Elaborate cellulosome architecture ofAcetivibrio cellulolyticusrevealed by selective screening of cohesin–dockerin interactions

Abstract

Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin–dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be “purified” via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin–dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome’s total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors

“…The residues that participate in dockerin recognition are mostly invariant in the three cohesins (data not shown), suggesting that ScaC can accommodate three ScaB proteins via the recognition of the C-terminal dockerin module. In addition, a second A. cellulolyticus protein termed ScaJ, accession number WP_010243813, contains a single N-terminal cohesin module that shares extensive similarity with the ScaC cohesins, primarily at the residues important for protein recognition (13,19). ScaJ also contains a C-terminal SLH domain, which suggests that, like ScaC, it constitutes an anchoring scaffoldin with the ability to recognize a single ScaB molecule.…”

“…Comparison of A. cellulolyticus with C. cellulolyticum and C. thermocellum Type I Coh-Doc Complexes-It was previously observed that the A. cellulolyticus type I DocScaB specifically recognizes type I cohesins of ScaC and will not bind type I cohesins of ScaA or type I cohesins from C. thermocellum or C. cellulolyticum (19). This lack of cross-specificity between CohDoc pairs of different species may assist accurate and efficient assembly of a coherent set of enzymes into each cellulosome.…”

“…In the type I ScaB A. cellulolyticus dockerin, the specificity residues at positions 11 and 12 of the two duplicated segments consist of a conserved Ile-Asn motif. Thus, a lack of cross-specificity between the type I-Doc interactions that modulate the binding of ScaB into ScaC or the cellulosomal enzymes into ScaA (9,19) may be related to differences at these key residues.…”

“…1). These two different type I subclasses do not cross-react (19,28). To identify the residues that modulate type I Coh-Doc specificity in A. cellulolyticus, a mutagenesis study, based on the crystal structure of DocScaB and the alignment of this dockerin with the primary consensus sequence of type I dockerins of A. cellulolyticus cellulosomal enzymes (ϳ100 primary sequences of A. cellulolyticus enzyme dockerins were used), was initiated (Fig.…”

Structure–function analyses generate novel specificities to assemble the components of multienzyme bacterial cellulosome complexes

“…Thus, Coh-contacting residues at positions 11 and 12 (numbering established considering the first Gly of each calcium binding loop as residue 1), which are thought to be the major specificity determinants of all type I Docs (3), are different in the ScaB and enzyme type I Docs. Differences at these key residues may explain why there is a lack of cross-specificity between the type I Doc interactions that modulate the binding of ScaB onto ScaC or the cellulosomal enzymes onto ScaA (10,21). It is possible, however, that other elements of the two type I Doc species confer their observed distinct specificities.…”