ORCID Profile
0000-0002-6468-4427
Current Organisation
Monash University
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Research Topics
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.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Organic Chemistry | Biochemistry and Cell Biology | Biological And Medical Chemistry | Organic Chemical Synthesis | Proteins and Peptides | Biochemistry And Cell Biology Not Elsewhere Classified | Characterisation of Biological Macromolecules | Medicinal and Biomolecular Chemistry | Organic Chemical Synthesis | Receptors and Membrane Biology | Systems Biology | Infectious Agents | Enzymes | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Protein Targeting And Signal Transduction | Cellular Immunology | Cellular Interactions (Incl. Adhesion, Matrix, Cell Wall) | Medicinal and Biomolecular Chemistry not elsewhere classified | Analytical Biochemistry
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences | Immune system and allergy | Cardiovascular system and diseases | Scientific instrumentation | Respiratory system and diseases (incl. asthma) | Treatments (e.g. chemicals, antibiotics) | Organs, diseases and abnormal conditions not elsewhere classified |
Related Links
Publications
Synergistic Interactions between Chemokine Receptor Elements in Recognition of Interleukin-8 by Soluble Receptor Mimics
Publisher: American Chemical Society (ACS)
Date: 30-01-2012
DOI: 10.1021/BI201615Y
Site-selective solid-phase synthesis of a CCR5 sulfopeptide library to interrogate HIV binding and entry
Publisher: American Chemical Society (ACS)
Date: 24-07-2014
DOI: 10.1021/CB500337R
Synthesis of 3,5-dihydroxyphenylglycine derivatives and the C-terminal dipeptide of vancomycin
Publisher: Elsevier BV
Date: 06-1991
The mechanism of an inhibitory antibody on TF-initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF·5G9 complex 1 1Edited by D. C. Rees
Publisher: Elsevier BV
Date: 02-1998
Abstract: The tissue factor (TF)-initiated blood coagulation protease cascade can be greatly inhibited in vivo by a potent anti-human-TF monoclonal antibody, 5G9. This antibody binds the carboxyl module of the extracellular domain of TF with a nanomolar binding constant and inhibits the formation of the TF.VIIa.X ternary initiation complex. We have determined the crystal structures of the extra-cellular modules of human TF, Fab 5G9, and their complex (TF.5G9) to 2.4 A, 2. 5 A, and 3.0 A, respectively, and measured the apparent inhibition constants of 5G9 on a panel of TF mutants. In our unliganded TF structure, a 7 degrees change in the relative orientation between the D1 and D2 modules was observed when compared with other published TF structures. Comparison of the free and bound Fab 5G9 indicates that small segmental and side chain variation of the antibody complementarity determining regions occurred on complexation with TF. The antibody-antigen recognition involves 18 TF antigen residues and 19 Fab residues from six CDR with one of the largest buried surface areas seen to date. A combination of structural and mutagenesis data indicate that Tyr156, Lys169, Arg200, and Lys201 play the major role in the antibody recognition. The TF. 5G9 structure provides insights into the mechanism by which the antibody 5G9 inhibits formation of the TF.VIIa.X ternary complex.
Regulation of Chemokine–Receptor Interactions and Functions
Publisher: MDPI AG
Date: 14-11-2017
DOI: 10.3390/IJMS18112415
Evasins: Tick Salivary Proteins that Inhibit Mammalian Chemokines
Publisher: Elsevier BV
Date: 02-2020
Engineering Broad-spectrum Inhibitors of Inflammatory Chemokines from a New Family of Tick Evasins
Publisher: Research Square Platform LLC
Date: 25-01-2023
DOI: 10.21203/RS.3.RS-2472929/V1
Abstract: Chemokines, the key regulators of leukocyte trafficking, are attractive targets for anti-inflammatory therapy. Evasins are anti-inflammatory, chemokine-binding proteins found in tick saliva, with important therapeutic potential. However, therapeutic application of evasins will require manipulation of their chemokine target selectivity. Here we describe a new family of evasins, class A3 evasins, that is unique to the tick genus Amblyomma and distinguished from “classical” class A1 evasins by an additional disulfide-bonded pair of cysteine residues near the chemokine recognition interface. The class A3 evasin EVA-AAM1001 (EVA-A) bound to CC chemokines and inhibited their receptor activation. However, unlike class A1 evasins, EVA-A did not utilise N- and C-terminal regions to differentiate chemokine targets. Instead, structures of EVA-A bound to four chemokines revealed a deep hydrophobic pocket, unique to class A3 evasins, that interacts with the residue immediately following the CC motif of the chemokine (the “CC+1” residue). The preference of EVA-A for chemokines with aliphatic CC+1 residues results from negative selection against binding of aromatic CC+1 residues into this pocket. Consequently, mutations to alleviate this negative selection yielded broad-spectrum chemokine inhibitors. This study illustrates that class A3 evasins are an excellent platform for engineering proteins with targeted chemokine binding selectivity for applications in research, diagnosis or anti-inflammatory therapy.
NMR characterization of cooperativity: fast ligand binding coupled to slow protein dimerization
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4SC00131A
Abstract: We describe a general approach for analysis of 2D NMR spectra to evaluate the cooperativity of ligand binding and protein dimerization in coupled systems.
Increased protein backbone conformational entropy upon hydrophobic ligand binding
Publisher: Springer Science and Business Media LLC
Date: 12-1999
DOI: 10.1038/70057
Abstract: For complexes between proteins and very small hydrophobic ligands, hydrophobic effects alone may be insufficient to outweigh the unfavorable entropic terms resulting from bimolecular association. NMR relaxation experiments indicate that the backbone flexibility of mouse major urinary protein increases upon binding the hydrophobic mouse pheromone 2-sec-butyl-4,5-dihydrothiazole. The associated increase in backbone conformational entropy of the protein appears to make a substantial contribution toward stabilization of the protein-pheromone complex. This term is likely comparable in magnitude to other important free energy contributions to binding and may represent a general mechanism to promote binding of very small ligands to macromolecules.
Glycosylation Regulates N-Terminal Proteolysis and Activity of the Chemokine CCL14
Publisher: American Chemical Society (ACS)
Date: 14-05-2021
Tyrosine sulfation: an increasingly recognised post-translational modification of secreted proteins
Publisher: Elsevier BV
Date: 06-2009
DOI: 10.1016/J.NBT.2009.03.011
Abstract: The post-translational sulfation of tyrosine residues occurs in numerous secreted and integral membrane proteins and, in many cases, plays a crucial role in controlling the interactions of these proteins with physiological binding partners as well as invading pathogens. Recent advances in our understanding of protein tyrosine sulfation have come about owing to the cloning of two human tyrosylprotein sulfotransferases (TPST-1 and TPST-2), the development of novel analytical and synthetic methodologies and detailed studies of proteins and peptides containing sulfotyrosine residues. In this article, we describe the TPST enzymes, review the major techniques available for studying the presence, location and function of tyrosine sulfation in proteins and discuss the biological functions and biochemical interactions of several proteins (or protein families) in which tyrosine sulfation influences the protein function. In particular, we describe the detailed evidence supporting the importance of tyrosine sulfation in the cellular adhesion function of P-selectin glycoprotein ligand-1, the leukocyte trafficking and pathogen invasion functions of chemokine receptors and the ligand binding and activation of other G-protein-coupled receptors by complement proteins, phospholipdis and glycoprotein hormones.
Phosphoproteomic characterization of the signaling network resulting from activation of the chemokine receptor CCR2
Publisher: Elsevier BV
Date: 05-2020
Overexpression of myoglobin and assignment of its amide, Cα and Cβ resonances
Publisher: Springer Science and Business Media LLC
Date: 11-1995
DOI: 10.1007/BF00197808
Molecular basis for methoxyamine initiated mutagenesis
Publisher: Elsevier BV
Date: 12-1991
DOI: 10.1016/0022-2836(91)90507-3
Abstract: In order to reach a more detailed understanding of the mechanism of the mutagenic action of methoxyamine and of N4-methoxycytidine and its 2'-deoxyribo-analogue, the solution structures of the self-complementary octanucleotide, d(CGAATTCG) and its analogues, d(CGAATCCG), d(CGAATMCG) and d(CGAATPCG) (designated 8mer-AT, 8mer-AC, 8mer-AM, and 8mer-AP, respectively), were investigated by 1H nuclear magnetic resonance spectroscopy M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2]oxazin-7-one, in which the N-O bond is held in the anti configuration with respect to N3 of the cytosine ring. Correlated spectroscopy and nuclear Overhauser spectroscopy allowed assignment of the base, anomeric and H2'/H2" protons in 8mers-AT, -AM and -AP, and showed that all three had features consistent with a regular B-DNA duplex structure. Duplex-to-coil transition temperatures were determined to be 52(+/- 2) degrees C (8mer-AT), 51(+/- 2) degrees C (8mer-AP), 32(+/- 2) degrees C (8mer-AM) on the chemical shift timescale, the melting transition was fast for 8mer-AT and 8mer-AP, but slow for 8mer-AM. Imino proton spectra were indicative of Watson-Crick base-pairing in 8mers-AT, -AP and -AM. The 8mer-AP duplex had a structure and melting characteristics virtually identical with those of the 8mer-AT duplex. The preferred syn configuration of the methoxyl group in M had a destabilising effect on the 8mer-AM duplex. At low temperatures, the A.M base-pair was in fast equilibrium between Watson-Crick and wobble configurations, with the methoxyl function anti-oriented, but the melting transition was accompanied by isomerization of the methoxyl group to the syn conformation. This syn-anti isomerization was the rate-determining step in the duplex-to-coil transition. The 8mer-AC oligomer did not form a stable duplex.
Erratum: Tyrosine sulfation of chemokine receptor CCR2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein-1 (MCP-1): (Journal of Biological Chemistry (2013) 288 (10024-10034))
Publisher: Elsevier BV
Date: 05-2014
Structural basis of chemokine and receptor interactions: Key regulators of leukocyte recruitment in inflammatory responses
Publisher: Wiley
Date: 24-10-2020
DOI: 10.1002/PRO.3744
Erratum: Ticks from diverse genera encode chemokine-inhibitory evasin proteins (J. Biol. Chem. (2018) 292 (15670–15680) DOI: 10.1074/jbc.M117.807255)
Publisher: Elsevier BV
Date: 10-2018
Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1
Publisher: Elsevier BV
Date: 03-2019
Semisynthesis of an evasin from tick saliva reveals a critical role of tyrosine sulfation for chemokine binding and inhibition
Publisher: Proceedings of the National Academy of Sciences
Date: 27-05-2020
Abstract: Blood-feeding arthropods produce antiinflammatory salivary proteins called evasins that function through inhibition of chemokine-receptor signaling in the host. Herein, we show that the evasin ACA-01 from the Amblyomma cajennense tick can be posttranslationally sulfated at two tyrosine residues, albeit as a mixture of sulfated variants. Homogenously sulfated variants of the proteins were efficiently assembled via a semisynthetic native chemical ligation strategy. Sulfation significantly improved the binding affinity of ACA-01 for a range of proinflammatory chemokines and enhanced the ability of ACA-01 to inhibit chemokine signaling through cognate receptors. Comparisons of evasin sequences and structural data suggest that tyrosine sulfation serves as a receptor mimetic strategy for recognizing and suppressing the proinflammatory activity of a wide variety of mammalian chemokines. As such, the incorporation of this posttranslational modification (PTM) or mimics thereof into evasins may provide a strategy to optimize tick salivary proteins for antiinflammatory applications.
Systematic Assessment of Chemokine Signaling at Chemokine Receptors CCR4, CCR7 and CCR10
Publisher: MDPI AG
Date: 19-04-2021
DOI: 10.3390/IJMS22084232
Abstract: Chemokines interact with chemokine receptors in a promiscuous network, such that each receptor can be activated by multiple chemokines. Moreover, different chemokines have been reported to preferentially activate different signalling pathways via the same receptor, a phenomenon known as biased agonism. The human CC chemokine receptors (CCRs) CCR4, CCR7 and CCR10 play important roles in T cell trafficking and have been reported to display biased agonism. To systematically characterize these effects, we analysed G protein- and β-arrestin-mediated signal transduction resulting from stimulation of these receptors by each of their cognate chemokine ligands within the same cellular background. Although the chemokines did not elicit ligand-biased agonism, the three receptors exhibited different arrays of signaling outcomes. Stimulation of CCR4 by either CC chemokine ligand 17 (CCL17) or CCL22 induced β-arrestin recruitment but not G protein-mediated signaling, suggesting that CCR4 has the potential to act as a scavenger receptor. At CCR7, both CCL19 and CCL21 stimulated G protein signaling and β-arrestin recruitment, with CCL19 consistently displaying higher potency. At CCR10, CCL27 and CCL28(4-108) stimulated both G protein signaling and β-arrestin recruitment, whereas CCL28(1-108) was inactive, suggesting that CCL28(4-108) is the biologically relevant form of this chemokine. These comparisons emphasize the intrinsic abilities of different receptors to couple with different downstream signaling pathways. Comparison of these results with previous studies indicates that differential agonism at these receptors may be highly dependent on the cellular context.
The evolutionary role of secondary metabolites — a review
Publisher: Elsevier BV
Date: 06-1992
DOI: 10.1016/0378-1119(92)90553-2
Abstract: It is argued that organisms have evolved the ability to biosynthesise secondary metabolites ('natural products') due to the selectional advantages they obtain as a result of the functions of the compounds. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes in Streptomyces, is interpreted in terms of the defense roles of antibiotics. The clustering together of antibiotic biosynthesis, regulation, and resistance genes, and in particular the staggering complexity shown in the case of the gene cluster for erythromycin A biosynthesis, implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature.
Regulation of Chemokine Recognition by Site-Specific Tyrosine Sulfation of Receptor Peptides
Publisher: Elsevier BV
Date: 02-2009
Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from nitrogen-15 NMR relaxation measurements
Publisher: American Chemical Society (ACS)
Date: 12-05-1992
DOI: 10.1021/BI00133A003
Abstract: The backbone dynamics of the uniformly 15N-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively however, a small number of residues exhibited significantly slower relaxation. Similarly, (1H)-15N NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs. The molecular rotational correlation time was 6.24 +/- 0.01 ns most residues had order parameters in the range 0.75-0.90 and tau e values of less than ca. 25 ps. Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins and the region from A146 to S149. All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure.
Structure-guided engineering of tick evasins for targeting chemokines in inflammatory diseases
Publisher: Proceedings of the National Academy of Sciences
Date: 25-02-2022
Abstract: Inflammatory diseases collectively account for numerous deaths and morbidity worldwide. New treatment approaches are needed. A central feature of inflammatory diseases is the recruitment of leukocytes to the affected tissues, which is stimulated by secreted proteins called chemokines. Effective suppression of leukocyte recruitment could be achieved by simultaneously targeting multiple chemokines, a natural molecular strategy used by tick salivary proteins called evasins. Here, we describe the structural and molecular features of a tick evasin that control its ability to bind and block a limited set of chemokines. By modifying these features, we demonstrate that evasins can be engineered to alter the array of chemokines that they target. Thus, this study establishes a structure-based paradigm for the development of antiinflammatory therapeutics.
Molecular Basis for Methoxyamine-initiated Mutagenesis: 1H Nuclear Magnetic Resonance Studies of Oligonucleotide Duplexes Containing Base-modified Cytosine Residues
Publisher: Elsevier BV
Date: 04-1993
Abstract: Methoxyamine, N4-methoxycytidine and its 2'-deoxyribo analogue are transition mutagens. The mechanism by which the latter acts after incorporation into or generation within DNA has been ascribed to the ability of the base analogue to pair effectively with both adenine and guanine. To obtain a detailed understanding of these interactions, the solution structures of the self-complementary octanucleotide d(CGGATCCG) and its analogues d(CGGATTCG), d(CGGATMCG) and d(CGGATPCG) (designated 8mer-GC, -GT, -GM and -GP, respectively) were investigated by 1H nuclear magnetic resonance spectroscopy M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2] oxazin-7-one. A variable temperature study showed the order of stability as 8mer GC > GP > GT > GM. Nuclear Overhauser spectroscopy permitted the assignment of the base, anomeric and H2'/H2" protons in these 8mers. All had spectra consistent with regular B-DNA duplex structures. Imino proton spectra showed that the 8mers GC, GP and GM involved Watson-Crick base-pairing but that the G.P and to a greater extent G.M base-pairs were in slow exchange on the nuclear magnetic resonance time-scale with the wobble configuration. Indeed, the G.M pair showed an additional exchange process interpreted in terms of the presence of syn and anti conformers of the methoxy group in the wobble pair. This accounts for the destabilization of M compared with the P-containing duplex. The observations are compared with those made earlier on the corresponding AT, AP and AM octamers. It is evident that M and P can form stable base-pairs with both A and G with essentially Watson-Crick geometry. This confirms the earlier, although unsubstantiated explanation for the transition mutational propenstty of methoxyamine which, in turn, was based on the fact that methoxycytosine bases have tautomeric constants (KT) much nearer to unity than the normal bases. The same general explanation for hydroxylamine and hydrazine-induced mutations is correspondingly rendered more certain.
Comparison of backbone and tryptophan side-chain dynamics of reduced and oxidized Escherichia coli thioredoxin using nitrogen-15 NMR relaxation measurements
Publisher: American Chemical Society (ACS)
Date: 19-01-1993
DOI: 10.1021/BI00053A007
Abstract: The backbone and tryptophan side-chain dynamics of both the reduced and oxidized forms of uniformly 15N-labeled Escherichia coli thioredoxin have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured for more than 90% of the protonated backbone nitrogen atoms and for the protonated indole nitrogen atoms of the two tryptophan residues. These data were analyzed by using a model free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The reduced and oxidized forms exhibit almost identical dynamic behavior on the picosecond to nanosecond time scale. The W31 side chain is significantly more mobile than the W28 side chain, consistent with the positions of W31 on the protein surface and W28 buried in the hydrophobic core. Backbone regions which are significantly more mobile than the average include the N-terminus, which is constrained in the crystal structure of oxidized thioredoxin by specific contacts with a Cu2+ ion, the C-terminus, residues 20-22, which constitute a linker region between the first alpha-helix and the second beta-strand, and residues 73-75 and 93-94, which are located adjacent to the active site. In contrast, on the microsecond to millisecond time scale, reduced thioredoxin exhibits considerable dynamic mobility in the residue 73-75 region, while oxidized thioredoxin exhibits no significant mobility in this region. The possible functional implications of the dynamics results are discussed.
Base pairing of cytosine analogues with adenine and guanine in oligonucleotide duplexes: evidence for exchange between Watson–Crick and wobble base pairs using1H NMR spectroscopy
Publisher: Royal Society of Chemistry (RSC)
Date: 1991
DOI: 10.1039/C39910001357
Why Are Secondary Metabolites (Natural Products) Biosynthesized?
Publisher: American Chemical Society (ACS)
Date: 11-1989
DOI: 10.1021/NP50066A001
Abstract: We adopt the definition of a natural product as a substance that has no known role in the internal economy of the producing organism. The literature abounds with conflicting views for the existence of such natural products. We propose that all such structures serve the producing organisms by improving their survival fitness. We argue that this conclusion is necessitated by the fact that natural products are normally complex structures, whose biosynthesis is programmed by many kilobases of DNA. If it were otherwise, the pressures of Darwinian natural selection would have precluded the expenditure of so much metabolic energy in their construction and the development of such complexity. We further conclude that a natural product improves the producer's survival fitness by acting at specific receptors in competing organisms. Current studies of natural products interacting with receptors support this view, in terms of both the sophistication of the molecule/molecule recognition and the mechanistic details of physiological action. By the application of Occam's razor and general weaknesses of other hypotheses, these other hypotheses are rejected. It is a consequence of our proposal that natural product/receptor interactions of sophistication comparable to enzyme/substrate interactions will be commonplace. Additionally, structures that are candidates to interact with known receptors (e.g., double helical DNA) can on occasion be suggested by inspection of the structures. A range of evidence to support the general conclusions is presented.
Divergent and Site‐Selective Solid‐Phase Synthesis of Sulfopeptides
Publisher: Wiley
Date: 20-04-2011
Influence of Chemokine N-Terminal Modification on Biased Agonism at the Chemokine Receptor CCR1
Publisher: MDPI AG
Date: 15-05-2019
DOI: 10.3390/IJMS20102417
Abstract: Leukocyte migration, a hallmark of the inflammatory response, is stimulated by the interactions between chemokines, which are expressed in injured or infected tissues, and chemokine receptors, which are G protein-coupled receptors (GPCRs) expressed in the leukocyte plasma membrane. One mechanism for the regulation of chemokine receptor signaling is biased agonism, the ability of different chemokine ligands to preferentially activate different intracellular signaling pathways via the same receptor. To identify features of chemokines that give rise to biased agonism, we studied the activation of the receptor CCR1 by the chemokines CCL7, CCL8, and CCL15(Δ26). We found that, compared to CCL15(Δ26), CCL7 and CCL8 exhibited biased agonism towards cAMP inhibition and away from β-Arrestin 2 recruitment. Moreover, N-terminal substitution of the CCL15(Δ26) N-terminus with that of CCL7 resulted in a chimera with similar biased agonism to CCL7. Similarly, N-terminal truncation of CCL15(Δ26) also resulted in signaling bias between cAMP inhibition and β-Arrestin 2 recruitment signals. These results show that the interactions of the chemokine N-terminal region with the receptor transmembrane region play a key role in selecting receptor conformations coupled to specific signaling pathways.
Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)
Publisher: Elsevier BV
Date: 04-2013
NMR Mapping of the Recombinant Mouse Major Urinary Protein I Binding Site Occupied by the Pheromone 2-sec-Butyl-4,5-dihydrothiazole
Publisher: American Chemical Society (ACS)
Date: 16-07-1999
DOI: 10.1021/BI990497T
Comparison of Protein Backbone Entropy and β-Sheet Stability: NMR-Derived Dynamics of Protein G B1 Domain Mutants
Publisher: American Chemical Society (ACS)
Date: 08-12-2000
DOI: 10.1021/JA003094L
Temperature-dependent spectral density analysis applied to monitoring backbone dynamics of major urinary protein-I complexed with the pheromone 2-sec-butyl-4,5-dihydrothiazole*
Publisher: Springer Science and Business Media LLC
Date: 04-2004
Molecular recognition by secondary metabolites
Publisher: Elsevier BV
Date: 07-1990
DOI: 10.1016/0006-2952(90)90174-J
Abstract: Aspects of molecular recognition based on the interaction between the vancomycin group of antibiotics and bacterial cell wall precursor analogues are discussed. The energetically unfavourable folding-in of the residue 1 sidechain in vancomycin and ristocetin A is discussed in terms of the favourable entropy associated with simultaneous release of solvent molecules. The effect of the sugar amino substituent on the strength of an adjacent hydrophobic interaction in the vancomycin/acetyl-D-Ala-D-Ala complex is rationalised as an intramolecular "salting-out" of hydrocarbon entities. The slow on-rate for dimerisation of the ristocetin A/N,N-diacetyl-L-Lys-D-Ala-D-Ala complex is attributed to the need for the relatively rigid peptide backbone of the antibiotic to be extensively desolvated before dimerisation can occur. Some of these concepts are then applied to understanding the interactions between antibiotics and the minor groove of double-helical DNA, the receptor site with which they have probably evolved to interact. Two structural motifs (pi-polarised aromatic rings and deoxy sugars) are postulated to be important in this recognition process. The possible roles of these structural features are discussed.
Sulfation of the Human Cytomegalovirus Protein UL22A Enhances Binding to the Chemokine RANTES
Publisher: Wiley
Date: 23-05-2017
Tyrosine Sulfation Influences the Chemokine Binding Selectivity of Peptides Derived from Chemokine Receptor CCR3
Publisher: American Chemical Society (ACS)
Date: 04-02-2011
DOI: 10.1021/BI101240V
Abstract: The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.
Discovery of Potent Cyclic Sulfopeptide Chemokine Inhibitors via Reprogrammed Genetic Code mRNA Display
Publisher: American Chemical Society (ACS)
Date: 24-04-2020
DOI: 10.1021/JACS.0C03152
Homogeneous Sulfopeptides and Sulfoproteins: Synthetic Approaches and Applications To Characterize the Effects of Tyrosine Sulfation on Biochemical Function
Publisher: American Chemical Society (ACS)
Date: 21-07-2015
DOI: 10.1021/ACS.ACCOUNTS.5B00255
Abstract: Post-translational modification of proteins plays critical roles in regulating structure, stability, localization, and function. Sulfation of the phenolic side chain of tyrosine residues to form sulfotyrosine (sTyr) is a widespread modification of extracellular and integral membrane proteins, influencing the activities of these proteins in cellular adhesion, blood clotting, inflammatory responses, and pathogen infection. Tyrosine sulfation commonly occurs in sequences containing clusters of tyrosine residues and is incomplete at each site, resulting in heterogeneous mixtures of sulfoforms. Purification of in idual sulfoforms is typically impractical. Therefore, the most promising approach to elucidate the influence of sulfation at each site is to prepare homogeneously sulfated proteins (or peptides) synthetically. This Account describes our recent progress in both development of such synthetic approaches and application of the resulting sulfopeptides and sulfoproteins to characterize the functional consequences of tyrosine sulfation. Initial synthetic studies used a cassette-based solid-phase peptide synthesis (SPPS) approach in which the side chain sulfate ester was protected to enable it to withstand Fmoc-based SPPS conditions. Subsequently, to address the need for efficient access to multiple sulfoforms of the same peptide, we developed a ergent solid-phase synthetic approach utilizing orthogonally side chain protected tyrosine residues. Using this methodology, we have carried out orthogonal deprotection and sulfation of up to three tyrosine residues within a given sequence, allowing access to all eight sulfoforms of a given target from a single solid-phase synthesis. With homogeneously sulfated peptides in hand, we have been able to probe the influence of tyrosine sulfation on biochemical function. Several of these studies focused on sulfated fragments of chemokine receptors, key mediators of leukocyte trafficking and inflammation. For the receptor CCR3, we showed that tyrosine sulfation enhances affinity and selectivity for binding to chemokine ligands, and we determined the structural basis of these affinity enhancements by NMR spectroscopy. Using a library of CCR5 sulfopeptides, we demonstrated the critical importance of sulfation at one specific site for supporting HIV-1 infection. Demonstrating the feasibility of producing homogeneously tyrosine-sulfated proteins, in addition to smaller peptides, we have used SPPS and native chemical ligation methods to synthesize the leech-derived antithrombotic protein hirudin P6, containing both tyrosine sulfation and glycosylation. Sulfation greatly enhanced inhibitory activity against thrombin, whereas addition of glycans to the sulfated protein decreased inhibition, indicating functional interplay between different post-translational modifications. In addition, the success of the ligation approach suggests that larger sulfoproteins could potentially be obtained by ligation of synthetic sulfopeptides to expressed proteins, using intein-based technology.
Unravelling G protein‐coupled receptor signalling networks using global phosphoproteomics
Publisher: Wiley
Date: 24-02-2023
DOI: 10.1111/BPH.16052
Abstract: G protein‐coupled receptor (GPCR) activation initiates signalling via a complex network of intracellular effectors that combine to produce erse cellular and tissue responses. Although we have an advanced understanding of the proximal events following receptor stimulation, the molecular detail of GPCR signalling further downstream often remains obscure. Unravelling these GPCR‐mediated signalling networks has important implications for receptor biology and drug discovery. In this context, phosphoproteomics has emerged as a powerful approach for investigating global GPCR signal transduction. Here, we provide a brief overview of the phosphoproteomic workflow and discuss current limitations and future directions for this technology. By highlighting some of the novel insights into GPCR signalling networks gained using phosphoproteomics, we demonstrate the utility of global phosphoproteomics to dissect GPCR signalling networks and to accelerate discovery of new targets for therapeutic development.
Mutational Mapping of Functional Residues in Tissue Factor: Identification of Factor VII Recognition Determinants in Both Structural Modules of the Predicted Cytokine Receptor Homology Domain
Publisher: American Chemical Society (ACS)
Date: 15-02-1994
DOI: 10.1021/BI00172A037
Abstract: Alanine scanning mutagenesis of tissue factor, the initiating receptor and cofactor molecule for the coagulation pathways, was used to define residue side chains with functional contributions. Approximately half of the residues were exchanged, and several stretches of functional residues throughout the entire extracellular domain were identified which contributed to overall coagulant function. Mutants were further characterized with respect to their affinity for binding of ligand, providing evidence that identified functional sequence spans are involved in ligand interaction. The tissue factor extracellular domain is suggested to adopt the folding pattern of the cytokine receptor homology unit, which is typically composed of two seven-beta-strand modules. Evaluation of the mutational analysis within this structural context suggests that functionally important residues are spatially proximate and clustered at the boundary of the predicted beta-strand modules. Residues contributing to ligand binding by tissue factor were identified in positions corresponding to ligand interactive residues in the growth hormone receptor and contact residues of other cytokine receptors, consistent with a conserved structural region for ligand interaction throughout the cytokine receptor family.
Structural basis of receptor sulfotyrosine recognition by a cc chemokine: The n-terminal region of CCR3 bound to CCL11/eotaxin-1
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.STR.2014.08.023
Abstract: Trafficking of leukocytes in immune surveillance and inflammatory responses is activated by chemokines engaging their receptors. Sulfation of tyrosine residues in peptides derived from the eosinophil chemokine receptor CCR3 dramatically enhances binding to cognate chemokines. We report the structural basis of this recognition and affinity enhancement. We describe the structure of a CC chemokine (CCL11/eotaxin-1) bound to a fragment of a chemokine receptor: residues 8–23 of CCR3, including two sulfotyrosine residues. We also show that intact CCR3 is sulfated and sulfation enhances receptor activity. The CCR3 sulfotyrosine residues form hydrophobic, salt bridge and cation-p interactions with residues that are highly conserved in CC chemokines. However, the orientation of the chemokine relative to the receptor N terminus differs substantially from those observed for two CXC chemokines, suggesting that initial binding of the receptor sulfotyrosine residues guides subsequent steps in receptor activation, thereby influencing the receptor conformational changes and signaling.
Phosphate modulates receptor sulfotyrosine recognition by the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2)
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4OB02262A
Abstract: Fluorescence anisotropy shows that the physiological buffer phosphate competes with a chemokine receptor sulfopeptide for binding to a cognate chemokine.
Ticks from diverse genera encode chemokine-inhibitory evasin proteins.
Publisher: Elsevier BV
Date: 09-2017
Proteomic Identification of Interferon-Induced Proteins with Tetratricopeptide Repeats as Markers of M1 Macrophage Polarization
Publisher: American Chemical Society (ACS)
Date: 06-03-2018
Backbone dynamics of the human CC-chemokine eotaxin
Publisher: Springer Science and Business Media LLC
Date: 1999
Abstract: Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. 15N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human cotaxin. 15N longitudinal (R1) and transverse (R2) auto relaxation rates, heteronuclear ¿1H¿-15N steady-state NOEs, and transverse cross-relaxation rates (eta xy) were obtained at 30 degrees C for all resolved backbone secondary amide groups using 1H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time (tau m) is 5.09 +/- 0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D parallel/D perpendicular) is 0.81 +/- 0.02. Residues with large litudes of subnanosecond motion are clustered in the N-terminal region (residues 1-19), the C-terminus (residues 68-73) and the loop connecting the first two beta-strands (residues 30-37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond-millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization.
The structural role of receptor tyrosine sulfation in chemokine recognition
Publisher: Wiley
Date: 27-02-2014
DOI: 10.1111/BPH.12455
Design and Receptor Interactions of Obligate Dimeric Mutant of Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)
Publisher: Elsevier BV
Date: 04-2012
Synthesis of polymers and nanoparticles bearing polystyrene sulfonate brushes for chemokine binding
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6OB00270F
Abstract: This paper describes the synthesis of polymers and silica nanoparticles, both bearing polystyrene sulfonate brushes, and the measurement of their binding affinity for the chemokine monocyte chemoattractant protein-1 (MCP-1) in monomeric and dimeric form.
Sulfation of the Human Cytomegalovirus Protein UL22A Enhances Binding to the Chemokine RANTES
Publisher: Wiley
Date: 23-05-2017
Abstract: UL22A is an 83 amino acid chemokine-binding protein produced by human cytomegalovirus that likely assists the virus in d ening the host antiviral response. We proposed that UL22A is sulfated on two tyrosine residues and tested this hypothesis through the chemical synthesis of a small library of differentially sulfated protein variants. The (sulfo)proteins were efficiently prepared using a novel β-selenoleucine motif to facilitate one-pot ligation-deselenization chemistry. Tyrosine sulfation of UL22A proved critical for RANTES binding, with the doubly sulfated variant exhibiting an improvement in binding of 2.5 orders of magnitude compared to the unmodified protein.
Engineering broad-spectrum inhibitors of inflammatory chemokines from subclass A3 tick evasins
Publisher: Springer Science and Business Media LLC
Date: 14-07-2023
DOI: 10.1038/S41467-023-39879-3
Abstract: Chemokines are key regulators of leukocyte trafficking and attractive targets for anti-inflammatory therapy. Evasins are chemokine-binding proteins from tick saliva, whose application as anti-inflammatory therapeutics will require manipulation of their chemokine target selectivity. Here we describe subclass A3 evasins, which are unique to the tick genus Amblyomma and distinguished from “classical” class A1 evasins by an additional disulfide bond near the chemokine recognition interface. The A3 evasin EVA-AAM1001 (EVA-A) bound to CC chemokines and inhibited their receptor activation. Unlike A1 evasins, EVA-A was not highly dependent on N- and C-terminal regions to differentiate chemokine targets. Structures of chemokine-bound EVA-A revealed a deep hydrophobic pocket, unique to A3 evasins, that interacts with the residue immediately following the CC motif of the chemokine. Mutations to this pocket altered the chemokine selectivity of EVA-A. Thus, class A3 evasins provide a suitable platform for engineering proteins with applications in research, diagnosis or anti-inflammatory therapy.
NMR Investigation of the Binding between Human Profilin I and Inositol 1,4,5-Triphosphate, the Soluble Headgroup of Phosphatidylinositol 4,5-Bisphosphate
Publisher: American Chemical Society (ACS)
Date: 26-11-2008
DOI: 10.1021/BI801535F
Abstract: Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is involved in the regulation of the actin cytoskeleton through interactions with a number of actin-binding proteins. We present here NMR titration experiments that monitor the interaction between the cytoskeletal protein profilin and inositol 1,4,5-triphosphate (IP(3)), the headgroup of PI(4,5)P(2). These experiments probe the interaction directly, at equilibrium, and with profilin in its native state. We show the binding between profilin and IP(3) can readily be observed at high concentrations, even though profilin does not bind to IP(3) under physiological conditions. Moreover, the titration data using wild-type profilin and an R88L mutant support the existence of at least three headgroup binding sites on profilin, consistent with previous experimentation with intact PI(4,5)P(2). This work suggests that various soluble inositol ligands can serve as effective probes to facilitate in vitro studies of PI-binding proteins that require membrane surfaces for high-affinity binding.
Active Monomeric and Dimeric Forms of Pseudomonas putida Glyoxalase I: Evidence for 3D Domain Swapping
Publisher: American Chemical Society (ACS)
Date: 29-03-0006
DOI: 10.1021/BI980868Q
Mechanisms of regulation of the chemokine-receptor network
Publisher: MDPI AG
Date: 07-02-2017
DOI: 10.3390/IJMS18020342
Key determinants of selective binding and activation by the monocyte chemoattractant proteins at the chemokine receptor CCR2.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 23-05-2017
DOI: 10.1126/SCISIGNAL.AAI8529
Abstract: Chemokines and their receptors collectively orchestrate the trafficking of leukocytes in normal immune function and inflammatory diseases. Different chemokines can induce distinct responses at the same receptor. In comparison to monocyte chemoattractant protein-1 (MCP-1 also known as CCL2), the chemokines MCP-2 (CCL8) and MCP-3 (CCL7) are partial agonists of their shared receptor CCR2, a key regulator of the trafficking of monocytes and macrophages that contribute to the pathology of atherosclerosis, obesity, and type 2 diabetes. Through experiments with chimeras of MCP-1 and MCP-3, we identified the chemokine amino-terminal region as being the primary determinant of both the binding and signaling selectivity of these two chemokines at CCR2. Analysis of CCR2 mutants showed that the chemokine amino terminus interacts with the major subpocket in the transmembrane helical bundle of CCR2, which is distinct from the interactions of some other chemokines with the minor subpockets of their receptors. These results suggest the major subpocket as a target for the development of small-molecule inhibitors of CCR2.
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