title: Intramolecular clasp of the cellulosomal Ruminococcus flavefaciens ScaA dockerin module confers structural stability.
abstract: The cellulosome is a large extracellular multi-enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme-integrating ScaA scaffoldin into the cohesin-bearing ScaB adaptor scaffoldin. In the absence of a high-resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N-terminal Trp and a C-terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NMR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca(2+)-bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well-defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module.
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Slutzki
authors.forename: Maroor K
authors.initials: MK
authors.lastname: Jobby
authors.forename: Seth
authors.initials: S
authors.lastname: Chitayat
authors.forename: Alon
authors.initials: A
authors.lastname: Karpol
authors.forename: Bareket
authors.initials: B
authors.lastname: Dassa
authors.forename: Yoav
authors.initials: Y
authors.lastname: Barak
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.forename: Steven P
authors.initials: SP
authors.lastname: Smith
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
keywords: ANS, 8-anilino-1-naphthalenesulfonate; CBM, carbohydrate-binding module family 3a from C. thermocellum; Cc, Clostridium cellulolyticum; Coh, cohesin; Cohesin; Ct, Clostridium thermocellum; Doc, dockerin; HBS, hepes-buffered saline; IPTG, isopropyl-1-thio-β-d-galactoside; Protein stability; Scaffoldin; Stacking interaction; TMB, 3,3′,5,5′-tetramethylbenzidine; Xyn, xylanase T6 from Geobacillus stearothemophilus; cELISA, competitive enzyme-linked interaction assay