Architecturally complex O-glycopeptidases are customized for mucin recognition and hydrolysis.

A challenge faced by peptidases is the recognition of highly diverse substrates. A feature of some peptidase families is the capacity to specifically use post-translationally added glycans present on their protein substrates as a recognition determinant. This is ultimately critical to enabling peptide bond hydrolysis. This class of enzyme is also frequently large and architecturally sophisticated. However, the molecular details underpinning glycan recognition by these O-glycopeptidases, the importance of these interactions, and the functional roles of their ancillary domains remain unclear. Here, using the Clostridium perfringens ZmpA, ZmpB, and ZmpC M60 peptidases as model proteins, we provide structural and functional insight into how these intricate proteins recognize glycans as part of catalytic and noncatalytic substrate recognition. Structural, kinetic, and mutagenic analyses support the key role of glycan recognition within the M60 domain catalytic site, though they point to ZmpA as an apparently inactive enzyme. Wider examination of the Zmp domain content reveals noncatalytic carbohydrate binding as a feature of these proteins. The complete three-dimensional structure of ZmpB provides rare insight into the overall molecular organization of a highly multimodular enzyme and reveals how the interplay of individual domain function may influence biological activity. O-glycopeptidases frequently occur in host-adapted microbes that inhabit or attack mucus layers. Therefore, we anticipate that these results will be fundamental to informing more detailed models of how the glycoproteins that are abundant in mucus are destroyed as part of pathogenic processes or liberated as energy sources during normal commensal lifestyles.

Structural evidence for a proline-specific glycopeptide recognition domain in an O-glycopeptidase.

The glycosylation of proteins is typically considered as a stabilizing modification, including resistance to proteolysis. A class of peptidases, referred to as glycopeptidases or O-glycopeptidases, circumvent the protective effect of glycans against proteolysis by accommodating the glycans in their active sites as specific features of substrate recognition. IMPa from Pseudomonas aeruginosa is such an O-glycopeptidase that cleaves the peptide bond immediately preceding a site of O-glycosylation, and through this glycoprotein-degrading function contributes to the host-pathogen interaction. IMPa, however, is a relatively large multidomain protein and how its additional domains may contribute to its function remains unknown. Here, through the determination of a crystal structure of IMPa in complex with an O-glycopeptide, we reveal that the N-terminal domain of IMPa, which is classified in Pfam as IMPa_N_2, is a proline recognition domain that also shows the properties of recognizing an O-linked glycan on the serine/threonine residue following the proline. The proline is bound in the center of a bowl formed by four functionally conserved aromatic amino acid side chains while the glycan wraps around one of the tyrosine residues in the bowl to make classic aromatic ring-carbohydrate CH-π interactions. This structural evidence provides unprecedented insight into how the ancillary domains in glycoprotein-specific peptidases can noncatalytically recognize specific glycosylated motifs that are common in mucin and mucin-like molecules.

Recognition of protein-linked glycans as a determinant of peptidase activity.

The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.

Noncellulosomal cohesin from the hyperthermophilic archaeon Archaeoglobus fulgidus.

The increasing numbers of published genomes has enabled extensive survey of protein sequences in nature. During the course of our studies on cellulolytic bacteria that produce multienzyme cellulosome complexes designed for efficient degradation of cellulosic substrates, we have investigated the intermodular cohesin-dockerin interaction, which provides the molecular basis for cellulosome assembly. An early search of the genome databases yielded the surprising existence of a dockerin-like sequence and two cohesin-like sequences in the hyperthermophilic noncellulolytic archaeon, Archaeoglobus fulgidus, which clearly contradicts the cellulosome paradigm. Here, we report a biochemical and biophysical analysis, which revealed particularly strong- and specific-binding interactions between these two cohesins and the single dockerin. The crystal structure of one of the recombinant cohesin modules was determined and found to resemble closely the type-I cohesin structure from the cellulosome of Clostridium thermocellum, with certain distinctive features: two of the loops in the archaeal cohesin structure are shorter than those of the C. thermocellum structure, and a large insertion of 27-amino acid residues, unique to the archaeal cohesin, appears to be largely disordered. Interestingly, the cohesin module undergoes reversible dimer and tetramer formation in solution, a property, which has not been observed previously for other cohesins. This is the first description of cohesin and dockerin interactions in a noncellulolytic archaeon and the first structure of an archaeal cohesin. This finding supports the notion that interactions based on the cohesin-dockerin paradigm are of more general occurrence and are not unique to the cellulosome system.

Cohesin diversity revealed by the crystal structure of the anchoring cohesin from Ruminococcus flavefaciens.

The cellulosome is an intriguing multienzyme complex found in cellulolytic bacteria that plays a key role in the degradation of plant cell-wall polysaccharides. In Ruminococcus flavefaciens, a predominant fiber-degrading bacterium found in ruminants, the cellulosome is anchored to the bacterial cell wall through a relatively short ScaE scaffoldin. Determination of the crystal structure of the lone type-III ScaE cohesin from R. flavefaciens (Rf-CohE) was initiated as a part of a structural effort to define cellulosome assembly. The structure was determined at 1.95 A resolution by single-wavelength anomalous diffraction. This is the first detailed description of a crystal structure for a type-III cohesin, and its features were compared with those of the known type-I and type-II cohesin structures. The Rf-CohE module folds into a nine-stranded beta-sandwich with jellyroll topology, typically observed for cohesins, and includes two beta-flaps in the midst of beta-strands 4 and 8, similar to the type-II cohesin structures. However, the presence in Rf-CohE of an additional 13-residue alpha-helix located between beta-strands 8 and 9 represents a dramatic divergence from other known cohesin structures. The prominent alpha-helix is enveloped by an extensive N-terminal loop, not observed in any other known cohesin, which embraces the helix presumably enhancing its stability. A planar surface at the upper portion of the front face of the molecule, bordered by beta-flap 8, exhibits plausible dimensions and exposed amino acid residues to accommodate the dockerin-binding site.

Crystallization and preliminary X-ray analysis of a cohesin-like module from AF2375 of the archaeon Archaeoglobus fulgidus.

A cohesin-like module of 160 amino-acid residues from the hypothetical protein AF2375 of the noncellulolytic, hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus was cloned, expressed, purified, crystallized and subjected to X-ray structural study in order to compare its structure with those of cellulolytic cohesins. The crystals had cubic symmetry, with unit-cell parameters a = b = c = 101.75 A in space group P4(3)32, and diffracted to 1.82 A resolution. The asymmetric unit contained a single cohesin molecule. A model assembled from six cohesin structures (PDB entries 1anu, 1aoh, 1g1k, 1qzn, 1zv9 and 1tyj) of very low sequence identity to the cohesin-like module was used in molecular-replacement attempts, producing a marginal solution.

Preliminary X-ray characterization of a novel type of anchoring cohesin from the cellulosome of Ruminococcus flavefaciens.

Ruminococcus flavefaciens is an anaerobic bacterium that resides in the gastrointestinal tract of ruminants. It produces a highly organized multi-enzyme cellulosome complex that plays a key role in the degradation of plant cell walls. ScaE is one of the critical structural components of its cellulosome that serves to anchor the complex to the cell wall. The seleno-L-methionine-labelled derivative of the ScaE cohesin module has been cloned, expressed, purified and crystallized. The crystals belong to space group C2, with unit-cell parameters a = 155.6, b = 69.3, c = 93.0 A, beta = 123.4 degrees, and contain four molecules in the asymmetric unit. Diffraction data were phased to 1.95 A using the anomalous signal from the Se atoms.

Crystallization and preliminary X-ray analysis of Acetivibrio cellulolyticus cellulosomal type II cohesin module: two versions having different linker lengths.

The second type II cohesin module of the cellulosomal scaffoldin polypeptide ScaB from Acetivibrio cellulolyticus (CohB2) was cloned into two constructs: one containing a short (five-residue) C-terminal linker (CohB2_S) and the second incorporating the full native 45-residue linker (CohB2_L). Both constructs encode proteins that also include the full native six-residue N-terminal linker. The CohB2_S and CohB2_L proteins were expressed, purified and crystallized in the orthorhombic crystal system, but with different unit cells and symmetries: space group P2(1)2(1)2(1) with unit-cell parameters a = 90.36, b = 68.65, c = 111.29 A for CohB2_S and space group P2(1)2(1)2 with unit-cell parameters a = 68.76, b = 159.22, c = 44.21 A for CohB2_L. The crystals diffracted to 2.0 and 2.9 A resolution, respectively. The asymmetric unit of CohB2_S contains three cohesin molecules, while that of CohB2_L contains two molecules.

Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction.

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

Crystal structure of a type-II cohesin module from the Bacteroides cellulosolvens cellulosome reveals novel and distinctive secondary structural elements.

The incorporation of enzymes into the multi-enzyme cellulosome complex and its anchoring to the bacterial cell surface are dictated by a set of binding interactions between two complementary protein modules: the cohesin and the dockerin. In this work, the X-ray crystal structure of a type-II cohesin from scaffoldin A of Bacteroides cellulosolvens has been determined to a resolution of 1.6 angstroms using molecular replacement. The type-II B. cellulosolvens cohesin (Bc-cohesin-II) is the first detailed description of a crystal structure for a type-II cohesin, and its features were compared with the known type-I cohesins from Clostridium thermocellum and Clostridium cellulolyticum (Ct-cohesin-I and Cc-cohesin-I, respectively). The overall jelly-roll topology of the type-II Bc-cohesin is very similar to that observed for the type-I cohesins with three additional secondary structures: an alpha-helix and two "beta-flaps" that disrupt the normal course of a beta-strand. In addition, beta-strand 5 is elevated by approximately 4 angstroms on the surface of the molecule, relative to the type-I Ct and Cc-cohesins. Like its type-I analogue, the hydrophobic/aromatic core of Bc-cohesin-II comprises an upper and lower core, but an additional aromatic patch and conserved tryptophan at the crown of the molecule serves to stabilize the alpha-helix of the type-II cohesin. Comparison of Bc-cohesin-II with the known type-I cohesin-dockerin heterodimer suggests that each of the additional secondary structural elements assumes a flanking position relative to the putative dockerin-binding surface. The raised ridge formed by beta-strand 5 confers additional distinctive topographic features to the proposed binding interface that collectively distinguish between the type-II and type-I cohesins.

The Details of Glycolipid Glycan Hydrolysis by the Structural Analysis of a Family 123 Glycoside Hydrolase from Clostridium perfringens.

Clostridium perfringens is an opportunistic pathogen of humans and animals whose genome encodes a wide variety of putative carbohydrate-hydrolyzing enzymes that are increasingly being shown to be directed toward the cleavage of host glycans. Among these putative enzymes is a member of glycoside hydrolase family 123. Here we show that the recombinant enzyme (referred to as CpNga123) encoded by the gene cloned from C. perfringens strain ATCC 13124 (locus tag CPF_1473) is a ß-N-acetylgalactosaminidase, similar to NgaP from Paenibacillus sp. TS12. Like NgaP, CpNga123 was able to cleave the terminal ß-D-GalNAc-(1→4)-D-Gal and ß-D-GalNAc-(1→3)-D-Gal motifs that would be found in glycosphigolipids. The X-ray crystal structure of CpNga123 revealed it to have an N-terminal ß-sandwich domain and a (ß/α)8-barrel catalytic domain with a C-terminal α-helical elaboration. The structures determined in complex with reaction products provide details of the -1 subsite architecture, catalytic residues, and a structural change in the active site that is likely required to enable hydrolysis of the glycosidic bond by promoting engagement of the substrate by the catalytic residues. The features of the active site support the likelihood of a substrate-assisted catalytic mechanism for this enzyme. The structures of an inactive mutant of CpNga123 in complex with intact GA2 and Gb4 glycosphingolipid motifs reveal insight into aglycon recognition and suggest that the kinked or pleated conformation of GA2 caused by the ß-1,4-linkage between N-acetylgalactosamine and galactose, and the accommodation of this conformation by the enzyme active site, may be responsible for greater activity on GA2.

Structural and functional characterization of a novel type-III dockerin from Ruminococcus flavefaciens.

Phylogenetic analysis of known dockerins in Ruminococcus flavefaciens revealed a novel subtype, type-III, in the scaffoldin proteins, ScaA, ScaB, ScaC and ScaE. In this study, we explored the Ca²âº-binding properties of the type-III dockerin from the ScaA scaffoldin (ScaADoc) using a battery of structural and biophysical approaches including circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, and nuclear magnetic resonance spectroscopy. Despite the lack of a second canonical Ca²âº-binding loop, the behaviour of ScaADoc is similar with respect to other dockerin protein modules in terms of its responsiveness to Ca²âº and affinity for the cohesin from the ScaB scaffoldin. Our results highlight the robustness of dockerin modules and how their Ca²âº-binding properties can be exploited in the construction of designer cellulosomes.

Modular arrangement of a cellulosomal scaffoldin subunit revealed from the crystal structure of a cohesin dyad.

The cellulosome complex is composed of a conglomerate of subunits, each of which comprises a set of interacting functional modules. Scaffoldin (Sca), a major cellulosomal subunit, is responsible for organizing the cellulolytic subunits into the complex. This is accomplished by the interaction of two complementary classes of modules-a cohesin (Coh) module on the Sca subunit and a dockerin module on each of the enzymatic subunits. Although individual Coh modules from different cellulosomal scaffoldins have been subjected to intensive structural investigation, the Sca subunit in its entirety has not, and there remains a paucity of information on the arrangement and interactions of Cohs within the Sca subunit. In the present work, we describe the crystal structure of a type II Coh dyad from the ScaB "adaptor" Sca of Acetivibrio cellulolyticus. The ScaB Cohs are oriented in an "antiparallel" manner relative to one another, with their dockerin-interacting surfaces (beta-strands 8-3-6-5) facing the same direction-aligned on the same plane. A set of extensive hydrophobic and hydrogen-bond contacts between the Cohs and the short interconnecting linker segment between them stabilizes the modular orientation. This Coh dyad structure provides novel information about Coh-Coh association and arrangement in the Sca and further insight into intermodular linker interactions. Putative structural arrangements of a hexamodular complex, composed of the Coh dyad bound to two X-dockerin modules, were suggested.

Intermodular linker flexibility revealed from crystal structures of adjacent cellulosomal cohesins of Acetivibrio cellulolyticus.

Cellulosome complexes comprise an intercalated set of multimodular dockerin-containing enzymatic subunits connected to cohesin-containing nonenzymatic subunits called scaffoldins. The adjoining modules in each cellulosomal subunit are interconnected by a variety of linker segments of different lengths and composition. The exact role of the cellulosomal linkers has yet to be described, although it is assumed that they contribute to the architecture and action of the cellulosome by providing the protein subunits with flexibility and by providing spacers between the enzymatic modules that could enhance interactions with the cellulose substrate. Here we present four crystal structures of Acetivibrio cellulolyticus cellulosomal type II cohesins with linker extensions. Two of the structures represent two different crystal forms (trigonal and orthorhombic) of the same N-terminal cohesin module (CohB1) together with its full (6-residue) native C-terminal linker, derived from scaffoldin B. The other two structures belong to the adjacent (second) cohesin module (CohB2), each of which was crystallized with the same 6-residue linker segment, but now positioned at the N-terminus and with either a truncated (5-residue) or a full-length (45-residue) C-terminal linker, respectively. Comparison between the two CohB1 structures revealed significant differences in the conformation of their equivalent C-terminal linker segment. In one crystal form a helical conformation was observed, as opposed to an extended conformation in the other. The CohB2 structures also displayed diverse conformations in their linker segments. In these structures, different linker conformations were observed in the individual molecules within the asymmetric unit of each structure. This conformational diversity implies that the linkers may adopt alternative conformations in their natural environment, consistent with varying environmental conditions. The findings suggest that linkers can play an important role in the assembly, dynamics and function of the cellulosomal components.

Preliminary X-ray characterization and phasing of a type II cohesin domain from the cellulosome of Acetivibrio cellulolyticus.

The N-terminal type II cohesin from the cellulosomal ScaB subunit of Acetivibrio cellulolyticus was crystallized in two different crystal systems: orthorhombic (space group P2(1)2(1)2(1)), with unit-cell parameters a = 37.455, b = 55.780, c = 87.912 A, and trigonal (space group P3(1)21), with unit-cell parameters a = 55.088, b = 55.088, c = 112.553 A. The two crystals diffracted to 1.2 and 1.9 A, respectively. A selenomethionine derivative was also crystallized and exhibited trigonal symmetry (space group P3(1)21), with unit-cell parameters a = 55.281, b = 55.281, c = 112.449 A and a diffraction limit of 1.97 A. Initial phasing of the trigonal crystals was successfully performed by the SIRAS method using Cu Kalpha radiation with the selenomethionine derivative as a heavy-atom derivative. The structure of the orthorhombic crystal form was solved by molecular replacement using the coordinates of the trigonal form.