Structural characterization of a novel autonomous cohesin fromRuminococcus flavefaciens

Abstract

PDB reference: cohesin from R. flavefaciens, 4n2oRuminococcus flavefaciens is a cellulolytic bacterium found in the rumen of herbivores and produces one of the most elaborate and variable cellulosome systems. The structure of an R. flavefaciens protein (RfCohG, ZP_06142108), representing a freestanding (non-cellulosomal) type III cohesin module, has been determined. A selenomethionine derivative with a C-terminal histidine tag was crystallized and diffraction data were measured to 2.44 Å resolution. Its structure was determined by single-wavelength anomalous dispersion, revealing eight molecules in the asymmetric unit. RfCohG exhibits the most complex among all known cohesin structures, possessing four -helical elements and a topographical protuberance on the putative dockerin-binding surface.

Standalone cohesin as a molecular shuttle in cellulosome assembly

“…The type III CohG:XDoc CttA was determined by the molecular replacement (MR) method, using CohG (4no2) [22] and XDoc CttA (4iu2) [15] as a combined search model. The crystals of the complex belonged to the space group P 2 1 2 1 2, producing a single CohG:XDoc CttA complex in the unit cell.…”

“…4 ). The opposite binding orientation could perhaps be dictated by the different topologies of the two different cohesin–dockerin binding surfaces [22]. In this context, a protuberance on CohG projects 11 Å from the planar surface of the 8‐3‐6‐5 β‐sheet (Fig.…”

“…The DNA encoding the cohesin module from the cohG scaffoldin gene of R. flavefaciens strain FD‐1 (accession number ZP_06142108, residue numbers 26–218) was cloned into the pET28a expression vector (Novagen, Madison, Wisconsin, USA) together with a sequence encoding C‐terminal hexahistidine (His) tag, as described in the previous paper [22]. The pETDuetACYC plasmid with the insert of XDoc dyad from the CttA scaffoldin gene of R. flavefaciens strain FD‐1 (gi:268610848) lacking the sequence encoding a hexa‐His tag, was kindly supplied by Orly Salama‐Alber [23].…”

“…In the first step, the coordinates of CohG (PDB code 4NO2) [22] were used as a search model for molecular replacement. The initial coordinates were subjected to several rounds of restrained refinement of positional and thermal parameters using REFMAC5 , followed by manual building of the non‐conserved side chains using Coot [29].…”

“…Recently, a peculiar and very interesting open reading frame, coding for only a signal peptide and an autonomous cohesin module (i.e., technically not part of a scaffoldin), was identified by bioinformatics analysis and termed CohG [22]. The structure of CohG was recently determined and described [22]; however, the interaction of the isolated cohesin module with various dockerins and its function remained unknown. In the present communication we shed light on the possible function of this unusual cohesin.…”