The CWB2 Cell Wall-Anchoring Module Is Revealed by the Crystal Structures of the Clostridium difficile Cell Wall Proteins Cwp8 and Cwp6

Abstract

Bacterial cell wall proteins play crucial roles in cell survival, growth, and environmental interactions. In Gram-positive bacteria, cell wall proteins include several types that are non-covalently attached via cell wall binding domains. Of the two conserved surface-layer (S-layer)-anchoring modules composed of three tandem SLH or CWB2 domains, the latter have so far eluded structural insight. The crystal structures of Cwp8 and Cwp6 reveal multi-domain proteins, each containing an embedded CWB2 module. It consists of a triangular trimer of Rossmann-fold CWB2 domains, a feature common to 29 cell wall proteins in Clostridium difficile 630. The structural basis of the intact module fold necessary for its binding to the cell wall is revealed. A comparison with previously reported atomic force microscopy data of S-layers suggests that C. difficile S-layers are complex oligomeric structures, likely composed of several different proteins.

Cwp2 from Clostridium difficile exhibits an extended three domain fold and cell adhesion in vitro

“…In the recently reported structure of Cwp8, to which we have shown that Cwp2 bears significant structural similarity, Usenik et al . demonstrated that domain 2 is likely to form the most surface exposed region of the protein, while domains 1 and 3 are more buried. This is also likely to be true for Cwp2 due to the high similarity between the two structures.…”

“…This study has determined the structure of the ‘functional’ region of the C. difficile S‐layer‐associated protein Cwp2. The protein bears a high degree of similarity to the recently reported three‐dimensional structure of Cwp8 , with an extended fold consisting of three domains. Domains 1 and 3 show a high degree of structural similarity, while domain 2 is separated by a hinge, shows the greatest degree of variation between the proteins and appears to be capable of assuming different orientations.…”

“…Data were indexed and integrated separately with dials and scaled together with aimless . Molecular replacement was performed with phaser using three polyalanine models based on the three functional domains of Cwp8 (PDB: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5J6Q) . Domains 1, 2 and 3 yielded translation function Z‐scores of 8.26, 7.11 and 8.46 respectively, indicating that the solutions were likely to be correct, however the resulting electron density was still ambiguous.…”

“…The structures of Cwp2, Cwp8 and LMW SLP were prepared for flexibility analysis using pymol (http://www.pymol.org) and molprobity . Normal mode eigenvectors were determined for the structures of the proteins using elnemo while first and froda were used to determine flexibility of the proteins.…”

Covalent attachment and Pro‐Pro endopeptidase (PPEP‐1)‐mediated release of Clostridium difficile cell surface proteins involved in adhesion

“…CWPs in Bacillus anthracis are non‐covalently bound to a pyruvylated secondary cell wall polysaccharide through pseudo‐trimeric arrangement of their S‐layer Homology (SLH) motif (Kern et al ., ). Recently, it was shown that, like the B. anthracis CWP SLH motif, C. difficile CWP CWB2 motifs form trimers (Usenik et al ., ), that are able to bind to PS‐II (Willing et al ., ). For further details on the C. difficile CWPs, we would like to refer to a recent excellent review by Kirk et al (Kirk et al ., ).…”