I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1218]
Abstract: The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-Å resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T1 in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-Å structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T1 refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni.
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Jun 2011
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I04-Macromolecular Crystallography
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Abstract: The enzymatic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The enzymes that catalyze this process include xylanases that degrade xylan, a β-1,4-xylose polymer that is decorated with various sugars. Although xylanases efficiently hydrolyze unsubstituted xylans, these enzymes are unable to access highly decorated forms of the polysaccharide, such as arabinoxylans that contain arabinofuranose decorations. Here, we show that a Clostridium thermocellum enzyme, designated CtXyl5A, hydrolyzes arabinoxylans but does not attack unsubstituted xylans. Analysis of the reaction products generated by CtXyl5A showed that all the oligosaccharides contain an O3 arabinose linked to the reducing end xylose. The crystal structure of the catalytic module (CtGH5) of CtXyl5A, appended to a family 6 noncatalytic carbohydrate-binding module (CtCBM6), showed that CtGH5 displays a canonical (α/β)8-barrel fold with the substrate binding cleft running along the surface of the protein. The catalytic apparatus is housed in the center of the cleft. Adjacent to the −1 subsite is a pocket that could accommodate an l-arabinofuranose-linked α-1,3 to the active site xylose, which is likely to function as a key specificity determinant. CtCBM6, which adopts a β-sandwich fold, recognizes the termini of xylo- and gluco-configured oligosaccharides, consistent with the pocket topology displayed by the ligand-binding site. In contrast to typical modular glycoside hydrolases, there is an extensive hydrophobic interface between CtGH5 and CtCBM6, and thus the two modules cannot function as independent entities.
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Jun 2011
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I22-Small angle scattering & Diffraction
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Abstract: The CCN (cyr61, ctgf, nov) proteins (CCN1-6) are an important family of matricellular regulatory factors involved in internal and external cell signaling. They are central to essential biological processes such as adhesion, proliferation, angiogenesis, tumorigenesis, wound healing, and modulation of the extracellular matrix. They possess a highly conserved modular structure with four distinct modules that interact with a wide range of regulatory proteins and ligands. However, at the structural level, little is known although their biological function(s) seems to require cooperation between individual modules. Here we present for the first time structural determinants of two of the CCN family members, CCN3 and CCN5 (expressed in Escherichia coli), using small angle x-ray scattering. The results provide a description of the overall molecular shape and possible general three-dimensional modular arrangement for CCN proteins. These data unequivocally provide insight of the nature of CCN protein(s) in solution and thus important insight into their structure-function relationships.
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May 2011
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1221]
Abstract: Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all ?-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric ?-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds.
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May 2011
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I02-Macromolecular Crystallography
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X.
Qi
,
F.
Loiseau
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W. L.
Chan
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Y.
Yan
,
Z.
Wei
,
L.-g.
Milroy
,
R. M.
Myers
,
S. V.
Ley
,
R. J.
Read
,
R. W.
Carrell
,
A.
Zhou
Diamond Proposal Number(s):
[6641]
Open Access
Abstract: The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the β-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the β-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr342 of the reactive loop and Tyr241 of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys243, which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg378. Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature.
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Apr 2011
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I02-Macromolecular Crystallography
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Ming
Yang
,
Wei
Ge
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Rasheduzzaman
Chowdhury
,
Timothy
Claridge
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Holger
Kramer
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Bernhard
Schmierer
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Michael A.
Mcdonough
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Lingzhi
Gong
,
Benedikt
Kessler
,
Peter
Ratcliffe
,
Mathew
Coleman
,
Christopher
Schofield
Abstract: Cytoskeleton, Post-translational Modification, Protein Stability, Protein Structure, Proteomics, Ankyrin Repeat Domain, AnkyrinR, Factor Inhibiting HIF, Hydroxylation, Hypoxia-inducible Factor
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Mar 2011
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I03-Macromolecular Crystallography
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Morten
Nielsen
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Michael
Suits
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Min
Yang
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Conor
Barry
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Carlos
Martinez-fleites
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Louise
Tailford
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James
Flint
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Claire
Dumon
,
Benjamin
Davis
,
Harry
Gilbert
,
Gideon
Davies
Abstract: The enzymatic transfer of the sugar mannose from activated sugar donors, is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to study catalytic mechanisms of glycosyltransfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of a-mannosyl-D-glycerate using GDP-mannose as the preferred donor species; a reaction that occurs with net retention of anomeric configuration. Past work has shown that the Rhodothermus marinus MGS, classified as a GT78 glycosyltransferase, displays a GT-A fold and performs catalysis in a metal-ion dependent manner. MGS shows very unusual metal-ion dependences with Mg2+, Ca2+and, to a varying extent, Mn2+, Ni2+, and Co2+, facilitating catalysis. Here, we probe these dependences through kinetic, and calorimetric analyses of wild-type and site-directed variants of the enzyme. Mutation of residues that interact with the guanine base of GDP are correlated with a higher kcat whilst substitution of H217, a key component of the metal-coordination site, results in a change in metal specificity to Mn2+. Structural analyses of MGS complexes not only provide insight into metal coordination but also into how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active centre and the subsequent coordination of the divalent metal-ion as key factors in MGS catalysis and metal-ion dependence. Furthermore, Y220, located on a flexible loop whose conformation is likely influenced by metal binding also plays a critical role in substrate binding.
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Feb 2011
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I02-Macromolecular Crystallography
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Alan
Cartmell
,
Lauren
Mckee
,
Maria
Pena
,
Johan
Larsbrink
,
Harry
Brumer
,
Satoshi
Kaneko
,
Hatomi
Ichinose
,
Rick
Lewis
,
Anders
Vikso-neilson
,
Harry
Gilbert
,
Jon
Marles-wright
Abstract: Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism- and structure-based families. Genomic data has shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, while B. thetaiotaomicron deploys a combination of endo and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific alpha-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave alpha-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific alpha-1,2-arabinofuranosidase, CjAbf43A displays a 5-bladed beta-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for alpha-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelf-like structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.
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Feb 2011
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I03-Macromolecular Crystallography
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Abstract: Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD) has an Fe(II) center in its active site that can be replaced with Mn(II) or Co(II). Whereas Mn-HPCD exhibits steady-state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD behaves somewhat differently, exhibiting significantly higher K\textM\textO 2 KMO2 and k cat. The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. Comparison of the X-ray crystal structures of the resting and substrate-bound forms of Fe-HPCD, Mn-HPCD, and Co-HPCD shows that metal substitution has no effect on the local ligand environment, the conformational integrity of the active site, or the overall protein structure, suggesting that the protein structure does not differentially tune the potential of the metal center. Analysis of the steady-state kinetics of Co-HPCD suggests that the Co(II) center alters the relative rate constants for the interconversion of intermediates in the catalytic cycle but still allows the dioxygenase reaction to proceed efficiently. When compared with the kinetic data for Fe-HPCD and Mn-HPCD, these results show that dioxygenase catalysis can proceed at high rates over a wide range of metal redox potentials. This is consistent with the proposed mechanism in which the metal mediates electron transfer between the catechol substrate and O2 to form the postulated [M(II)(semiquinone)superoxo] reactive species. These kinetic differences and the spectroscopic properties of Co-HPCD provide new tools with which to explore the unique O2 activation mechanism associated with the extradiol dioxygenase family.
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Feb 2011
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6388]
Abstract: Heme enzymes are ubiquitous in biology and catalyze a vast array of biological redox processes. The formation of high valent ferryl intermediates of the heme iron (known as Compounds I and Compound II) is implicated for a number of catalytic heme enzymes, but these species are formed only transiently and thus have proved somewhat elusive. In consequence, there has been conflicting evidence as to the nature of these ferryl intermediates in a number of different heme enzymes, in particular the precise nature of the bond between the heme iron and the bound oxygen atom. In this work, we present high resolution crystal structures of both Compound I and Compound II intermediates in two different heme peroxidase enzymes, cytochrome c peroxidase and ascorbate peroxidase, allowing direct and accurate comparison of the bonding interactions in the different intermediates. A consistent picture emerges across all structures, showing lengthening of the ferryl oxygen bond (and presumed protonation) on reduction of Compound I to Compound II. These data clarify long standing inconsistencies on the nature of the ferryl heme species in these intermediates.
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Jan 2011
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