The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile.

William J Bradshaw,K Ravi Acharya,Jonathan M Kirby,Nethaji Thiyagarajan,Christopher J Chambers,Clifford C Shone,April K Roberts,Abigail H Davies

doi:10.1107/s1399004714009997

William J Bradshaw, K Ravi Acharya + Show 6 more

Open Access

https://doi.org/10.1107/s1399004714009997

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Abstract

Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhoea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The mechanism of C. difficile surface-layer (S-layer) biogenesis is also largely unknown but involves the post-translational cleavage of a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits by Cwp84, a surface-located cysteine protease. Here, the first crystal structure of the surface protein Cwp84 is described at 1.4 Å resolution and the key structural components are identified. The truncated Cwp84 active-site mutant (amino-acid residues 33-497; C116A) exhibits three regions: a cleavable propeptide and a cysteine protease domain which exhibits a cathepsin L-like fold followed by a newly identified putative carbohydrate-binding domain with a bound calcium ion, which is referred to here as a lectin-like domain. This study thus provides the first structural insights into Cwp84 and a strong base to elucidate its role in the C. difficile S-layer maturation mechanism.

Highlights

Disruption of the normally protective gut flora results in the extensive colonization and growth of Clostridium difficile (Guarner & Malagelada, 2003), a predominantly nosocomially acquired Gram-positive, spore-forming bacterium
Cwp84 active-site mutant, residues 33–497, which comprises Tyr63, we were able to interpret this part of the the propeptide, the cysteine protease domain and the newly structure with a fair degree of certainty; little to no density was identified ‘lectin-like’ domain (Fig. 1)
We have determined the structure of the Cwp84 cysteine protease domain with its bound propeptide and a newly discovered lectin-like domain

Summary

Introduction

Disruption of the normally protective gut flora results in the extensive colonization and growth of Clostridium difficile (Guarner & Malagelada, 2003), a predominantly nosocomially acquired Gram-positive, spore-forming bacterium. C. difficile expresses a self-assembling paracrystalline protein array on its outermost surface, known as an S-layer. The HMW SLP contains three putative cell-wall binding/ anchoring domains (CWBDs; Pfam 04122) which are thought to mediate noncovalent binding to the bacterial cell surface via a currently unknown mechanism. A total of 28 S-layer paralogues, including Cwp, containing three Pfam 04122 repeats at either the N-terminal or C-terminus with a ‘functional’ domain at the other end, have been identified in the C. difficile genome (Calabi et al, 2001; Fagan et al, 2011; Monot et al, 2011; Sebaihia et al, 2006)

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