Dataset Information

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.

SUBMITTER: Slutzki M

PROVIDER: S-EPMC3821032 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Intramolecular clasp of the cellulosomal Ruminococcus flavefaciens ScaA dockerin module confers structural stability.

Slutzki Michal M Jobby Maroor K MK Chitayat Seth S Karpol Alon A Dassa Bareket B Barak Yoav Y Lamed Raphael R Smith Steven P SP Bayer Edward A EA

FEBS open bio 20130925

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 ...[more]

PMID: 24251102

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Project description:BACKGROUND: The cellulosome is a multi-enzyme machine, which plays a key role in the breakdown of plant cell walls in many anaerobic cellulose-degrading microorganisms. Ruminococcus flavefaciens FD-1, a major fiber-degrading bacterium present in the gut of herbivores, has the most intricate cellulosomal organization thus far described. Cellulosome complexes are assembled through high-affinity cohesin-dockerin interactions. More than two-hundred dockerin-containing proteins have been identified in the R. flavefaciens genome, yet the reason for the expansion of these crucial cellulosomal components is yet unknown. METHODOLOGY/PRINCIPAL FINDINGS: We have explored the full spectrum of 222 dockerin-containing proteins potentially involved in the assembly of cellulosome-like complexes of R. flavefaciens. Bioinformatic analysis of the various dockerin modules showed distinctive conservation patterns within their two Ca(2+)-binding repeats and their flanking regions. Thus, we established the conceptual framework for six major groups of dockerin types, according to their unique sequence features. Within this framework, the modular architecture of the parent proteins, some of which are multi-functional proteins, was evaluated together with their gene expression levels. Specific dockerin types were found to be associated with selected groups of functional components, such as carbohydrate-binding modules, numerous peptidases, and/or carbohydrate-active enzymes. In addition, members of other dockerin groups were linked to structural proteins, e.g., cohesin-containing proteins, belonging to the scaffoldins. CONCLUSIONS/SIGNIFICANCE: This report profiles the abundance and sequence diversity of the R. flavefaciens FD-1 dockerins, and provides the molecular basis for future understanding of the potential for a wide array of cohesin-dockerin specificities. Conserved differences between dockerins may be reflected in their stability, function or expression within the context of the parent protein, in response to their role in the rumen environment.

Project description:BackgroundRuminococcus flavefaciens is one of the predominant fiber-degrading bacteria found in the rumen of herbivores. Bioinformatic analysis of the recently sequenced genome indicated that this bacterium produces one of the most intricate cellulosome systems known to date. A distinct ORF, encoding for a multi-modular protein, RflaF_05439, was discovered during mining of the genome sequence. It is composed of two tandem modules of currently undefined function that share 45% identity and a C-terminal X-dockerin modular dyad. Gaining insight into the diversity, architecture and organization of different types of proteins in the cellulosome system is essential for broadening our understanding of a multi-enzyme complex, considered to be one of the most efficient systems for plant cell wall polysaccharide degradation in nature.Methodology/principal findingsFollowing bioinformatic analysis, the second tandem module of RflaF_05439 was cloned and its selenium-labeled derivative was expressed and crystallized. The crystals belong to space group P21 with unit-cell parameters of a?=?65.81, b?=?60.61, c?=?66.13 Å, ??=?107.66° and contain two protein molecules in the asymmetric unit. The crystal structure was determined at 1.38-Å resolution by X-ray diffraction using the single-wavelength anomalous dispersion (SAD) method and was refined to Rfactor and Rfree of 0.127 and 0.152 respectively. The protein molecule mainly comprises a ?-sheet flanked by short ?-helixes, and a globular ?-helical domain. The structure was found to be structurally similar to members of the NlpC/P60 superfamily of cysteine peptidases.Conclusions/significanceThe 3D structure of the second repeat of the RflaF_05439 enabled us to propose a role for the currently undefined function of this protein. Its putative function as a cysteine peptidase is inferred from in silico structural homology studies. It is therefore apparent that cellulosomes integrate proteins with other functions in addition to the classic well-defined carbohydrate active enzymes.

Dataset Information

Intramolecular clasp of the cellulosomal Ruminococcus flavefaciens ScaA dockerin module confers structural stability.

Publications

Intramolecular clasp of the cellulosomal Ruminococcus flavefaciens ScaA dockerin module confers structural stability.

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