ARDC Research Link Australia

Publication

Publisher: Springer Science and Business Media LLC

Date: 04-1997

DOI: 10.1007/BF02443517

Publication

Publisher: Elsevier BV

Date: 03-2014

DOI: 10.1016/J.BMC.2015.01.023

Abstract: We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1-15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST-FOG1(1-45).

Publication

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding

Publisher: Elsevier BV

Date: 2018

DOI: 10.1111/JTH.13900

Abstract: Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred in iduals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected in iduals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected in iduals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.

Publication

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Publisher: MDPI AG

Date: 18-11-2020

DOI: 10.3390/MOLECULES25225392

Abstract: Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29–766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

Publication

Cross-linking mass spectrometry discovers, evaluates, and corroborates structures and protein–protein interactions in the human cell

Publisher: Proceedings of the National Academy of Sciences

Date: 18-04-2023

DOI: 10.1073/PNAS.2219418120

Abstract: Significant recent advances in structural biology, particularly in the field of cryoelectron microscopy, have dramatically expanded our ability to create structural models of proteins and protein complexes. However, many proteins remain refractory to these approaches because of their low abundance, low stability, or—in the case of complexes—simply not having yet been analyzed. Here, we demonstrate the power of using cross-linking mass spectrometry (XL-MS) for the high-throughput experimental assessment of the structures of proteins and protein complexes. This included those produced by high-resolution but in vitro experimental data, as well as in silico predictions based on amino acid sequence alone. We present the largest XL-MS dataset to date, describing 28,910 unique residue pairs captured across 4,084 unique human proteins and 2,110 unique protein–protein interactions. We show that models of proteins and their complexes predicted by AlphaFold2, and inspired and corroborated by the XL-MS data, offer opportunities to deeply mine the structural proteome and interactome and reveal mechanisms underlying protein structure and function.

Publication

Dissection of the Contributions toward Dimerization of Glycopeptide Antibiotics

Publisher: American Chemical Society (ACS)

Date: 06-1994

DOI: 10.1021/JA00090A005

Publication

Glycopeptide Antibiotic Activity and the Possible Role of Dimerization: A Model for Biological Signaling

Publisher: American Chemical Society (ACS)

Date: 06-1994

DOI: 10.1021/JA00090A006

Publication

Characterization of the conserved interaction between GATA and FOG family proteins

Publisher: Elsevier BV

Date: 09-2002

DOI: 10.1074/JBC.M204663200

Publication

Bromodomain protein Brd3 associates with acetylated GATA1 to promote its chromatin occupancy at erythroid target genes

Publisher: Proceedings of the National Academy of Sciences

Date: 02-05-2011

DOI: 10.1073/PNAS.1102140108

Abstract: Acetylation of histones triggers association with bromodomain-containing proteins that regulate erse chromatin-related processes. Although acetylation of transcription factors has been appreciated for some time, the mechanistic consequences are less well understood. The hematopoietic transcription factor GATA1 is acetylated at conserved lysines that are required for its stable association with chromatin. We show that the BET family protein Brd3 binds via its first bromodomain (BD1) to GATA1 in an acetylation-dependent manner in vitro and in vivo. Mutation of a single residue in BD1 that is involved in acetyl-lysine binding abrogated recruitment of Brd3 by GATA1, demonstrating that acetylation of GATA1 is essential for Brd3 association with chromatin. Notably, Brd3 is recruited by GATA1 to both active and repressed target genes in a fashion seemingly independent of histone acetylation. Anti-Brd3 ChIP followed by massively parallel sequencing in GATA1-deficient erythroid precursor cells and those that are GATA1 replete revealed that GATA1 is a major determinant of Brd3 recruitment to genomic targets within chromatin. A pharmacologic compound that occupies the acetyl-lysine binding pockets of Brd3 bromodomains disrupts the Brd3-GATA1 interaction, diminishes the chromatin occupancy of both proteins, and inhibits erythroid maturation. Together these findings provide a mechanism for GATA1 acetylation and suggest that Brd3 “reads” acetyl marks on nuclear factors to promote their stable association with chromatin.

Publication

Structure and Inhibition of Orotidine 5′-Monophosphate Decarboxylase from Plasmodium falciparum

Publisher: American Chemical Society (ACS)

Date: 02-03-2009

DOI: 10.1021/BI900043P

Publication

Publisher: Springer Science and Business Media LLC

Date: 04-2011

DOI: 10.1038/NSMB0411-516E

Publication

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

Publisher: Springer Science and Business Media LLC

Date: 1999

DOI: 10.1023/A:1008309602929

Abstract: Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional 1H/15N NMR spectroscopy, and we use 1H/15N spin relaxation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NF. Notably, this face is well removed from the putative DNA-binding face of the NF, an observation which is suggestive of simultaneous roles for the NF that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.

Publication

Protein-protein interactions: Analysis of a false positive GST pulldown result

Publisher: Wiley

Date: 02-06-2011

DOI: 10.1002/PROT.23068

Abstract: One of the most common ways to demonstrate a direct protein-protein interaction in vitro is the glutathione-S-transferse (GST)-pulldown. Here we report the detailed characterization of a putative interaction between two transcription factor proteins, GATA-1 and Krüppel-like factor 3 (KLF3/BKLF) that show robust interactions in GST-pulldown experiments. Attempts to map the interaction interface of GATA-1 on KLF3 using a mutagenic screening approach did not yield a contiguous binding face on KLF3, suggesting that the interaction might be non-specific. NMR experiments showed that the proteins do not interact at protein concentrations of 50-100 μM. Rather, the GST tag can cause part of KLF3 to misfold. In addition to misfolding, the fact that both proteins are DNA-binding domains appears to introduce binding artifacts (possibly nucleic acid bridging) that cannot be resolved by the addition of nucleases or ethidium bromide (EtBr). This study emphasizes the need for caution in relying on GST-pulldown results and related methods, without convincing confirmation from different approaches.

Publication

¹H, ¹³C and ¹⁵N resonance assignments of a C-terminal domain of human CHD1

Publisher: Springer Science and Business Media LLC

Date: 19-08-2016

DOI: 10.1007/S12104-015-9631-1

Abstract: Chromatin remodelling proteins are an essential family of eukaryotic proteins. They harness the energy from ATP hydrolysis and apply it to alter chromatin structure in order to regulate all aspects of genome biology. Chromodomain helicase DNA-binding protein 1 (CHD1) is one such remodelling protein that has specialised nucleosome organising abilities and is conserved across eukaryotes. CHD1 possesses a pair of tandem chromodomains that directly precede the core catalytic Snf2 helicase-like domain, and a C-terminal SANT-SLIDE DNA-binding domain. We have identified an additional conserved domain in the C-terminal region of CHD1. Here, we report the backbone and side chain resonance assignments for this domain from human CHD1 at pH 6.5 and 25 °C (BMRB No. 25638).

Publication

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1016/J.JSB.2004.01.004

Publication

Publisher: Wiley

Date: 16-11-2017

DOI: 10.1111/MPP.12597

Publication

Arabinosylation Modulates the Growth-Regulating Activity of the Peptide Hormone CLE40a from Soybean

Publisher: Elsevier BV

Publisher: Elsevier BV

Date: 06-2003

DOI: 10.1074/JBC.M301896200

Publication

Thermostable small-molecule inhibitor of angiogenesis and vascular permeability that suppresses a pERK-FosB/ΔFosB-VCAM-1 axis.

Publisher: American Association for the Advancement of Science (AAAS)

Date: 31-07-2020

DOI: 10.1126/SCIADV.AAZ7815

Abstract: A thermostable drug suppresses a pERK-FosB/ΔFosB-VCAM-1 axis, endothelial activation, angiogenesis, and retinal permeability.

Publication

Publisher: Wiley

Date: 13-11-2017

DOI: 10.1111/FEBS.14301

Publication

Analysis of disease-causing GATA1 mutations in murine gene complementation systems

Publisher: American Society of Hematology

Date: 27-06-2013

DOI: 10.1182/BLOOD-2013-03-488080

Abstract: Disease-causing mutations in GATA1 impair binding to the cofactors FOG1 or TAL1 but not DNA. Different substitutions at the same residue selectively disrupt FOG1 or TAL1 binding leading to distinct disease phenotypes.

Publication

Macromolecular NMR spectroscopy for the non-spectroscopist: Beyond macromolecular solution structure determination

Publisher: Wiley

Date: 28-01-2011

DOI: 10.1111/J.1742-4658.2011.08005.X

Abstract: A strength of NMR spectroscopy is its ability to monitor, on an atomic level, molecular changes and interactions. In this review, which is intended for non-spectroscopist, we describe major uses of NMR in protein science beyond solution structure determination. After first touching on how NMR can be used to quickly determine whether a mutation induces structural perturbations in a protein, we describe the unparalleled ability of NMR to monitor binding interactions over a wide range of affinities, molecular masses and solution conditions. We discuss the use of NMR to measure the dynamics of proteins at the atomic level and over a wide range of timescales. Finally, we outline new and expanding areas such as macromolecular structure determination in multicomponent systems, as well as in the solid state and in vivo.

Publication

Date: 04-2014

DOI: 10.1016/J.JMB.2013.12.010

Publication

Solution structures of two CCHC zinc fingers from the FOG family protein U-shaped that mediate protein-protein interactions

Publisher: Elsevier BV

Date: 11-2000

DOI: 10.1016/S0969-2126(00)00527-X

Abstract: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.

Publication

Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability

Publisher: Oxford University Press (OUP)

Date: 24-11-2017

DOI: 10.1093/NAR/GKW1014

Publication

Publisher: Public Library of Science (PLoS)

Date: 13-10-2014

DOI: 10.1371/JOURNAL.PONE.0109108

Publication

1H, 15N and 13C assignments of an intramolecular LMO4-LIM1/CtIP complex

Publisher: Springer Science and Business Media LLC

Date: 04-06-2012

DOI: 10.1007/S12104-011-9319-0

Abstract: LMO4 is a broadly expressed LIM-only protein that is involved in neural tube development and implicated in breast cancer. Here we report backbone and side chain NMR assignments for an engineered intramolecular complex of the N-terminal LIM domain from LMO4 tethered to residues 641-685 of C-terminal binding protein interacting protein (CtIP/RBBP8).

Publication

Characterization of a Family of RanBP2-Type Zinc Fingers that Can Recognize Single-Stranded RNA

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.JMB.2010.12.041

Abstract: The recognition of single-stranded RNA (ssRNA) is an important aspect of gene regulation, and a number of different classes of protein domains that recognize ssRNA in a sequence-specific manner have been identified. Recently, we demonstrated that the RanBP2-type zinc finger (ZnF) domains from the human splicing factor ZnF Ran binding domain-containing protein 2 (ZRANB2) can bind to a sequence containing the consensus AGGUAA. Six other human proteins, namely, Ewing's sarcoma (EWS), translocated in liposarcoma (TLS)/FUS, RNA-binding protein 56 (RBP56), RNA-binding motif 5 (RBM5), RNA-binding motif 10 (RBM10) and testis-expressed sequence 13A (TEX13A), each contains a single ZnF with homology to the ZRANB2 ZnFs, and several of these proteins have been implicated in the regulation of mRNA processing. Here, we show that all of these ZnFs are able to bind with micromolar affinities to ssRNA containing a GGU motif. NMR titration data reveal that binding is mediated by the corresponding surfaces on each ZnF, and we also show that sequence selectivity is largely limited to the GGU core motif and that substitution of the three flanking adenines that were selected in our original selection experiment has a minimal effect on binding affinity. These data establish a subset of RanBP2-type ZnFs as a new family of ssRNA-binding motifs.

Publication

TC-1 Is a Novel Tumorigenic and Natively Disordered Protein Associated with Thyroid Cancer

Publisher: American Association for Cancer Research (AACR)

Date: 15-04-2004

DOI: 10.1158/0008-5472.CAN-03-2093

Abstract: A novel gene, thyroid cancer 1 (TC-1), was found recently to be overexpressed in thyroid cancer. TC-1 shows no homology to any of the known thyroid cancer-associated genes. We have produced stable transformants of normal thyroid cells that express the TC-1 gene, and these cells show increased proliferation rates and anchorage-independent growth in soft agar. Apoptosis rates also are decreased in the transformed cells. We also have expressed recombinant TC-1 protein and have undertaken a structural and functional characterization of the protein. The protein is monomeric and predominantly unstructured under conditions of physiologic salt and pH. This places it in the category of natively disordered proteins, a rapidly expanding group of proteins, many members of which play critical roles in cell regulation processes. We show that the protein can be phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, and the activity of both of these kinases is up-regulated when cells are stably transfected with TC-1. These results suggest that overexpression of TC-1 may be important in thyroid carcinogenesis.

Publication

Comparative structure-function analysis of bromodomain and extraterminal motif (BET) proteins in a gene-complementation system

Publisher: Elsevier BV

Date: 02-2020

DOI: 10.1074/JBC.RA119.010679

Publication

Crystal and Solution Structures of a Superantigen from Yersinia pseudotuberculosis Reveal a Yelly-Roll Fold

Publisher: Elsevier BV

Date: 03-2004

DOI: 10.1016/J.STR.2003.12.002

Abstract: Superantigens are a class of microbial proteins with the ability to excessively activate T cells by binding to the T cell receptor. The staphylococcal and streptococcal superantigens are closely related in structure and possess an N-terminal domain that resembles an OB fold and a C-terminal domain similar to a beta-grasp fold. Yersinia pseudotuberculosis produces superantigens, YPMa, YPMb, and YPMc, which have no significant amino acid similarity to other proteins. We have determined the crystal and solution structures of YPMa, which show that the protein has a jelly-roll fold. The closest structural neighbors to YPMa are viral capsid proteins and members of the tumor necrosis factor superfamily. In the crystal structure, YPMa packs as a trimer, another feature shared with viral capsid proteins and TNF superfamily proteins. However, in solution YPMa behaves as a monomer, and any functional relevance of the trimer observed in the crystals is yet to be established.

Publication

Publisher: Informa UK Limited

Date: 17-11-2017

DOI: 10.1080/21505594.2017.1385692

Publication

Transcriptional cofactors of the FOG family interact with GATA proteins by means of multiple zinc fingers.

Publisher: Wiley

Date: 11-02-1970

DOI: 10.1093/EMBOJ/18.10.2812

Publication

The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity

Publisher: Elsevier BV

Publisher: American Chemical Society (ACS)

Date: 11-1998

DOI: 10.1021/BI981163R

Publication

Key residues characteristic of GATA N-fingers are recognized by FOG

Publisher: Elsevier BV

Date: 12-1998

DOI: 10.1074/JBC.273.50.33595

Publication

Structural and biophysical analysis of the DNA binding properties of myelin transcription factor 1

Publisher: Elsevier BV

Date: 02-2008

DOI: 10.1074/JBC.M703772200

Publication

The role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families

Publisher: Springer Science and Business Media LLC

Date: 06-12-2022

DOI: 10.1038/S41467-022-35002-0

Abstract: CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by ~150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.

Publication

The Nucleosome Remodeling and Deacetylase Complex Has an Asymmetric, Dynamic, and Modular Architecture.

Publisher: Elsevier BV

Date: 12-2020

DOI: 10.1016/J.CELREP.2020.108450

Publication

A structural analysis of DNA binding by myelin transcription factor 1 double zinc fingers

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1074/JBC.M113.482075

Publication

The yeast transcription elongation factor Spt4/5 is a sequence-specific RNA binding protein.

Publisher: Wiley

Date: 15-07-2016

DOI: 10.1002/PRO.2976

Publication

Assessment of the robustness of a serendipitous zinc binding fold: Mutagenesis and protein grafting

Publisher: Elsevier BV

Date: 02-2005

DOI: 10.1016/J.STR.2004.12.007

Abstract: Zinc binding motifs have received much attention in the area of protein design. Here, we have tested the suitability of a recently discovered nonnative zinc binding structure as a protein design scaffold. A series of multiple alanine mutants was created to investigate the minimal requirements for folding, and solution structures of these mutants showed that the original fold was maintained, despite changes in approximately 50% of the sequence. We next attempted to transplant binding faces from chosen bimolecular interactions onto one of these mutants, and many of the resulting "chimeras" were shown to adopt a native-like fold. These results both highlight the robust nature of small zinc binding domains and underscore the complexity of designing functional proteins, even using such small, highly ordered scaffolds as templates.

Publication

Structural Analysis of MED-1 Reveals Unexpected Diversity in the Mechanism of DNA Recognition by GATA-type Zinc Finger Domains

Publisher: Elsevier BV

Date: 02-2009

DOI: 10.1074/JBC.M808712200

Publication

The C-terminal Domain of Eos Forms a High Order Complex in Solution

Publisher: Elsevier BV

Date: 10-2003

DOI: 10.1074/JBC.M306817200

Publication

GATAD2B-associatedneurodevelopmental disorder (GAND): clinical and molecular insights into a NuRD-relateddisorder

Publisher: Elsevier BV

Date: 05-2020

DOI: 10.1038/S41436-019-0747-Z

Publication

The Structure of a Truncated Phosphoribosylanthranilate Isomerase Suggests a Unified Model for Evolution of the (βα)8 Barrel Fold

Publisher: Elsevier BV

Date: 04-2011

DOI: 10.1016/J.JMB.2011.02.048

Abstract: The (βα)(8) barrel is one of the most common protein folds, and enzymes with this architecture display a remarkable range of catalytic activities. Many of these functions are associated with ancient metabolic pathways, and phylogenetic reconstructions suggest that the (βα)(8) barrel was one of the very first protein folds to emerge. Consequently, there is considerable interest in understanding the evolutionary processes that gave rise to this fold. In particular, much attention has been focused on the plausibility of (βα)(8) barrel evolution from homodimers of half barrels. However, we previously isolated a three-quarter-barrel-sized fragment of a (βα)(8) barrel, termed truncated phosphoribosylanthranilate isomerase (trPRAI), that is soluble and almost as thermostable as full-length N-(5'-phosphoribosyl)anthranilate isomerase (PRAI). Here, we report the NMR-derived structure of trPRAI. The subdomain is monomeric, is well ordered and adopts a native-like structure in solution. Side chains from strands β(1) (Glu3 and Lys5), β(2) (Tyr25) and β(6) (Lys122) of trPRAI repack to shield the hydrophobic core from the solvent. This result demonstrates that three-quarter barrels were viable intermediates in the evolution of the (βα)(8) barrel fold. We propose a unified model for (βα)(8) barrel evolution that combines our data, previously published work and plausible scenarios for the emergence of (initially error-prone) genetic systems. In this model, the earliest proto-cells contained erse pools of part-barrel subdomains. Combinatorial assembly of these subdomains gave rise to many distinct lineages of (βα)(8) barrel proteins, that is, our model excludes the possibility that there was a single (βα)(8) barrel from which all present ex les are descended.

Publication

Publisher: Oxford University Press (OUP)

Date: 07-2016

DOI: 10.1093/NAR/GKW590

Publication

Publisher: Elsevier BV

Date: 04-1996

DOI: 10.1016/0040-4020(96)00198-6

Publication

The identification and structure of an N-terminal PR domain show that FOG1 is a member of the PRDM family of proteins

Publisher: Public Library of Science (PLoS)

Date: 27-08-2014

DOI: 10.1371/JOURNAL.PONE.0106011

Publication

A class of zinc fingers involved in protein-protein interactions

Publisher: Wiley

Date: 02-2000

DOI: 10.1046/J.1432-1327.2000.01095.X

Abstract: Zinc fingers (ZnFs) are extremely common protein domains. Several classes of ZnFs are distinguished by the nature and spacing of their zinc-coordinating residues. While the structure and function of some ZnFs are well characterized, many others have been identified only through their amino acid sequence. A number of proteins contain a conserved C-X2-C-X12-H-X1-5-C sequence, which is similar to the spacing observed for the 'classic' CCHH ZnFs. Although these domains have been implicated in protein-protein (and not protein-nucleic acid) interactions, nothing is known about their structure or function at a molecular level. Here, we address this problem through the expression and biophysical characterization of several CCHC-type zinc fingers from the erythroid transcription factor FOG and the related Drosophila protein U-shaped. Each of these domains does indeed fold in a zinc-dependent fashion, coordinating the metal in a tetrahedral manner through the sidechains of one histidine and three cysteine residues, and forming extremely thermostable structures. Analysis of CD spectra suggests an overall fold similar to that of the CCHH fingers, and indeed a point mutant of FOG-F1 in which the final cysteine residue is replaced by histidine remains capable of folding. However, the CCHC (as opposed to CCHH) motif is a prerequisite for GATA-1 binding activity, demonstrating that CCHC and CCHH topologies are not interchangeable. This demonstration that members of a structurally distinct subclass of genuine zinc finger domains are involved in the mediation of protein-protein interactions has implications for the prediction of protein function from nucleotide sequences.

Publication

Solution structure of the LIM-homeodomain transcription factor complex Lhx3/Ldb1 and the effects of a pituitary mutation on key Lhx3 interactions.

Publisher: Public Library of Science (PLoS)

Date: 25-07-2012

DOI: 10.1371/JOURNAL.PONE.0040719

Publication

A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin

Publisher: Elsevier BV

Date: 10-2009

DOI: 10.1074/JBC.M109.027045

Publication

Publisher: Wiley

Date: 25-09-2012

DOI: 10.1002/PRO.2153

Publication

Plant Homeodomain (PHD) Fingers of CHD4 Are Histone H3-binding Modules with Preference for Unmodified H3K4 and Methylated H3K9

Publisher: Elsevier BV

Date: 04-2011

DOI: 10.1074/JBC.M110.208207

Publication

Cyclic peptides can engage a single binding pocket through highly divergent modes

Publisher: Proceedings of the National Academy of Sciences

Date: 12-10-2020

DOI: 10.1073/PNAS.2003086117

Abstract: Large DNA-encoded libraries of cyclic peptides are emerging as powerful sources of molecules to tackle challenging drug targets. The structural and functional ersity contained within these libraries is, however, little explored. Here we demonstrate that one such library contains members that use unexpectedly erse mechanisms to recognize the same surface on the same target proteins with high affinity and specificity. This range of binding modes is much larger than observed in natural ligands of the same proteins, demonstrating the power and versatility of the technology. Our data also reveal opportunities for the development of more sophisticated approaches to achieving specificity when trying to selectively target one member of a family of closely related proteins.

Publication

Publisher: Elsevier BV

Date: 11-2011

DOI: 10.1074/JBC.M111.287300

Publication

Publisher: Elsevier BV

Date: 07-2003

DOI: 10.1074/JBC.M211146200

Publication

Publisher: Elsevier BV

Date: 08-2007

DOI: 10.1086/519530

Publication

Binding of the CHD4 PHD2 finger to histone H3 is modulated by covalent modifications

Publisher: Portland Press Ltd.

Date: 25-09-2009

DOI: 10.1042/BJ20090870

Abstract: CHD4 (chromodomain helicase DNA-binding protein 4) ATPase is a major subunit of the repressive NuRD (nucleosome remodelling and deacetylase) complex, which is involved in transcriptional regulation and development. CHD4 contains two PHD (plant homeodomain) fingers of unknown function. Here we show that the second PHD finger (PHD2) of CHD4 recognizes the N-terminus of histone H3 and that this interaction is facilitated by acetylation or methylation of Lys9 (H3K9ac and H3K9me respectively) but is inhibited by methylation of Lys4 (H3K4me) or acetylation of Ala1 (H3A1ac). An 18 μM binding affinity toward unmodified H3 rises to 0.6 μM for H3K9ac and to 0.9 μM for H3K9me3, whereas it drops to 2.0 mM for H3K4me3, as measured by tryptophan fluorescence and NMR. A peptide library screen further shows that phosphorylation of Thr3, Thr6 or Ser10 abolishes this interaction. A model of the PHD2–H3 complex, generated using a combination of NMR, data-driven docking and mutagenesis data, reveals an elongated site on the PHD2 surface where the H3 peptide is bound. Together our findings suggest that the PHD2 finger plays a role in targeting of the CHD4/NuRD complex to chromatin.

Publication

Is there a telltale RH fingerprint in zinc fingers that recognizes methylated CpG dinucleotides?

Publisher: Elsevier BV

Date: 09-2013

DOI: 10.1016/J.TIBS.2013.06.005

Publication

Publisher: American Society for Clinical Investigation

Date: 15-11-2004

DOI: 10.1172/JCI21982

Publication

Evolution of Quaternary Structure in a Homotetrameric Enzyme

Publisher: Elsevier BV

Date: 07-2008

DOI: 10.1016/J.JMB.2008.05.038

Abstract: Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits.

Publication

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Publisher: MDPI AG

Date: 17-11-2020

DOI: 10.20944/PREPRINTS202009.0390.V2

Abstract: Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity & U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS-CoV, SARS-CoV-2 does not bind human DPP4.

Publication

Publisher: Elsevier BV

Date: 12-2000

DOI: 10.1074/JBC.M004264200

Publication

A mass spectrometric investigation of the ability of metal complexes to modulate transcription factor activity

Publisher: Royal Society of Chemistry (RSC)

Date: 2009

DOI: 10.1039/B904751D

Abstract: ESI mass spectrometry was used to assess the ability of metal complexes to inhibit binding of a transcription factor to a DNA molecule containing its recognition sequence.

Publication

Zinc fingers are known as domains for binding DNA and RNA. Do they also mediate protein-protein interactions?

Publisher: Wiley

Date: 12-2006

DOI: 10.1080/15216540600868445

Publication

Crystal structures of flax rust avirulence proteins AvrL567-A and -D reveal details of the structural basis for flax disease resistance specificity

Publisher: Oxford University Press (OUP)

Date: 09-2007

DOI: 10.1105/TPC.107.053611

Abstract: The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Mel sora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of in idual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

Publication

Type I shorthorn sculpin antifreeze protein: Recombinant synthesis, solution conformation, and ice growth inhibition studies

Publisher: Elsevier BV

Date: 07-2002

DOI: 10.1074/JBC.M200307200

Publication

Publisher: Elsevier BV

Date: 03-2014

DOI: 10.1074/JBC.M113.545566

Joel Mackay

Researcher

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Publications

Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from Arabidopsis thaliana

Structure and inhibition of orotidine 5′-monophosphate decarboxylase from Plasmodium falciparum

Two-timing zinc finger transcription factors liaising with RNA

It takes two to tango: The structure and function of LIM, RING, PHD and MYND domains

Zipping up transcription factors: Rational design of anti-Jun and anti-Fos peptides

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis

The NuRD complex and macrocephaly associated neurodevelopmental disorders

Molecular mechanism of AHSP-mediated stabilization of α-hemoglobin

The Chromatin Remodelling Protein CHD1 Contains a Previously Unrecognised C-Terminal Helical Domain

Solution structure of a hydrophobic analogue of the winter flounder antifreeze protein

Two‐state conformational equilibrium in the Par‐4 leucine zipper domain

Engineering specificity changes on a RanBP2 zinc finger that binds single-stranded RNA

Molecular architecture of nucleosome remodeling and deacetylase sub-complexes by integrative structure determination

Late-stage modification of peptides and proteins at cysteine with diaryliodonium salts

Fibroblast-expressed LRRC15 suppresses SARS-CoV-2 infection and controls antiviral and antifibrotic transcriptional programs

The H2A.Z and NuRD associated protein HMG20A controls early head and heart developmental transcription programs

Semisynthesis of an evasin from tick saliva reveals a critical role of tyrosine sulfation for chemokine binding and inhibition

Development of a sensitive peptide-based immunoassay: Application to detection of the Jun and Fos oncoproteins

A Novel Haem-binding Interface in the 22 kDa Haem-binding Protein p22HBP

Discovery and Structure of a Potent and Highly Specific Blocker of Insect Calcium Channels

CSL: A notch above the rest

Interdomain orientation of cardiac Troponin C characterized by paramagnetic relaxation enhancement NMR reveals a compact state

GATA-1: One protein, many partners

A rapid method for assessing the RNA-binding potential of a protein

Ikaros: a key regulator of haematopoiesis

The N-terminal Region of Chromodomain Helicase DNA-binding Protein 4 (CHD4) Is Essential for Activity and Contains a High Mobility Group (HMG) Box-like-domain That Can Bind Poly(ADP-ribose)

Role of Alpha Hemoglobin‐Stabilizing Protein in Normal Erythropoiesis and β‐Thalassemia

A peptide affinity reagent for isolating an intact and catalytically active multi-protein complex from mammalian cells

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Cross-linking mass spectrometry discovers, evaluates, and corroborates structures and protein–protein interactions in the human cell

Dissection of the Contributions toward Dimerization of Glycopeptide Antibiotics

Glycopeptide Antibiotic Activity and the Possible Role of Dimerization: A Model for Biological Signaling

Characterization of the conserved interaction between GATA and FOG family proteins

Bromodomain protein Brd3 associates with acetylated GATA1 to promote its chromatin occupancy at erythroid target genes

Structure and Inhibition of Orotidine 5′-Monophosphate Decarboxylase from Plasmodium falciparum

DMA-binding studies of XSPTSPSZ, derivatives of the intercalating heptad repeat of RNA polymerase II

The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components

AHSP (α-haemoglobin-stabilizing protein) stabilizes apo-α-haemoglobin in a partially folded state

The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5′ splice site-like sequences

Erratum: The prospects for designer single-stranded RNA-binding proteins

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

Protein-protein interactions: Analysis of a false positive GST pulldown result

1H, 13C and 15N resonance assignments of a C-terminal domain of human CHD1

Response to Chatr-aryamontri et al.: Protein interactions: to believe or not to believe?

1H, 15N and 13C assignments of FLIN2, an intramolecular LMO2:ldb1 complex [2]

The hydrophobic domain 26 of human tropoelastin is unstructured in solution

Protein interactions: is seeing believing?

The characterization of protein interactions – what, how and how much?

The transactivation domain within cysteine/histidine-rich region 1 of CBP comprises two novel zinc-binding modules

Peppy: A virtual reality environment for exploring the principles of polypeptide structure

Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: Application to myosin light chain 2

Nuclear Magnetic Resonance Characterization of the Jun Leucine Zipper Domain: Unusual Properties of Coiled-Coil Interfacial Polar Residues

Presence of transient helical segments in the galanin-like peptide evident from 1H NMR, circular dichroism, and prediction studies

NMR spectroscopy as a tool for the rapid assessment of the conformation of GST‐fusion proteins

The Detection and Quantitation of Protein Oligomerization

Rapid Elaboration of Fragments into Leads Applied to Bromodomain-3 Extra Terminal Domain

Rapid Elaboration of Fragments into Leads Applied to Bromodomain-3 Extra Terminal Domain

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P

Arabinosylation Modulates the Growth-Regulating Activity of the Peptide Hormone CLE40a from Soybean

Structural Basis and Specificity of Acetylated Transcription Factor GATA1 Recognition by BET Family Bromodomain Protein Brd3

Implementing the LIM code: The structural basis for cell type-specific assembly of LIM-homeodomain complexes

TUNE: Tree Mining-Based Approach for Dual-Fuel Engine Performance Management of Heavy Duty Trucks

Pathogenic human variant that dislocates GATA2 zinc fingers disrupts hematopoietic gene expression and signaling networks

Insights into Association of the NuRD Complex with FOG-1 from the Crystal Structure of an RbAp48·FOG-1 Complex

Structural basis of simultaneous recruitment of the transcriptional regulators LMO2 and FOG1/ZFPM1 by the transcription factor GATA1

Effects of Calcium Binding and the Hypertrophic Cardiomyopathy A8V Mutation on the Dynamic Equilibrium between Closed and Open Conformations of the Regulatory N-Domain of Isolated Cardiac Troponin C

Solution structure of a recombinant type I sculpin antifreeze protein

Backbone and side-chain assignments of a tethered complex between LMO4 and DEAF-1

The Structure of the Zinc Finger Domain from Human Splicing Factor ZNF265 Fold

Thermostable small-molecule inhibitor of angiogenesis and vascular permeability that suppresses a pERK-FosB/ΔFosB-VCAM-1 axis.

CHD4 Is a Peripheral Component of the Nucleosome Remodeling and Deacetylase Complex

¹H, ¹³C and ¹⁵N resonance assignments of a C-terminal domain of human CHD1

Analysis of the Structure and Function of the Transcriptional Coregulator HOP^,

TNP Analogues Inhibit the Virulence Promoting IP_3-4 Kinase Arg1 in the Fungal Pathogen Cryptococcus neoformans.