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Structure–function analyses generate novel specificities to assemble the components of multi-enzyme bacterial cellulosome complexes

DOI: 10.1074/jbc.RA117.001241 DOI Help

Authors: Pedro Bule (CIISA - Faculdade de Medicina Veterinária, Universidade de Lisboa) , Kate Cameron (Universidade de Lisboa) , José A. M. Prates (CIISA - Faculdade de Medicina Veterinária, Universidade de Lisboa) , Luís M. A. Ferreira (Universidade de Lisboa) , Steven Philip Smith (Queen's University, Canada) , Harry J. Gilbert (Newcastle University) , Edward A. Bayer (The Weizmann Institute of Science) , Shabir Najmudin (CIISA - Faculdade de Medicina Veterinária, Universidade de Lisboa) , Carlos M. G A. Fontes (3CIISA - Faculdade de Medicina Veterinária, Universidade de Lisboa) , Victor Alves (Universidade de Lisboa)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: January 2018
Diamond Proposal Number(s): 8425

Open Access Open Access

Abstract: The 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 ScaA-enzyme 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.

Journal Keywords: cellulase; protein engineering; protein-protein interaction; cellulosome; cellulose

Diamond Keywords: Enzymes; Bacteria

Subject Areas: Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Other Facilities: European Synchrotron Radiation Facility (ESRF)

Added On: 08/02/2018 11:45


Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)