Architecturally complex -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.

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.

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.

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 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.

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 Ca²⁺-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 Ca²⁺-binding loop, the behaviour of ScaADoc is similar with respect to other dockerin protein modules in terms of its responsiveness to Ca²⁺ and affinity for the cohesin from the ScaB scaffoldin.

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.

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.

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.

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.

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.

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.

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.

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.

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.