MOLECULAR CLAMP OF THE RUMINOCOCCUS FLAVEFACIENS SCAA DOCKERIN TERMINI

Yoav Barak ¹ Michal Slutzki ² Alon Karpol ² Maroor Jobby ³ Bareket Dassa ² Steven Smith ³ Raphael Lamed ⁴ Edward A. Bayer ²

¹Chemical Research Support, Weizmann Institute of Science, Rehovot
²Biological Chemistry, Weizmann Institute of Science, Rehovot
³Biochemistry, Queen’s University, Kingston
⁴Molecular Microbiology and Biotechnology, Tel Aviv University, Tel-Aviv

The cellulosome is a large extracellular, multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and non-catalytic subunits, interconnected by the tenacious Ca²⁺-dependent cohesin-dockerin interaction. In the cellulosome of the rumen bacterium, Ruminococcus flavefaciens, at least four distinct types of cohesin-dockerin interaction have been observed and were found to differ from other known cohesin and dockerin modules. In this study, we concentrated on the singular ScaA dockerin, which incorporates the primary enzyme-integrating ScaA scaffoldin into the ScaB adaptor scaffoldin. In the absence of a solved structure for the ScaA dockerin module, we have generated a model using the I-TASSER server, in which we identified a stacking interaction between the dockerin termini (W9 and P85). Upon review of available dockerin structures from other species, we discovered a potential conservation of a terminal clamp either as a stacking or electrostatic interaction. We suggest that this thematic interaction may play a role in structure stabilization of the dockerin. To challenge this hypothesis experimentally, the participating residues were mutated to alanine, and the properties of the resultant mutants were compared to those of the wild-type dockerin. The characteristic blue shift in tryptophan fluorescence was lacking in the P85A mutant upon Ca²⁺-binding, leading us to conclude that in contrast to the wild type, the tryptophan failed to undergo quenching in this mutant. The interaction of the mutants with the cohesin partner was also impaired, thus supporting the importance of an interaction between the tryptophan and the eliminated proline. Sequence analysis of different dockerins in R. flavefaciens and other microorganisms revealed a trend of aromatic residues upstream of the first Ca²⁺-binding loop and a proline residue at the C-terminus of the molecule. We suggest a dockerin clamp to be a major characteristic of dockerins together with the Ca²⁺-binding motif and internal symmetry.