that FcRn interactions were directly affected by the amino acid substitutions. The C1q-binding site mutant Fc was nearly identical with the wild-type Fc. Surprisingly, several hu3S193 Fc variants showed large changes in global structure compared with wild-type Fc. All hu3S193 Fc mutants had similar antibody-dependent cellular cytotoxicity, despite some with conformations expected to diminish Fc gamma receptor binding. Several hu3S193 variants displayed altered CDC, but there was no correlation with the different Fc conformations. All versions of hu3S193, except the C1q-binding site mutant, bound C1q, suggesting that the altered CDC of some variants could result from different propensities to form IgG hexamers after engaging Ley on target cells. Overall, our findings support the concept that the antibody Fc is both flexible and mobile in solution. Structure-based design approaches should take into account the conformational plasticity of the Fc when engineering antibodies with optimal effector properties.
Publisher: Elsevier BV
Date: 12-12-2019
Abstract: Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5–2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.MOLIMM.2015.03.255
Abstract: Monoclonal antibodies are being developed as therapeutics to complement drugs and vaccines or to fill the gap where no drugs or vaccines exist. These therapeutic antibodies (ThAb) may be especially important for infectious diseases in which there is antibiotic resistance, toxin-mediated pathogenesis, or for emerging pathogens. The unique structure of antibodies determines the specific nature of the effector function, so when developing ThAb, the desired effector functions need to be considered and integrated into the design and development processes to ensure maximum efficacy and safety. Antibody subclass is a critical consideration, but it is noteworthy that almost all ThAb that are licenced or currently in development utilise an IgG1 backbone. This review outlines the major structural properties that vary across subclasses, how these properties affect functional immunity, and discusses the various approaches used to study subclass responses to infectious diseases. We also review the factors associated with the selection of antibody subclasses when designing ThAb and highlight circumstances where different subclass properties might be beneficial when applied to particular infectious diseases. These approaches are critical to the future design of ThAb and to the study of naturally-acquired and vaccine-induced immunity.
Publisher: Public Library of Science (PLoS)
Date: 17-10-2019
Publisher: Wiley
Date: 30-08-2011
DOI: 10.1111/J.1365-2893.2011.01504.X
Abstract: The NS2 protein of hepatitis C virus (HCV) plays a critical role in virus morphogenesis and infectivity. The crystal structure of the C-terminus of the NS2 protein (NS2(Pro)) from the H77 strain indicates that NS2(Pro) forms a homodimer. In this study, using computational modelling, we identified residues at the NS2(Pro) dimer interface that have a role in dimerization and confirmed their capacity to influence dimerization by expression studies. Our modelling analysis identified 22 residues at the NS2(Pro) dimer interface that may be important for dimer formation. Based on the free binding energy, we selected the top five ranked mutations (V162A, M170A, I175A, D186A and I201A) for further study. Western blot analysis revealed that M170A, I175A, I201A, D186A and V162A resulted in a 4.0-, 3.2-, 3.0-, 2.8- and 1.5-fold increase, respectively, in the monomer/dimer ratio compared to wild type, confirming a role in homodimer formation or stability. Japanese Fulminant Hepatitis type 1 mutants expressing M170A, I175A, D186A and I201A demonstrated increasing defects in both RNA replication and the production of infectious virus compared to wild type. This study identified residues at the NS2(Pro) dimer interface that modulate NS2(Pro) homodimerization and demonstrated that abrogation of NS2(Pro) homodimerization results in defects in HCV replication and release of infectious virus.
Publisher: Elsevier BV
Date: 05-2017
DOI: 10.1111/JTH.13666
Abstract: Essentials Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies. Background Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods PC-based nanocapsules (2.8 × 10
Publisher: Elsevier BV
Date: 06-2020
DOI: 10.1016/J.SBI.2019.12.001
Abstract: Because of the ongoing increase in antibiotic-resistant microbes, new strategies such as therapeutic antibodies and effective vaccines are required. Bacterial carbohydrates are known to be particularly antigenic, and several monoclonal antibodies that target bacterial polysaccharides have been generated, with more in current development. This review examines the known 3D crystal structures of anti-bacterial antibodies and the structural basis for carbohydrate recognition and explores the potential mechanisms for antibody-dependent bacterial cell death. Understanding the key interactions between an antibody and its polysaccharide target on the surface of bacteria or in biofilms can provide essential information for the development of more specific and effective antibody therapeutics as well as carbohydrate-based vaccines.
Publisher: Informa UK Limited
Date: 30-03-2016
Publisher: Oxford University Press (OUP)
Date: 07-2015
DOI: 10.1093/CID/CIV525
Publisher: Elsevier BV
Date: 07-2004
Publisher: Elsevier BV
Date: 03-2001
Publisher: Elsevier BV
Date: 06-2004
Publisher: Wiley
Date: 09-2002
DOI: 10.1002/JMR.595
Abstract: Mcg light chain dimer has been extensively studied by crystallography and peptide binding studies to investigate its peptide cross-reactivity as well as to use it as a model system for designing space filling peptide ligands. Here we extend these investigations by utilizing automated docking. Mcg light chain dimer is an ideal model system for such study due to the availability of experimental data for both the native structure and the 14 complexes with various peptide ligands. We show the ability of the docking approach to reproduce the experimental structures and discuss the limitations associated with such outcomes. We demonstrate the usefulness of the docking approach in generating structural information otherwise not available from the experiment.
Publisher: Frontiers Media SA
Date: 02-06-2015
Publisher: Wiley
Date: 09-2002
DOI: 10.1002/JMR.596
Abstract: The VH domains of two human monoclonal antibodies, designated Mcg IgG1(lambda) and Yvo IgM(kappa), were particularly intractable to standard protein sequencing protocols. Peptides liberated from the VH domains of these proteins, using standard enzymatic or chemical cleavages, invariably precipitated during the procedures. Boiling in SDS containing buffers dissolved precipitates and the peptides were separated using SDS-PAGE. Fully overlapped VH sequences were obtained with a series of 'in-gel' cleavages, followed by passive/differential transfers of peptides onto PVDF membranes. Both the in-gel cleavages and passive transfers could be applied to 'wet' or 'dry' gels so that gels could be archived and used at a later date to obtain additional sequence information from a fragment of interest. Repetitive yields of even the most insoluble peptides were such that the sequences of various peptides from relatively complex mixtures of peptides could be assigned with confidence. Despite the overall success of the sequencing, we occasionally referred to electron density maps, calculated for crystals of the Fab of Yvo IgM, to resolve particular sequences and confirm ambiguous amino acid assignments. Methods we describe in this report should be generally useful for obtaining sequences of proteins with intractable cores and may find many applications in the 'post genomic era'.
Publisher: Wiley
Date: 21-10-2005
DOI: 10.1111/J.1440-1711.2005.01374.X
Abstract: Lewis system carbohydrate antigens have been shown to be expressed at high levels in many cancers of epithelial cell origin, including those of colon, breast, lung, prostate and ovary. The type 1 (Le(a) and Le(b)) antigens are important histo-blood groups, while type 2 (Le(x) and Le(y)) antigens in healthy in iduals are only expressed, at relatively low levels, by a few tissues, including some epithelial cells. Thus, the type 2 antigens are considered to be tumour-associated antigens and are promising targets for cancer treatment, including antibody-based immunotherapy. In this review, we discuss the conformational characteristics of the free and bound forms of Lewis oligosaccharides and the 3D structures of antibodies in complex with Le(y) and Le(x) antigens. Collectively, the structural studies have demonstrated that the Lewis determinants are rigid structures, which generally maintain the same conformation in the free and bound states. The rigid nature and similarities in shape of type 1 and 2 Lewis oligosaccharides appear to make them perfectly suited to driving a structurally convergent immune response (at least in the case of Le(y) specific antibodies) toward a highly specific recognition of in idual carbohydrate determinants, which is a goal in the development of effective antibody-based cancer treatments.
Publisher: CRC Press
Date: 27-09-2012
DOI: 10.1201/B12965
Publisher: Wiley
Date: 21-03-2015
DOI: 10.1002/JMR.2471
Abstract: Molecular docking is a computational method for predicting the placement of ligands in the binding sites of their receptor(s). In this review, we discuss the methodological developments that occurred in the docking field in 2012 and 2013, with a particular focus on the more difficult aspects of this computational discipline. The main challenges and therefore focal points for developments in docking, covered in this review, are receptor flexibility, solvation, scoring, and virtual screening. We specifically deal with such aspects of molecular docking and its applications as selection criteria for constructing receptor ensembles, target dependence of scoring functions, integration of higher-level theory into scoring, implicit and explicit handling of solvation in the binding process, and comparison and evaluation of docking and scoring methods.
Publisher: Public Library of Science (PLoS)
Date: 29-10-2015
Publisher: Wiley
Date: 08-2005
DOI: 10.1111/J.1440-1711.2005.01367.X
Abstract: Carbohydrate antigens have a central role in the hyperacute rejection of animal-to-human organ grafts (xenotransplantation) and they are emerging in importance in the immunotherapy of cancer. This article traces the historical origins of the discovery of key carbohydrate antigens and explores the future impact of recent technological advances of the field of glycobiology as it relates to xenotransplantation and cancer.
Publisher: Proceedings of the National Academy of Sciences
Date: 18-09-2007
Abstract: Infection by Staphylococcus aureus can result in severe conditions such as septicemia, toxic shock, pneumonia, and endocarditis with antibiotic resistance and persistent nasal carriage in normal in iduals being key drivers of the medical impact of this virulent pathogen. In both virulent infection and nasal colonization, S. aureus encounters the host immune system and produces a wide array of factors that frustrate host immunity. One in particular, the prototypical staphylococcal superantigen-like protein SSL7, potently binds IgA and C5, thereby inhibiting immune responses dependent on these major immune mediators. We report here the three-dimensional structure of the complex of SSL7 with Fc of human IgA1 at 3.2 Å resolution. Two SSL7 molecules interact with the Fc (one per heavy chain) primarily at the junction between the Cα2 and Cα3 domains. The binding site on each IgA chain is extensive, with SSL7 shielding most of the lateral surface of the Cα3 domain. However, the SSL7 molecules are positioned such that they should allow binding to secretory IgA. The key IgA residues interacting with SSL7 are also bound by the leukocyte IgA receptor, FcαRI (CD89), thereby explaining how SSL7 potently inhibits IgA-dependent cellular effector functions mediated by FcαRI, such as phagocytosis, degranulation, and respiratory burst. Thus, the ability of S. aureus to subvert IgA-mediated immunity is likely to facilitate survival in mucosal environments such as the nasal passage and may contribute to systemic infections.
Publisher: Wiley
Date: 25-11-2009
DOI: 10.1038/ICB.2008.82
Abstract: The interaction of immune complexes with the human Fc receptor, FcgammaRIIa, initiates the release of inflammatory mediators and is implicated in the pathogenesis of human autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, so this FcR is a potential target for therapy. We have used the three-dimensional structure of an FcgammaRIIa dimer to design small molecule inhibitors, modeled on a distinct groove and pocket created by receptor dimerization, adjacent to the ligand-binding sites. These small chemical entities (SCEs) blocked immune complex-induced platelet activation and aggregation and tumor necrosis factor secretion from macrophages in a human cell line and transgenic mouse macrophages. The SCE appeared specific for FcgammaRIIa, as they inhibited only immune complex-induced responses and had no effect on responses to stimuli unrelated to FcR, for ex le platelet stimulation with arachidonic acid. In vivo testing of the SCE in FcgammaRIIa transgenic mice showed that they inhibited the development and stopped the progression of collagen-induced arthritis (CIA). The SCEs were more potent than methotrexate and anti-CD3 in sustained suppression of CIA. Thus, in vitro and in vivo activity of these SCE FcgammaRIIa receptor antagonists demonstrated their potential as anti-inflammatory agents for autoimmune diseases involving immune complexes.
Publisher: Elsevier BV
Date: 2007
DOI: 10.1016/J.MOLIMM.2007.06.152
Abstract: A disulphide-constrained peptide that binds to the low affinity Fc receptor, FcgammaRIIa (CD32) has been identified and its structure solved by NMR. Linear (7-mer and 12-mer) and disulphide-constrained (7-mer) phage display peptide libraries were panned on recombinant soluble FcgammaRIIa genetically fused to HSA (HSA-FcgammaRIIa). Peptides were isolated only from the constrained peptide library and these contained the consensus sequence, CWPGWxxC. Phage clones displaying variants of the peptide consensus sequence bound to FcgammaRIIa and the strongest binding clone C7C1 (CWPGWDLNC) competed with IgG for binding to FcgammaRIIa and was inhibited from binding to FcgammaRIIa by the FcgammaRIIa-blocking antibody, IV.3, suggesting that C7C1 and IgG share related binding sites on FcgammaRIIa. A synthetic disulphide-constrained peptide, pep-C7C1 bound to FcgammaRIIa by biosensor analysis, albeit with low affinity (KD approximately 100microM). It was significant that the FcgammaRIIa consensus peptide sequence contained a Proline (Pro3), which when substituted with alanine abrogated FcgammaRIIa binding, consistent with Pro3 contributing to receptor binding. Upon binding of IgG and IgE to their respective Fc receptors (FcgammaRs and FcepsilonRI) Pro329 in the Fc makes a critical interaction with two highly conserved Trp residues (Trp90 and Trp113) of the FcRs. The NMR structure of pep-C7C1 revealed a stabilizing type II beta-turn between Trp2 and Trp5, with Pro3 solvent exposed. Modelling of the pep-C7C1 structure in complex with FcgammaRIIa suggests that Pro3 of C7C1 binds to FcgammaRIIa by inserting between Trp90 and Trp113 of FcgammaRIIa thereby mimicking the molecular interaction made between FcgammaRIIa and IgG.
Publisher: Wiley
Date: 03-08-2006
Publisher: Springer Science and Business Media LLC
Date: 22-08-2019
Publisher: No publisher found
Date: 2013
DOI: 10.1021/JP309109K
Abstract: Cold-induced precipitation of a monoclonal IgM cryoglobulin isolated from a patient with Waldenström's macroglobulinemia was observed to have a negative activation enthalpy. The rate of the reaction increased, as the temperature decreased. Differential scanning calorimetry of the monoclonal IgM showed precipitation as an inverted peak during a downward temperature scan. The transition temperature was between 14 and 15 °C and was possibly concentration dependent. At temperatures below the transition the precipitation was best described by second-order kinetics. The difference in change in enthalpy between precipitation and disassociation suggests that cold-induced precipitation had a fast precipitation stage followed by a slower consolidation reaction. Negligible curvature of the Eyring plot suggested the precipitation reaction was dominated by van der Waal forces and hydrogen bonding. Conversely, during an upward temperature scan, disassociation was observed as a positive enthalpy peak. This reaction had two stages, a reaction undoing consolidation followed by heat-induced disassociation that had first-order kinetics.
Publisher: Elsevier BV
Date: 2020
Publisher: Public Library of Science (PLoS)
Date: 23-08-2010
Publisher: Elsevier BV
Date: 02-2011
DOI: 10.1016/J.VIROL.2010.12.010
Abstract: CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally erse R5 envelope (Env) clones (n=16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.
Publisher: Wiley
Date: 2011
DOI: 10.1002/BIP.21427
Abstract: Carbohydrate-antibody interactions mediate many cellular processes and immune responses. Carbohydrates expressed on the surface of cells serve as recognition elements for particular cell types, for ex le, in the ABO(H) blood group system. Antibodies that recognize host-incompatible ABO(H) system antigens exist in the bloodstream of all in iduals (except AB in iduals), preventing blood transfusion and organ transplantation between incompatible donors and recipients. A similar barrier exists for cross-species transplantation (xenotransplantation), in particular for pig-to-human transplantation. All humans express antibodies against the major carbohydrate xenoantigen, Galalpha (1,3)Gal (alphaGal), preventing successful xenotransplantation. Although antibody binding sites are precisely organized so as to selectively bind a specific antigen, many antibodies recognize molecules other than their native antigen. A range of peptides have been identified that can mimic carbohydrates and inhibit anti-alphaGal antibodies. However, the structural basis of how the peptides achieved this was not known. Previously, we developed an in silico method which we used to investigate carbohydrate recognition by a panel of anti-alphaGal antibodies. The method involves molecular docking of carbohydrates to antibodies and uses the docked carbohydrate poses to generate maps of th antibody binding sites in terms of prevalent hydrogen bonding and van der Waals interactions. We have applied this method to investigate peptide recognition by the anti-alphaGal antibodies. It was found that the site maps of the peptides and the carbohydrates were similar, indicating that the peptides interact with the same residues as those involved in carbohydrate recognition. This study demonstrates the potential for "design by mapping" of anti-carbohydrate antibody inhibitors.
Location: United States of America
Start Date: 2017
End Date: 2017
Funder: Australian Research Council
View Funded ActivityStart Date: 2014
End Date: 2016
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2011
End Date: 2013
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2015
End Date: 2017
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2017
End Date: 2017
Funder: Australian Research Council
View Funded ActivityStart Date: 1992
End Date: 2013
Funder: National Center for Advancing Translational Sciences
View Funded ActivityStart Date: 2023
End Date: 12-2025
Amount: $407,033.00
Funder: Australian Research Council
View Funded ActivityStart Date: 04-2017
End Date: 12-2017
Amount: $850,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 10-2017
Amount: $325,000.00
Funder: Australian Research Council
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