I02-Macromolecular Crystallography
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Diana O.
Ribeiro
,
Aldino
Viegas
,
Virgínia M. R.
Pires
,
João
Medeiros‐silva
,
Pedro
Bule
,
Wengang
Chai
,
Filipa
Marcelo
,
Carlos M. G. A.
Fontes
,
Eurico J.
Cabrita
,
Angelina S.
Palma
,
Ana Luisa
Carvalho
Diamond Proposal Number(s):
[16609]
Abstract: Understanding the specific molecular interactions between proteins and β1,3‐1,4‐mixed‐linked d‐glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate‐binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3‐1,4‐mixed‐linked over β1,4‐linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3‐1,4‐linked glucose oligosaccharides showed that both the chain length and the position of the β1,3‐linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site‐directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3‐1,4‐mixed‐linked glucans. This is mediated by a conformation–selection mechanism of the ligand in the binding cleft through CH‐π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3‐linkage at the reducing end and at specific positions along the β1,4‐linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed‐linked β‐glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture.
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Dec 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[16609]
Abstract: The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.
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Jun 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8425]
Open Access
Abstract: The cellulosome is a remarkably intricate multienzyme nanomachine produced by anaerobic bacteria to degrade plant cell wall polysaccharides. Cellulosome assembly is mediated through binding of enzyme-borne dockerin modules to cohesin modules of the primary scaffoldin subunit. The anaerobic bacterium Acetivibrio cellulolyticus produces a highly intricate cellulosome comprising an adaptor scaffoldin, ScaB, whose cohesins interact with the dockerin of the primary scaffoldin (ScaA) that integrates the cellulosomal enzymes. The ScaB dockerin selectively binds to cohesin modules in ScaC that anchors the cellulosome onto the cell surface. Correct cellulosome assembly requires distinct specificities displayed by structurally related type I cohesin-dockerin pairs that mediate ScaC-ScaB and ScaA-enzyme assemblies. To explore the mechanism by which these two critical protein interactions display their required specificities, we determined the crystal structure of the dockerin of a cellulosomal enzyme in complex with a ScaA cohesin. The data revealed that the enzyme-borne dockerin binds to the ScaA cohesin in two orientations, indicating two identical cohesin-binding sites. Combined mutagenesis experiments served to identify amino acid residues that modulate type I cohesin-dockerin specificity in A. cellulolyticus. Rational design was used to test the hypothesis that the ligand-binding surfaces of ScaA- and ScaB-associated dockerins mediate cohesin recognition, independent of the structural scaffold. Novel specificities could thus be engineered into one, but not both of the ligand-binding sites of ScaB, while attempts at manipulating the specificity of the enzyme-associated dockerin were unsuccessful. These data indicate that dockerin specificity requires critical interplay between the ligand-binding surface and the structural scaffold of these modules.
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Jan 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8425]
Open Access
Abstract: Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.
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Apr 2017
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I04-Macromolecular Crystallography
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Abstract: The recent division of the large glycoside hydrolase family 43 (GH43) into subfamilies offers a renewed opportunity to develop structure–function studies aimed at clarifying the molecular determinants of substrate specificity in carbohydrate-degrading enzymes. α-L-Arabinofuranosidases (EC 3.2.1.55) remove arabinose side chains from heteropolysaccharides such as xylan and arabinan. However, there is some evidence suggesting that arabinofuranosidases are substrate-specific, being unable to display a debranching activity on different polysaccharides. Here, the structure of Clostridium thermocellum arabinofuranosidase 43A (CtAbf43A), which has been shown to act in the removal of arabinose side chains from arabinoxylan but not from pectic arabinan, is reported. CtAbf43A belongs to GH43 subfamily 16, the members of which have a restricted capacity to attack xylans. The crystal structure of CtAbf43A comprises a five-bladed β-propeller fold typical of GH43 enzymes. CtAbf43A displays a highly compact architecture compatible with its high thermostability. Analysis of CtAbf43A along with the other member of GH43 subfamily 16 with known structure, the Bacillus subtilis arabinofuranosidase BsAXH-m2,3, suggests that the specificity of subfamily 16 for arabinoxylan is conferred by a long surface substrate-binding cleft that is complementary to the xylan backbone. The lack of a curved-shaped carbohydrate-interacting platform precludes GH43 subfamily 16 enzymes from interacting with the nonlinear arabinan scaffold and therefore from deconstructing this polysaccharide.
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Dec 2016
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8425]
Abstract: Glucuronoxylan endo-β-1,4-xylanases cleave the xylan chain specifically at sites containing 4-O-methylglucuronic acid substitutions. These enzymes have recently received considerable attention owing to their importance in the cooperative hydrolysis of heteropolysaccharides. However, little is known about the hydrolysis of glucuronoxylans in extreme environments. Here, the structure of a thermostable family 30 glucuronoxylan endo-β-1,4-xylanase (CtXyn30A) from Clostridium thermocellum is reported. CtXyn30A is part of the cellulosome, a highly elaborate multi-enzyme complex secreted by the bacterium to efficiently deconstruct plant cell-wall carbohydrates. CtXyn30A preferably hydrolyses glucuronoxylans and displays maximum activity at pH 6.0 and 70°C. The structure of CtXyn30A displays a (β/α)8 TIM-barrel core with a side-associated β-sheet domain. Structural analysis of the CtXyn30A mutant E225A, solved in the presence of xylotetraose, revealed xylotetraose-cleavage oligosaccharides partially occupying subsites −3 to +2. The sugar ring at the +1 subsite is held in place by hydrophobic stacking interactions between Tyr139 and Tyr200 and hydrogen bonds to the OH group of Tyr227. Although family 30 glycoside hydrolases are retaining enzymes, the xylopyranosyl ring at the −1 subsite of CtXyn30A-E225A appears in the α-anomeric configuration. A set of residues were found to be strictly conserved in glucuronoxylan endo-β-1,4-xylanases and constitute the molecular determinants of the restricted specificity displayed by these enzymes. CtXyn30A is the first thermostable glucuronoxylan endo-β-1,4-xylanase described to date. This work reveals that substrate recognition by both thermophilic and mesophilic glucuronoxylan endo-β-1,4-xylanases is modulated by a conserved set of residues.
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Nov 2016
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8425]
Open Access
Abstract: The assembly of one of Nature most elaborate multi-enzyme complexes, the cellulosome, results from the binding of enzyme-borne dockerins to reiterated cohesin domains located in a non-catalytic primary scaffoldin. Generally, dockerins present two similar cohesin binding interfaces that support a dual binding mode. The dynamic integration of enzymes in cellulosomes, afforded by the dual binding mode, is believed to incorporate additional flexibility in highly populated multi-enzyme complexes. Ruminococcus flavefaciens, the primary degrader of plant structural carbohydrates in the rumen of mammals, uses a portfolio of more than 220 different enzymes to assemble the most intricate cellulosome known to date. A sequence-based analysis organized R. flavefaciens dockerins into six groups. Strikingly, a subset of R. flavefaciens cellulosomal enzymes, comprising dockerins of groups 3 and 6, were shown to be indirectly incorporated into primary scaffoldins, via an adaptor scaffoldin termed ScaC. Here we report the crystal structure of a group 3 R. flavefaciens dockerin, Doc3, in complex with ScaC cohesin. Doc3 is unusual as it presents a large cohesin-interacting surface that lacks the structural symmetry required to support a dual binding mode. In addition, dockerins of groups 3 and 6, which bind exclusively to ScaC cohesin, display a conserved mechanism of protein recognition that is similar to Doc3. Group 3 and 6 dockerins are predominantly appended to hemicellulose degrading enzymes. Thus, single binding mode dockerins interacting with adaptor scaffoldins exemplify an evolutionary pathway developed by R. flavefaciens to recruit hemicellulases to the sophisticated cellulosomes acting on the gastro intestinal tract of mammals.
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Nov 2016
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Aurore
Labourel
,
Lucy I.
Crouch
,
Joana L. A.
Brás
,
Adam
Jackson
,
Artur
Rogowski
,
Joseph
Gray
,
Madhav P.
Yadav
,
Bernard
Henrissat
,
Carlos M. G. A.
Fontes
,
Harry J.
Gilbert
,
Shabir
Najmudin
,
Arnaud
Basle
,
Fiona
Cuskin
Diamond Proposal Number(s):
[1960, 7854, 9948]
Open Access
Abstract: The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.
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Aug 2016
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
Data acquisition
Detectors
Diagnostics
Health Physics
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Immacolata
Venditto
,
Ana S.
Luis
,
Maja
Rydahl
,
Julia
Schückel
,
Vânia O.
Fernandes
,
Silvia
Vidal-Melgosa
,
Pedro
Bule
,
Arun
Goyal
,
Virginia M. R.
Pires
,
Catarina G.
Dourado
,
Luís M. A.
Ferreira
,
Pedro M.
Coutinho
,
Bernard
Henrissat
,
J. Paul
Knox
,
Arnaud
Baslé
,
Shabir
Najmudin
,
Harry J.
Gilbert
,
William G. T.
Willats
,
Carlos M. G. A.
Fontes
Diamond Proposal Number(s):
[9948]
Abstract: The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBMfamilies that targeted beta-glucans, beta-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize beta-glucans and beta-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.
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Jun 2016
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I03-Macromolecular Crystallography
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Abstract: A number of anaerobic microorganisms produce multi-modular, multi-enzyme complexes termed cellulosomes. These extracellular macromolecular nanomachines are designed for the efficient degradation of plant cell-wall carbohydrates to smaller sugars that are subsequently used as a source of carbon and energy. Cellulolytic strains from the rumens of mammals, such as Ruminococcus flavefaciens, have been shown to have one of the most complex cellulosomal systems known. Cellulosome assembly requires the binding of dockerin modules located in cellulosomal enzymes to cohesin modules located in a macromolecular scaffolding protein. Over 220 genes encoding dockerin-containing proteins have been identified in the R. flavefaciens genome. The dockerin-containing enzymes can be incorporated into the primary scaffoldin (ScaA), which in turn can bind to adaptor scaffoldins (ScaB or ScaC) and subsequently to anchoring scaffoldin (ScaE), thereby attaching the whole complex to the cell surface. However, unlike other cellulosomes such as that from Clostridium thermocellum, the Ruminococcus species lack a specific carbohydrate-binding module (CBM) on ScaA which recruits the entire complex onto the surface of the substrate. Instead, a cellulose-binding protein, CttA, comprising two putative tandem novel carbohydrate-binding modules and a C-terminal X-dockerin module, which can bind to the cohesin of ScaE, may mediate the attachment of bacterial cells to cellulose. Here, the expression, purification and crystallization of the carbohydrate-binding modular part of the CttA from R. flavefaciens are described. X-ray data have been collected to resolutions of 3.23 and to 1.61 Å in space groups P3121 or P3221 and P21, respectively. The structure was phased using bound iodide from the crystallization buffer by SAD experiments.
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Jun 2015
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