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

Abstract

Data deposition: Coordinates and observed structure factor amplitudes have been deposited in the Protein Data Bank with the wwPDB entry codes 5NRK (AcCohScaB6-DocCel5 M1) and 5NRM (AcCohScaB6-DocCel5 M2). ABSTRACTThe cellulosome is a remarkably intricate multienzyme nanomachine produced by anaerobic bacteria to degrade plant cell wall polysaccharides. Cellulosome assembly is mediated through binding of enzyme-borne dockerin modules to cohesin modules of the primary scaffoldin subunit. The anaerobic bacterium Acetivibrio cellulolyticus produces a highly intricate cellulosome comprising an adaptor scaffoldin, ScaB, whose cohesins interact with the dockerin of the primary scaffoldin (ScaA) that integrates the cellulosomal enzymes. The ScaB dockerin selectively binds to cohesin modules in ScaC that anchors the cellulosome onto the cell surface. Correct cellulosome assembly requires distinct specificities displayed by structurally related type I cohesin-dockerin pairs that mediate ScaC-ScaB and ScaAenzyme assemblies. To explore the mechanism by which these two critical protein interactions display their required specificities, we determined the crystal structure of the dockerin of a cellulosomal enzyme in complex with a ScaA cohesin. The data revealed that the enzyme-borne dockerin binds to the ScaA cohesin in two orientations, indicating two identical cohesin-binding sites. Combined mutagenesis experiments served to identify amino acid residues that modulate type I cohesin-dockerin specificity in A. cellulolyticus. Rational design was used to test the hypothesis that the ligand-binding surfaces of ScaA-and ScaB-associated dockerins mediate cohesin recognition, independent of the structural scaffold. Novel specificities could thus be engineered into one, but not both of the ligand-binding sites of ScaB, while attempts at manipulating the specificity of the enzyme-associated dockerin were unsuccessful. These data indicate that dockerin specificity requires critical interplay between the ligand-binding surface and the structural scaffold of these modules.Plant cell wall polysaccharides, primarily cellulose and hemicelluloses, are a major reservoir of carbon and energy (1), and the recycling of these complex carbohydrates by microorganisms is integral to the carbon cycle. Furthermore, as the demand for renewable sources of energy and novel molecules for the http://www.jbc.org/cgi

Discovery and mechanism of a pH-dependent dual-binding-site switch in the interaction of a pair of protein modules

“…The pH-dependent switch should reflect the difference in interaction between CaCohA2 and the two orientations of the dockerin, since CaDoc0917 has two asymmetric cohesin-binding sites. By comparing the structures of the complexes for each orientation at low or high pH, we observed similar hydrophobic interactions between CaCohA2 and Leu 51 / Ile 18 of CaDoc0917 and three major differences at the binding interface, which are the interactions between CaCohA2 and the asymmetric Glu 53 /Gln 23 , Met 52 /Val 19 , and Ile 55 /Arg 22 of CaDoc0917 (Fig. 3, C and D).…”

Self‐assembling protein nanocages for modular enzyme assembly by orthogonal bioconjugation

“…27 This self-assembled enzyme cascade brings multiple cellulases in close proximity to the substrate using a cellulose-binding domain (CBD) for efficient cellulose hydrolysis. 28 We have prior success in using DNA scaffolds to create artificial cellulosome structures to achieve improved cellulose hydrolysis. The overall activity can be further enhanced by using a longer DNA template generated by rolling circle amplification, supporting a direct correlation between local enzyme density and the efficiency of cellulose hydrolysis.…”

Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization

“…The immense size of cellulosomes, the presence of flexible intermodular linkers, and potential unspecific binding of enzymes to any position on the scaffoldin subunit make the task of unveiling its structure and regulation challenging. Although the structure of whole cellulosomes remains unknown, several cellulosomal fragments have been solved [7][8][9][15][16][17][18][19][20][21][22][23][24][25][26] , and useful insights has been provided by small-angle X-ray scattering [27][28][29] , cryo-electron microscopy 30 , molecular dynamics simulations 31,32 and single molecule experiments 33 . It is crucial to get a detailed picture of their structural organization to understand the synergistic effects encountered in cellulosomes, and, as a key element in cellulosome self-assembly, the role of cohesin-dockerin interaction in its fine structure and regulation.…”