Expression, purification, crystallization and preliminary X-ray analysis of CttA, a putative cellulose-binding protein from Ruminococcus flavefaciens

DOI: 10.1107/S2053230X15008249
PMID: 26057813

Authors: Immacolata Venditto (CIISA, Faculdade de Medicina Veterinaria, Universidade de Lisboa) , Pedro Bule (CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa) , Andrew Thompson (Synchrotron SOLEIL) , Juan Sanchez-Weatherby (Diamond Light Source) , James Sandy (Diamond Light Source) , Luis M. A. Ferreira (CIISA, Universidade de Lisboa) , Carlos M. G. A. Fontes (CIISA, Universidade de Lisboa) , Shabir Najmudin (Universidade Técnica de Lisboa)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section F Structural Biology Communications , VOL 71 , PAGES 784 - 789

State: Published (Approved)
Published: June 2015

Abstract: A number of anaerobic microorganisms produce multi-modular, multi-enzyme complexes termed cellulosomes. These extracellular macromolecular nano­machines are designed for the efficient degradation of plant cell-wall carbohydrates to smaller sugars that are subsequently used as a source of carbon and energy. Cellulolytic strains from the rumens of mammals, such as Ruminococcus flavefaciens, have been shown to have one of the most complex cellulosomal systems known. Cellulosome assembly requires the binding of dockerin modules located in cellulosomal enzymes to cohesin modules located in a macromolecular scaffolding protein. Over 220 genes encoding dockerin-containing proteins have been identified in the R. flavefaciens genome. The dockerin-containing enzymes can be incorporated into the primary scaffoldin (ScaA), which in turn can bind to adaptor scaffoldins (ScaB or ScaC) and subsequently to anchoring scaffoldin (ScaE), thereby attaching the whole complex to the cell surface. However, unlike other cellulosomes such as that from Clostridium thermocellum, the Ruminococcus species lack a specific carbohydrate-binding module (CBM) on ScaA which recruits the entire complex onto the surface of the substrate. Instead, a cellulose-binding protein, CttA, comprising two putative tandem novel carbohydrate-binding modules and a C-terminal X-dockerin module, which can bind to the cohesin of ScaE, may mediate the attachment of bacterial cells to cellulose. Here, the expression, purification and crystallization of the carbohydrate-binding modular part of the CttA from R. flavefaciens are described. X-ray data have been collected to resolutions of 3.23 and to 1.61 Å in space groups P3121 or P3221 and P21, respectively. The structure was phased using bound iodide from the crystallization buffer by SAD experiments.

Journal Keywords: X-Dockerin; Cellulosome; Ctta; Ruminococcus Flavefaciens; Cell Surface Attachment; Cellulose-Binding Protein.

Diamond Keywords: Bacteria

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography

Other Facilities: ESRF

Added On: 30/05/2015 16:46

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)