Crystallization and preliminary X-ray characterization of a type III cohesin-dockerin complex from the cellulosome system of Ruminococcus flavefaciens

Abstract

In Ruminococcus flavefaciens, a predominant fibre-degrading bacterium found in ruminants, cellulosomal proteins are anchored to the bacterial cell wall through a relatively small ScaE scaffoldin which includes a single type III cohesin. The cotton-binding protein CttA consists of two cellulose-binding modules and a C-terminal modular pair (XDoc) comprising an X-module and a contiguous dockerin, which exhibits high affinity towards the ScaE cohesin. Seleno-L-methionine-labelled derivatives of the ScaE cohesin module and the XDoc from CttA have been expressed, copurified and cocrystallized. The crystals belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 78.7, c = 203.4 Å, and the unit cell contains a single cohesin-XDoc complex in the asymmetric unit. The diffraction data were phased to 2.0 Å resolution using the anomalous signal of the Se atoms.

Figure 1

Schematic representation of the intermodular interaction in the R. flavefaciens FD-­1 cellulosome system. ScaE fulfils a key role in the cell-attachment and substrate-binding functions of the R. flavefaciens cellulosome system via the interaction of its cohesin module with the XDoc dyad of the cotton-binding protein CttA. CttA contains two putative CBMs, which are associated with binding to cellulosic substrates (e.g. cotton). The RfCohE–XDoc complex is circled.

Figure 2

SDS–PAGE gel (12%). The left column contains molecular-weight markers (kDa). Lanes 1 and 2 contain purified CohE and XDoc, respectively. Lane 3 contains the purified RfCohE–XDoc complex.

Figure 3

Crystals of the SeMet-labelled RfCohE–XDoc complex.

Figure 4

X-ray diffraction pattern from an ordered RfCohE–XDoc crystal.