Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module.

ABSTRACT: Activation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

A dual cohesin-dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome.

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin-dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.

Molecular organization and protein stability of the Clostridium thermocellum glucuronoxylan endo-ß-1,4-xylanase of family 30 glycoside hydrolase in solution.

Glucuronoxylan-ß-1,4-xylanohydrolase from Clostridium thermocellum (CtXynGH30) hydrolyzes ß-1,4-xylosidic linkages in 4-O-Methyl-D-glucuronoxylan. CtXynGH30 comprises an N-terminal catalytic domain, CtXyn30A, joined by a typical linker sequence to a family 6 carbohydrate-binding module, termed CtCBM6. ITC, mass spectrometric and enzyme activity analyses of CtXyn30A:CtCBM6 (1:1 M ratio), CtXyn30A and CtXynGH30 showed that the linker peptide plays a key role in connecting and orienting CtXyn30A and CtCBM6 modules resulting in the enhanced activity of CtXynGH30. To visualize the disposition of the two protein domains of CtXynGH30, SAXS analysis revealed that CtXynGH30 is monomeric and has a boot-shaped molecular envelope in solution with a Dmax of 18 nm and Rg of 3.6 nm. Kratky plot displayed the protein in a fully folded and flexible state. The ab initio derived dummy atom model of CtXynGH30 superposed well with the modelled structure.

Structure-function analyses generate novel specificities to assemble the components of multienzyme bacterial cellulosome complexes.

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, whereas 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.

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition.

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 CBM families that targeted ß-glucans, ß-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 ß-glucans and ß-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.

Stability and Ligand Promiscuity of Type A Carbohydrate-binding Modules Are Illustrated by the Structure of Spirochaeta thermophila StCBM64C.

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

Target highlights from the first post-PSI CASP experiment (CASP12, May-August 2016).

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.

Single Binding Mode Integration of Hemicellulose-degrading Enzymes via Adaptor Scaffoldins in Ruminococcus flavefaciens Cellulosome.

The assembly of one of Nature's most elaborate multienzyme 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 multienzyme complexes. Ruminococcus flavefaciens, the primary degrader of plant structural carbohydrates in the rumen of mammals, uses a portfolio of more than 220 different dockerins 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. Groups 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 in the gastrointestinal tract of mammals.

The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans.

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 ß-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-ß-1,4-Xylp-ß-1,4-Xylp-[α-1,3-Araf]-ß-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.

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and ß-1,3-1,4-Glucans through Distinct Mechanisms.

Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with ß-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a ß-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to ß-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to ß-1,3-1,4-glucans while substantially decreasing the affinity for decorated ß-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified ß-1,3-1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant.

Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors.

Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼10(12) M). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes.

Combined Crystal Structure of a Type I Cohesin: MUTATION AND AFFINITY BINDING STUDIES REVEAL STRUCTURAL DETERMINANTS OF COHESIN-DOCKERIN SPECIFICITIES.

Cohesin-dockerin interactions orchestrate the assembly of one of nature's most elaborate multienzyme complexes, the cellulosome. Cellulosomes are produced exclusively by anaerobic microbes and mediate highly efficient hydrolysis of plant structural polysaccharides, such as cellulose and hemicellulose. In the canonical model of cellulosome assembly, type I dockerin modules of the enzymes bind to reiterated type I cohesin modules of a primary scaffoldin. Each type I dockerin contains two highly conserved cohesin-binding sites, which confer quaternary flexibility to the multienzyme complex. The scaffoldin also bears a type II dockerin that anchors the entire complex to the cell surface by binding type II cohesins of anchoring scaffoldins. In Bacteroides cellulosolvens, however, the organization of the cohesin-dockerin types is reversed, whereby type II cohesin-dockerin pairs integrate the enzymes into the primary scaffoldin, and type I modules mediate cellulosome attachment to an anchoring scaffoldin. Here, we report the crystal structure of a type I cohesin from B. cellulosolvens anchoring scaffoldin ScaB to 1.84-Å resolution. The structure resembles other type I cohesins, and the putative dockerin-binding site, centered at ß-strands 3, 5, and 6, is likely to be conserved in other B. cellulosolvens type I cohesins. Combined computational modeling, mutagenesis, and affinity-based binding studies revealed similar hydrogen-bonding networks between putative Ser/Asp recognition residues in the dockerin at positions 11/12 and 45/46, suggesting that a dual-binding mode is not exclusive to the integration of enzymes into primary cellulosomes but can also characterize polycellulosome assembly and cell-surface attachment. This general approach may provide valuable structural information of the cohesin-dockerin interface, in lieu of a definitive crystal structure.

Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan.

Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind ß-glucan chains often display broad specificity recognizing ß1,4-glucans (cellulose), ß1,3-ß1,4-mixed linked glucans and xyloglucan, a ß1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the ß1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of ß-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a ß-sandwich fold. The ligand binding site comprises the ß-sheet that forms the concave surface of the proteins. Binding to the backbone chains of ß-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize ß-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln(106) is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which ß-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated ß-glucans.

ArsC3 from Desulfovibrio alaskensis G20, a cation and sulfate-independent highly efficient arsenate reductase.

Desulfovibrio alaskensis G20, a sulfate-reducing bacterium, contains an arsRBC2C3 operon that encodes two putative arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3. In this study, resistance assays in E. coli transformed with plasmids containing either of the two recombinant arsenate reductases, showed that only DaG20_ArsC3 is functional and able to confer arsenate resistance. Kinetic studies revealed that this enzyme uses thioredoxin as electron donor and therefore belongs to Staphylococcus aureus plasmid pI258 and Bacillus subtilis thioredoxin-coupled arsenate reductases family. Both enzymes from this family contain a potassium-binding site, but only in Sa_ArsC does potassium actually binds resulting in a lower K m. Important differences between the S. aureus and B. subtilis enzymes and DaG20_ArsC3 are observed. DaG20_ArsC3 contains only two (Asn10, Ser33) of the four (Asn10, Ser33, Thr63, Asp65) conserved amino acid residues that form the potassium-binding site and the kinetics is not significantly affected by the presence of either potassium or sulfate ions. Isothermal titration calorimetry measurements confirmed nonspecific binding of K(+) and Na(+), corroborating the non-relevance of these cations for catalysis. Furthermore, the low K m and high k cat values determined for DaG20_ArsC3 revealed that this enzyme is the most catalytically efficient potassium-independent arsenate reductase described so far and, for the first time indicates that potassium binding is not essential to have low K m, for Trx-arsenate reductases.

Novel Clostridium thermocellum type I cohesin-dockerin complexes reveal a single binding mode.

Protein-protein interactions play a pivotal role in a large number of biological processes exemplified by the assembly of the cellulosome. Integration of cellulosomal components occurs through the binding of type I cohesin modules located in a non-catalytic molecular scaffold to type I dockerin modules located at the C terminus of cellulosomal enzymes. The majority of type I dockerins display internal symmetry reflected by the presence of two essentially identical cohesin-binding surfaces. Here we report the crystal structures of two novel Clostridium thermocellum type I cohesin-dockerin complexes (CohOlpC-Doc124A and CohOlpA-Doc918). The data revealed that the two dockerins, Doc918 and Doc124A, are unusual because they lack the structural symmetry required to support a dual binding mode. Thus, in both cases, cohesin recognition is dominated by residues located at positions 11, 12, and 19 of one of the dockerin binding surfaces. The alternative binding mode is not possible (Doc918) or highly limited (Doc124A) because residues that assume the critical interacting positions, when dockerins are reoriented by 180°, make steric clashes with the cohesin. In common with a third dockerin (Doc258) that also presents a single binding mode, Doc124A directs the appended cellulase, Cel124A, to the surface of C. thermocellum and not to cellulosomes because it binds preferentially to type I cohesins located at the cell envelope. Although there are a few exceptions, such as Doc918 described here, these data suggest that there is considerable selective pressure for the evolution of a dual binding mode in type I dockerins that direct enzymes into cellulosomes.

Overproduction, purification, crystallization and preliminary X-ray characterization of the C-terminal family 65 carbohydrate-binding module (CBM65B) of endoglucanase Cel5A from Eubacterium cellulosolvens.

The rumen anaerobic cellulolytic bacterium Eubacterium cellulosolvens produces a large range of cellulases and hemicellulases responsible for the efficient hydrolysis of plant cell wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises a tandemly repeated carbohydrate-binding module (CBM65) fused to a glycoside hydrolase family 5 (Cel5A) catalytic domain, joined by flexible linker sequences. The second carbohydrate-binding module located at the C-terminus side of the endoglucanase (CBM65B) has been co-crystallized with either cellohexaose or xyloglucan heptasaccharide. The crystals belong to the hexagonal space group P6(5) and tetragonal space group P4(3)2(1)2, containing a single molecule in the asymmetric unit. The structures of CBM65B have been solved by molecular replacement.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum.

The modular carbohydrate-active enzyme belonging to glycoside hydrolase family 30 (GH30) from Clostridium thermocellum (CtXynGH30) is a cellulosomal protein which plays an important role in plant cell-wall degradation. The full-length CtXynGH30 contains an N-terminal catalytic module (Xyn30A) followed by a family 6 carbohydrate-binding module (CBM6) and a dockerin at the C-terminus. The recombinant protein has a molecular mass of 45 kDa. Preliminary structural characterization was carried out on Xyn30A crystallized in different conditions. All tested crystals belonged to space group P1 with one molecule in the asymmetric unit. Molecular replacement has been used to solve the Xyn30A structure.

Aqua-(4-cyano-pyridine-κN (4))(5,10,15,20-tetra-phenyl-porphyrinato-κ(4) N)magnesium.

In the title complex, [Mg(C44H28N4)(C6H4N2)(H2O)], the Mg(2+) cation is octa-hedrally coordinated and lies on an inversion center with the axially located 4-cyano-pyridine and aqua ligands exhibiting 50% substitutional disorder. The cyano-bound 4-cyano-pyridine mol-ecule also is disordered across the inversion centre. The four N atoms of the pyrrole rings of the dianionic 5,10,15,20-tetra-phenyl-porphyrin ligand occupy the equatorial sites of the octa-hedron [Mg-N = 2.0552 (10) and 2.0678 (11) Å] and the axial Mg-(N,O) bond length is 2.3798 (12) Å. The crystal packing is stabilized by weak inter-molecular C-H⋯π inter-actions.

Ultra-high resolution X-ray structure of orthorhombic bovine pancreatic Ribonuclease A at 100K.

The crystal structure of orthorhombic Bovine Pancreatic Ribonuclease A has been determined to 0.85 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16000 keV (λ = 0.77 Å). This is the first ultra-high-resolution structure of a native form of Ribonuclease A to be reported. Refinement carried out with anisotropic displacement parameters, stereochemical restraints, inclusion of H atoms in calculated positions, five [Formula: see text] moieties, eleven ethanol molecules and 293 water molecules, converged with final R values of R1(Free) = 0.129 (4279 reflections) and R1 = 0.112 (85,346 reflections). The refined structure was deposited in the Protein Data Bank as structure 7p4r. Conserved waters, using four high resolution structures, have been investigated. Cluster analysis identified clusters of water molecules that are associated with the active site of Bovine Ribonuclease A. Particular attention has been paid to making detailed comparisons between the present structure and other high quality Bovine Pancreatic Ribonuclease A X-ray crystal structures with special reference to the deposited classic monoclinic structure 3RN3 Howlin et al. (Acta Crystallogr A 45:851-861, 1989). Detailed studies of various aspects of hydrogen bonding and conformation have been carried out with particular reference to active site residues Lys-1, Lys-7, Gln-11, His-12, Lys-41, Asn-44, Thr-45, Lys-66, His-119 and Ser-123. For the two histidine residues in the active site the initial electron density map gives a clear confirmation that the position of His-12 is very similar in the orthorhombic structure to that in 3RN3. In 3RN3 His-119 exhibited poor electron density which was modelled and refined as two distinct sites, A (65%) and B (35%) but with respect to His-119 in the present ultra-high resolution orthorhombic structure there is clear electron density which was modelled and refined as a single conformation distinct from either conformation A or B in 3RN3. Other points of interest include Serine-32 which is disordered at the end of the sidechain in the present orthorhombic form but has been modelled as a single form in 3RN3. Lysine-66: there is density indicating a possible conformation for this residue. However, the density is relatively weak, and the conformation is unclear. Three types of amino acid representation in the ultra-high resolution electron density are examined: (i) sharp with very clearly resolved features, for example Lys-37; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry, for example Tyr-76; (iii) poor density and difficult or impossible to model, an example is Lys-31 for which density is missing except for Cß. The side chains of Gln-11, His-12, Lys-41, Thr-45 and His-119 are generally recognised as being closely involved in the enzyme activity. It has also been suggested that Lys-7, Asp-44, Lys-66, Phe-120, Asp-121 and Ser-123 may also have possible roles in this mechanism. A molecular dynamics study on both structures has investigated the conformations of His-119 which was modelled as two conformations in 3RN3 but is observed to have a single clearly defined conformation in the present orthorhombic structure. MD has also been used to investigate Lys-31, Lys-41 and Ser32. The form of the Ribonuclease A enzyme used in both the present study and in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851-861, 1989) includes a sulphate anion which occupies approximately the same location as the [Formula: see text] phosphate group in protein nucleotide complexes (Borkakoti et al. in J Mol Biol 169:743-755, 1983). The present structure contains 5 [Formula: see text] groups SO41151-SO41155 two of which, SO41152 and SO41153 are disordered, SO41152 being in the active site, and 11 EtOH molecules, EOH A 201-EOH A 211 all of which have good geometry. H atoms were built into the EtOH molecules geometrically. Illustrations of these features in the present structure are included here. The sulphates are presumably present in the material purchased for use in the present study. 293 water molecules are included in the present structure compared to 134 in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851-861, 1989).

Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies.

The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.