Overexpression, crystallization and preliminary X-ray characterization of Ruminococcus flavefaciens scaffoldin C cohesin in complex with a dockerin from an uncharacterized CBM-containing protein

DOI: 10.1107/S2053230X14012667
PMID: 25084382

Authors: Pedro Bule (Universidade de Lisboa) , Vered Ruimy-Israeli (The Weizmann Institute of Science) , Vânia Cardoso (Universidade de Lisboa) , Edward A. Bayer (The Weizmann Institute of Science) , Carlos M. G. A. Fontes (Universidade de Lisboa) , Shabir Najmudin (Universidade de Lisboa)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section F Structural Biology Communications , VOL 70 , PAGES 1061 - 1064

State: Published (Approved)
Published: August 2014

Abstract: Cellulosomes are massive cell-bound multienzyme complexes tethered by macromolecular scaffolds that coordinate the efforts of many anaerobic bacteria to hydrolyze plant cell-wall polysaccharides, which are a major untapped source of carbon and energy. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between cohesin modules, located in the scaffold, and dockerin modules, found in the enzymes and other cellulosomal proteins. The proposed cellulosomal architecture for Ruminococcus flavefaciens strain FD-1 consists of a major scaffoldin (ScaB) that acts as the backbone to which other components attach. It has nine cohesins and a dockerin with a fused X-module that binds to the cohesin on ScaE, which in turn is covalently attached to the cell wall. The ScaA dockerin binds to ScaB cohesins allowing more carbohydrate-active modules to be assembled. ScaC acts as an adaptor that binds to both ScaA and selected ScaB cohesins, thereby increasing the repertoire of dockerin-bearing proteins that integrate into the complex. In previous studies, a screen for novel cohesin-dockerin complexes was performed which led to the identification of a total of 58 probable cohesin-dockerin pairs. Four were selected for subsequent structural and biochemical characterization based on the quality of their expression and the diversity in their specificities. One of these is C12D22, which comprises the cohesin from the adaptor ScaC protein bound to the dockerin of a CBM-containing protein. This complex has been purified and crystallized, and data were collected to resolutions of 2.5 Å (hexagonal, P65), 2.16 Å (orthorhombic, P212121) and 2.4 Å (orthorhombic, P21212) from three different crystalline forms.

Journal Keywords: cellulosome; cohesin; dockerin; Ruminococcus flavefaciens; scaffoldin C

Diamond Keywords: Bacteria

Subject Areas: Biology and Bio-materials


Instruments: I04-Macromolecular Crystallography

Added On: 25/02/2015 08:19

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)