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ABSTRACT: The B3 DNA binding domain is a plant-specific domain, found throughout the plant kingdom from the alga Chlamydomonas to grasses and flowering plants. Over 100 B3 domain-containing proteins are found in the model plant Arabidopsis thaliana and one of these is critical for accelerating flowering in response to prolonged cold treatment; an epigenetic process called vernalization. Despite the specific phenotype of genetic vrn1 mutants, the VERNALIZATION1 (VRN1) protein localizes throughout the nucleus and in vitro shows sequence non-specific binding. In this work we used a dominant repressor tag that overcomes genetic redundancy to show that VRN1 is involved in processes beyond vernalization that are essential for Arabidopsis development. To understand its sequence non-specific binding we crystallized VRN1208-341 and solved its crystal structure to 1.6 Å resolution using Se-SAD methods. The crystallized construct comprises the second VRN1 B3 domain and a preceding region conserved among VRN1 orthologs, but absent in other B3 domains. We established the DNA binding face using NMR then mutated positively charged residues on this surface with a series of 16 Ala and Glu substitutions ensuring the protein fold was not disturbed using HSQC NMR spectra. A triple mutant R249E R289E R296E was almost completely incapable of DNA binding in vitro. Thus, we have revealed that although VRN1 is sequence non-specific in DNA binding, it has a defined DNA binding surface.
Journal of Biological Chemistry 12/2012; · 4.77 Impact Factor
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Eugene Valkov,
Anna Stamp,
Frank Dimaio,
David Baker,
Brett Verstak,
Pietro Roversi,
Stuart Kellie,
Matthew J Sweet,
Ashley Mansell,
Nicholas J Gay, Jennifer L Martin,
Bostjan Kobe
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Gordon J King,
Jacqueline Stoeckli,
Shu-Hong Hu,
Brit Winnen,
Wilko G A Duprez,
Christopher C Meoli,
Jagath R Junutula,
Russell J Jarrott,
David E James,
Andrew E Whitten, Jennifer L Martin
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ABSTRACT: The APPL1 and APPL2 proteins (APPL=adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain and leucine zipper containing protein) are localised to their own endosomal sub-compartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction, through their ability to act as Rab effectors (Rabs are a family of Ras GTP-ases involved in membrane trafficking). Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterised, but little is known about APPL2. Here we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains, and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL:Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a Kd of 140 nM. Together with other biophysical data we conclude the stoichiometry of the complex is 2:2.
Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
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ABSTRACT: The enzyme TcpG is a periplasmic protein produced by the Gram-negative pathogen Vibrio cholerae. TcpG is essential for the production of ToxR-regulated proteins, including virulence-factor pilus proteins and cholera toxin, and is therefore a target for the development of a new class of anti-virulence drugs. Here, the 1.2 Å resolution crystal structure of TcpG is reported using a cryocooled crystal. This structure is compared with a previous crystal structure determined at 2.1 Å resolution from data measured at room temperature. The new crystal structure is the first DsbA crystal structure to be solved at a sufficiently high resolution to allow the inclusion of refined H atoms in the model. The redox properties of TcpG are also reported, allowing comparison of its oxidoreductase activity with those of other DSB proteins. One of the defining features of the Escherichia coli DsbA enzyme is its destabilizing disulfide, and this is also present in TcpG. The data presented here provide new insights into the structure and redox properties of this enzyme, showing that the binding mode identified between E. coli DsbB and DsbA is likely to be conserved in TcpG and that the β5-α7 loop near the proposed DsbB binding site is flexible, and suggesting that the tense oxidized conformation of TcpG may be the consequence of a short contact at the active site that is induced by disulfide formation and is relieved by reduction.
Acta crystallographica. Section D, Biological crystallography 10/2012; 68(Pt 10):1290-302. · 12.67 Impact Factor
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Michelle P Christie,
Andrew E Whitten,
Gordon J King,
Shu-Hong Hu,
Russell J Jarrott,
Kai-En Chen,
Anthony P Duff,
Philip Callow,
Brett M Collins,
David E James, Jennifer L Martin
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ABSTRACT: When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode.
Proceedings of the National Academy of Sciences 06/2012; 109(25):9816-21. · 9.68 Impact Factor
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ABSTRACT: Fam96a mRNA, which encodes a mammalian DUF59 protein, is enriched in macrophages. Recombinant human Fam96a forms stable monomers and dimers in solution. Crystal structures of these two forms revealed that each adopts a distinct type of domain-swapped dimer, one of which is stabilized by zinc binding. Two hinge loops control Fam96a domain swapping; both are flexible and highly conserved, suggesting that domain swapping may be a common feature of eukaryotic but not bacterial DUF59 proteins. The derived monomer fold of Fam96a diverges from that of bacterial DUF59 counterparts in that the C-terminal region of Fam96a is much longer and is positioned on the opposite side of the N-terminal core fold. The putative metal-binding site of bacterial DUF59 proteins is not conserved in Fam96a, but Fam96a interacts tightly in vitro with Ciao1, the cytosolic iron-assembly protein. Moreover, Fam96a and Ciao1 can be co-immunoprecipitated, suggesting that the interaction also occurs in vivo. Although predicted to have a signal peptide, it is shown that Fam96a is cytoplasmic. The data reveal that eukaryotic DUF59 proteins share intriguing characteristics with amyloidogenic proteins.
Acta crystallographica. Section D, Biological crystallography 06/2012; 68(Pt 6):637-48. · 12.67 Impact Factor
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ABSTRACT: Proteins containing a domain of unknown function 59 (DUF59) appear to have a variety of physiological functions, ranging from iron-sulfur cluster assembly to DNA repair. DUF59 proteins have been found in bacteria, archaea and eukaryotes, however Fam96a and Fam96b are the only mammalian proteins predicted to contain a DUF59 domain. Fam96a is an 18 kDa protein comprised primarily of a DUF59 domain (residues 31-157) and an N-terminal signal peptide (residues 1-27). Interestingly, the DUF59 domain of Fam96a exists as monomeric and dimeric forms in solution, and X-ray crystallography studies of both forms unexpectedly revealed two different domain-swapped dimer structures. Here we report the backbone resonance assignments and secondary structure of the monomeric form of the 127 residue DUF59 domain of human Fam96a. This study provides the basis for further understanding the structural variability exhibited by Fam96a and the mechanism for domain swapping.
Biomolecular NMR Assignments 05/2012; · 0.72 Impact Factor
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ABSTRACT: ARHGAP22 is a RhoGAP protein comprising an N-terminal PH domain, a RhoGAP domain and a C-terminal coiled-coil domain. It has recently been identified as an Akt substrate that binds 14-3-3 proteins in response to treatment with growth factors involved in cell migration. We used a range of biophysical techniques to investigate the weak interaction between 14-3-3 and a truncated form of ARHGAP22 lacking the coiled-coil domain. This weak interaction could be stabilized by chemical cross-linking which we used to show that: a monomer of ARHGAP22 binds a dimer of 14-3-3; the ARHGAP22 PH domain is required for the 14-3-3 interaction; the RhoGAP domain is unlikely to participate in the interaction; Ser16 is the more important of two predicted 14-3-3 binding sites; and, phosphorylation of Ser16 may not be necessary for 14-3-3 interaction under the conditions we used. Small angle X-ray scattering and cross-link information were used to generate solution structures of the isolated proteins and of the cross-linked ARHGAP22:14-3-3 complex, showing that no major rearrangement occurs in either protein upon binding, and supporting a role for the PH domain and N-terminal peptide of ARHGAP22 in the 14-3-3 interaction. Small-angle X-ray scattering measurements of mixtures of ARHGAP22 and 14-3-3 were used to establish that the affinity of the interaction is ∼30 µM.
PLoS ONE 01/2012; 7(8):e41731. · 4.09 Impact Factor
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Eugene Valkov,
Anna Stamp,
Frank Dimaio,
David Baker,
Brett Verstak,
Pietro Roversi,
Stuart Kellie,
Matthew J Sweet,
Ashley Mansell,
Nicholas J Gay, Jennifer L Martin,
Bostjan Kobe
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ABSTRACT: Initiation of the innate immune response requires agonist recognition by pathogen-recognition receptors such as the Toll-like receptors (TLRs). Toll/interleukin-1 receptor (TIR) domain-containing adaptors are critical in orchestrating the signal transduction pathways after TLR and interleukin-1 receptor activation. Myeloid differentiation primary response gene 88 (MyD88) adaptor-like (MAL)/TIR domain-containing adaptor protein (TIRAP) is involved in bridging MyD88 to TLR2 and TLR4 in response to bacterial infection. Genetic studies have associated a number of unique single-nucleotide polymorphisms in MAL with protection against invasive microbial infection, but a molecular understanding has been hampered by a lack of structural information. The present study describes the crystal structure of MAL TIR domain. Significant structural differences exist in the overall fold of MAL compared with other TIR domain structures: A sequence motif comprising a β-strand in other TIR domains instead corresponds to a long loop, placing the functionally important "BB loop" proline motif in a unique surface position in MAL. The structure suggests possible dimerization and MyD88-interacting interfaces, and we confirm the key interface residues by coimmunoprecipitation using site-directed mutants. Jointly, our results provide a molecular and structural basis for the role of MAL in TLR signaling and disease protection.
Proceedings of the National Academy of Sciences 09/2011; 108(36):14879-84. · 9.68 Impact Factor
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ABSTRACT: Since its discovery in 1991, the bacterial periplasmic oxidative folding catalyst DsbA has been the focus of intense research. Early studies addressed why it is so oxidizing and how it is maintained in its less stable oxidized state. The crystal structure of Escherichia coli DsbA (EcDsbA) revealed that the oxidizing periplasmic enzyme is a distant evolutionary cousin of the reducing cytoplasmic enzyme thioredoxin. Recent significant developments have deepened our understanding of DsbA function, mechanism, and interactions: the structure of the partner membrane protein EcDsbB, including its complex with EcDsbA, proved a landmark in the field. Studies of DsbA machineries from bacteria other than E. coli K-12 have highlighted dramatic differences from the model organism, including a striking divergence in redox parameters and surface features. Several DsbA structures have provided the first clues to its interaction with substrates, and finally, evidence for a central role of DsbA in bacterial virulence has been demonstrated in a range of organisms. Here, we review current knowledge on DsbA, a bacterial periplasmic protein that introduces disulfide bonds into diverse substrate proteins and which may one day be the target of a new class of anti-virulence drugs to treat bacterial infection.
Antioxidants & Redox Signaling 01/2011; 14(9):1729-60. · 8.20 Impact Factor
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ABSTRACT: Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed-open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1-N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.
Proceedings of the National Academy of Sciences 01/2011; 108(3):1040-5. · 9.68 Impact Factor
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ABSTRACT: CNS (central nervous system) adrenaline (epinephrine) is implicated in a wide range of physiological and pathological conditions. PNMT (phenylethanolamine N-methyltransferase) catalyses the final step in the biosynthesis of adrenaline, the conversion of noradrenaline (norepinephrine) to adrenaline by methylation. To help elucidate the role of CNS adrenaline, and to develop potential drug leads, potent, selective and CNS-active inhibitors are required. The fragment screening approach has advantages over other lead discovery methods including high hit rates, more efficient hits and the ability to sample chemical diversity more easily. In the present study we applied fragment-based screening approaches to the enzyme PNMT. We used crystallography as the primary screen and identified 12 hits from a small commercial library of 384 drug-like fragments. The hits include nine chemicals with two fused rings and three single-ring chemical systems. Eight of the hits come from three chemical classes: benzimidazoles (a known class of PNMT inhibitor), purines and quinolines. Nine of the hits have measurable binding affinities (~5-700 μM) as determined by isothermal titration calorimetry and all nine have ligand efficiencies of 0.39 kcal/mol per heavy atom or better (1 kcal≈4.184 kJ). We synthesized five elaborated benzimidazole compounds and characterized their binding to PNMT, showing for the first time how this class of inhibitors interact with the noradrenaline-binding site. Finally, we performed a pilot study with PNMT for fragment-based screening by MS showing that this approach could be used as a fast and efficient first-pass screening method prior to characterization of binding mode and affinity of hits.
Biochemical Journal 10/2010; 431(1):51-61. · 4.90 Impact Factor
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Markus Muttenthaler,
Simon T Nevin,
Anton A Grishin,
Shyuan T Ngo,
Peng T Choy,
Norelle L Daly,
Shu-Hong Hu,
Christopher J Armishaw,
Ching-I A Wang,
Richard J Lewis, Jennifer L Martin,
Peter G Noakes,
David J Craik,
David J Adams,
Paul F Alewood
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ABSTRACT: Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.
Journal of the American Chemical Society 02/2010; 132(10):3514-22. · 9.91 Impact Factor
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ABSTRACT: Current dogma dictates that bacterial proteins with misoxidized disulfide bonds are shuffled into correctly oxidized states by DsbC. There are two proposed mechanisms for DsbC activity. The first involves a DsbC-only model of substrate disulfide rearrangement. The second invokes cycles of reduction and oxidation of substrate disulfide bonds by DsbC and DsbA respectively. Here, we addressed whether the second mechanism is important in vivo by identifying whether a periplasmic reductase could complement DsbC. We screened for naturally occurring periplasmic reductases in Bacteroides fragilis, a bacterium chosen because we predicted it encodes reductases and has a reducing periplasm. We found that the B. fragilis periplasmic protein TrxP has a thioredoxin fold with an extended N-terminal region; that it is a very active reductase but a poor isomerase; and that it fully complements dsbC. These results provide direct in vivo evidence that correctly folded protein is achievable via cycles of oxidation and reduction.
Molecular Microbiology 12/2009; 75(1):13-28. · 5.01 Impact Factor
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Conan K. Wang,
Shu-Hong Hu, Jennifer L. Martin,
Tove Sjögren,
Janos Hajdu,
Lars Bohlin,
Per Claeson,
Ulf Göransson,
K. Johan Rosengren,
Jun Tang,
Ning-Hua Tan,
David J. Craik
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ABSTRACT: Cyclotides are a family of plant defense proteins that are highly resistant to adverse chemical, thermal, and enzymatic treatment.
Here, we present the first crystal structure of a cyclotide, varv F, from the European field pansy, Viola arvensis, determined at a resolution of 1.8 Å. The solution state NMR structure was also determined and, combined with measurements
of biophysical parameters for several cyclotides, provided an insight into the structural features that account for the remarkable
stability of the cyclotide family. The x-ray data confirm the cystine knot topology and the circular backbone, and delineate
a conserved network of hydrogen bonds that contribute to the stability of the cyclotide fold. The structural role of a highly
conserved Glu residue that has been shown to regulate cyclotide function was also determined, verifying its involvement in
a stabilizing hydrogen bond network. We also demonstrate that varv F binds to dodecylphosphocholine micelles, defining the
binding orientation and showing that its structure remains unchanged upon binding, further demonstrating that the cyclotide
fold is rigid. This study provides a biological insight into the mechanism by which cyclotides maintain their native activity
in the unfavorable environment of predator insect guts. It also provides a structural basis for explaining how a cluster of
residues important for bioactivity may be involved in self-association interactions in membranes. As well as being important
for their bioactivity, the structural rigidity of cyclotides makes them very suitable as a stable template for peptide-based
drug design.
Journal of Biological Chemistry 04/2009; 284(16):10672-10683. · 4.77 Impact Factor
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ABSTRACT: VERNALIZATION1 (VRN1) is required in the model plant Arabidopsis thaliana for the epigenetic suppression of the floral repressor FLC by prolonged cold treatment. Stable suppression of FLC accelerates flowering, a physiological process known as vernalization. VRN1 is a 341-residue DNA-binding protein that contains two plant-specific B3 domains (B3a and B3b), a putative nuclear localization sequence (NLS) and two putative PEST domains. VRN1(208-341) includes the second B3 domain and a region upstream that is highly conserved in the VRN1 orthologues of other dicotyledonous plants. VRN1(208-341) was crystallized by the hanging-drop method in 0.05 M sodium acetate pH 6.0 containing 1.0 M NaCl and 18%(w/v) PEG 3350. Preliminary X-ray diffraction data analysis revealed that the VRN1(208-341) crystal diffracted to 2.1 A and belonged to space group C2, with unit-cell parameters a = 105.2, b = 47.9, c = 61.2 A, alpha = 90.0, beta = 115.4, gamma = 90.0 degrees . Assuming that two molecules occupy the asymmetric unit, a Matthews coefficient of 2.05 A(3) Da(-1) and a solvent content of 40.1% were calculated.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2009; 65(Pt 3):291-4. · 0.51 Impact Factor
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ABSTRACT: If DNA is the information of life, then proteins are the machines of life--but they must be assembled and correctly folded to function. A key step in the protein-folding pathway is the introduction of disulphide bonds between cysteine residues in a process called oxidative protein folding. Many bacteria use an oxidative protein-folding machinery to assemble proteins that are essential for cell integrity and to produce virulence factors. Although our current knowledge of this machinery stems largely from Escherichia coli K-12, this view must now be adjusted to encompass the wider range of disulphide catalytic systems present in bacteria.
Nature Reviews Microbiology 03/2009; 7(3):215-25. · 21.18 Impact Factor
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Guoping Ren,
Daniel Stephan,
Zhaohui Xu,
Ying Zheng,
Danming Tang,
Rosemary S Harrison,
Mareike Kurz,
Russell Jarrott,
Stephen R Shouldice,
Annie Hiniker, Jennifer L Martin,
Begoña Heras,
James C A Bardwell
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ABSTRACT: The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.
Journal of Biological Chemistry 02/2009; 284(15):10150-9. · 4.77 Impact Factor
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ABSTRACT: Crystallography is commonly used for studying the structures of protein-protein complexes. However, a crystal structure does not define a unique protein-protein interface, and distinguishing a 'biological interface' from 'crystal contacts' is often not straightforward. A number of computational approaches exist for distinguishing them, but their error rate is high, emphasizing the need to obtain further data on the biological interface using complementary structural and functional approaches. In addition to reviewing the computational and experimental approaches for addressing this problem, we highlight two relevant examples. The first example from our laboratory involves the structure of acyl-CoA thioesterase 7, where each domain of this two-domain protein was crystallized separately, but both yielded a non-functional assembly. The structure of the full-length protein was uncovered using a combination of complementary approaches including chemical cross-linking, analytical ultracentrifugation and mutagenesis. The second example involves the platelet glycoprotein Ibalpha-thrombin complex. Two groups reported the crystal structures of this complex, but all the interacting interfaces differed between the two structures. Our computational analysis did not fully resolve the reasons for the discrepancies, but provided interesting insights into the system. This review highlights the need to complement crystallographic studies with complementary experimental and computational approaches.
Biochemical Society Transactions 01/2009; 36(Pt 6):1438-41. · 3.71 Impact Factor
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ABSTRACT: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate.
We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised.
Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.
PLoS ONE 01/2009; 4(11):e7851. · 4.09 Impact Factor
