Overexpression, crystallization and preliminary X-ray characterization ofRuminococcus flavefaciensscaffoldin C cohesin in complex with a dockerin from an uncharacterized CBM-containing protein

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, P6 5 ), 2.16 Å (orthorhombic, P2 1 2 1 2 1 ) and 2.4 Å (orthorhombic, P2 1 2 1 2) from three different crystalline forms.

A dual cohesin–dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome

“…Docs are inherently unstable when produced in E. coli . To promote Doc stability, B. cellulosolvens Doc of protein WP_050753099 (residues 683–752) was coexpressed in vivo with the 11th Coh of ScaA1, Bc CohScaA1 11 (AAG01230; residues 2073–2242) ( 29 , 30 ). The immediate binding of Bc DocCel48 to Bc CohScaA1 11 is believed to confer the necessary Doc stabilization.…”

Overexpression, purification, crystallization and preliminary X-ray characterization of the fourth scaffoldin A cohesin fromAcetivibrio cellulolyticusin complex with a dockerin from a family 5 glycoside hydrolase

“…The cohesin-encoding gene was positioned at the 3 0 end and the dockerin-encoding gene at the 5 0 end. Between the two, the sequences of the T7 terminator (to terminate transcription of the dockerin gene) and the T7 promoter (to control transcription of the cohesin gene) were added (Cameron et al, 2014;Bule et al, 2014). This construct also contained specifically tailored NheI and NcoI recognition sites at the 5 0 end and XhoI and SalI at the 3 0 end to allow subcloning into pET-28a (Novagen) such that the sequence encoding a six-residue His tag could be introduced either at the N-terminus of the dockerin (by cutting with NheI-SalI; the primary sequence of the N-terminal engineered tag is MGSSHHHHHHSSGLVPRGSHMAS) or at the C-terminus of the cohesin (by cutting with NcoI and XhoI; the primary sequence of the C-terminal engineered tag is LEHHHHHH).…”