Crystal Structure of an Uncommon Cellulosome-Related Protein Module from Ruminococcus flavefaciens That Resembles Papain-Like Cysteine Peptidases

Abstract

Background Ruminococcus flavefaciens is one of the predominant fiber-degrading bacteria found in the rumen of herbivores. Bioinformatic analysis of the recently sequenced genome indicated that this bacterium produces one of the most intricate cellulosome systems known to date. A distinct ORF, encoding for a multi-modular protein, RflaF_05439, was discovered during mining of the genome sequence. It is composed of two tandem modules of currently undefined function that share 45% identity and a C-terminal X-dockerin modular dyad. Gaining insight into the diversity, architecture and organization of different types of proteins in the cellulosome system is essential for broadening our understanding of a multi-enzyme complex, considered to be one of the most efficient systems for plant cell wall polysaccharide degradation in nature.Methodology/Principal FindingsFollowing bioinformatic analysis, the second tandem module of RflaF_05439 was cloned and its selenium-labeled derivative was expressed and crystallized. The crystals belong to space group P21 with unit-cell parameters of a = 65.81, b = 60.61, c = 66.13 Å, β = 107.66° and contain two protein molecules in the asymmetric unit. The crystal structure was determined at 1.38-Å resolution by X-ray diffraction using the single-wavelength anomalous dispersion (SAD) method and was refined to Rfactor and Rfree of 0.127 and 0.152 respectively. The protein molecule mainly comprises a β-sheet flanked by short α-helixes, and a globular α-helical domain. The structure was found to be structurally similar to members of the NlpC/P60 superfamily of cysteine peptidases.Conclusions/SignificanceThe 3D structure of the second repeat of the RflaF_05439 enabled us to propose a role for the currently undefined function of this protein. Its putative function as a cysteine peptidase is inferred from in silico structural homology studies. It is therefore apparent that cellulosomes integrate proteins with other functions in addition to the classic well-defined carbohydrate active enzymes.

Biochemical Characterization of Streptomyces sp. I1.2 Secretome Reveals the Presence of Multienzymatic Complexes Containing Cellulases and Accessory Enzymes

“…I1.2 is particularly intriguing, since biomass-modifying enzymes are directly involved in the deconstruction of lignocellulosic biomass. However, recent studies have suggested the presence of peptidases and proteases as members of Ruminococcus flavefaciens cellulossome, although the functional relevance of these enzymes is still not clear [27]. These observations are consistent with the detection of one Clostridium thermocellum serine protease and two serine proteases inhibitors (serpins), both containing one dockerin module [28,29].…”

Standalone cohesin as a molecular shuttle in cellulosome assembly

“…1 ). Sequence data and structural analysis have revealed that the dockerins of ScaB, CttA and a cysteine peptidase‐like scaffoldin [20] are different from the conventional symmetric dockerins, described earlier. They possess three insertions, one of which is positioned inside the second calcium‐binding loop [15].…”

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome

“…Examples of frequent components include an N-terminal signal peptide, a MurNAc amidase, and one or multiple targeting domains, such as the LysM domain, the peptydoglycan binding domain (PGBD), the choline binding domain (CGD), and the bacterial SH3b domain34. Recently, the crystal structure of a CHAP domain has been characterized as a cellulosome-related module, indicating that this family of cysteine peptidases might modulate processes other than cell-wall hydrolysis46. CHAP domains have been unified under the peptidase families C40 and C51 in the MEROPS database23, under Pfam47 domains NlpC/P60 (PF00877) and CHAP (PF05257), and under the COG database48 entries COG0791 ‘cell-wall-associated hydrolases (invasion-associated proteins)’ and COG3942 ‘surface antigen’.…”

“…CHAP superfamily members also share structural similarities that cannot be detected at the sequence level46. Beyond the catalytic core, the structures are highly divergent through deletions and insertions of structural elements.…”

“…In this regard, the PGBD and SH3 domain from PCP may form a bridge between the bacterial cell wall and the cell wall of plant substrates via their respective binding to these organelles. This correlation is also strengthened by the discovery of a CHAP domain acting as module in a cellulosome46. Recently, Xu and coworker have shed a light on the differences between substrate specificity of one turnover and two recycling cysteine proteases64.…”