Crystallization and preliminary X-ray analysis ofAcetivibrio cellulolyticuscellulosomal type II cohesin module: two versions having different linker lengths

Abstract

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 = 90.36, b = 68.65, c = 111.29 Å for CohB2_S and space group P2 1 2 1 2 with unitcell parameters a = 68.76, b = 159.22, c = 44.21 Å for CohB2_L. The crystals diffracted to 2.0 and 2.9 Å resolution, respectively. The asymmetric unit of CohB2_S contains three cohesin molecules, while that of CohB2_L contains two molecules.

Intermodular Linker Flexibility Revealed from Crystal Structures of Adjacent Cellulosomal Cohesins of Acetivibrio cellulolyticus

“…The respective recombinant proteins were expressed, purified and crystallized as previously described. 17 CohB2_S crystallized in the P2 1 2 1 2 1 space group, whereas CohB2_L crystallized in the P2 1 2 1 2 space group. A molecular replacement strategy employing structural models of CohB1-t and CohB2_S was used for structure determination of CohB2_S and CohB2_L, respectively.…”

“…15,17 Molecular replacement program MOLREP 45 as implemented in CCP4 46 was employed for structure determination, except for the SeMet CohB1-t structure that was isomorphous to the previously reported CohB1-t structure 15 and was thus refined directly. All models were corrected manually with the program COOT 47 and refined with REFMAC5 48 (implemented at the CCP4 suite).…”

Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: structural and functional significance of the intermodular linker

“…The three modules are separated by distinctive linker sequences. Such intermodular linker segments were proposed to be important for the physical association of the modules in the space, and to promote intermodular and/or intersubunit protein–protein interactions ( Bayer et al, 1998 ; Bayer et al, 2009 ; Noach et al, 2008 ).…”

Genome-wide analysis of Acetivibrio cellulolyticus provides a blueprint of an elaborate cellulosome system

“…It is bound to the cell surface via its C-terminal X-module/dockerin dyad (XDoc) to at least two additional scaffoldins. Thus, ScaA can either interact directly with the ScaD surface-anchoring scaffoldin or it may bind to the ScaC scaffoldin indirectly through a ScaB adaptor scaffoldin [ 18 , 20 , 21 ]. ScaC and ScaD serve as anchoring scaffoldins, owing to their C- terminal S-layer homology (SLH) modules, but unlike any other scaffolding yet described, the ScaD protein harbors two different types of cohesin (types I and II), which exhibit two divergent dockerin-binding specificities [ 19 ].…”