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

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, whereas 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.

Keywords: carbohydrate-degrading enzyme; cellulase; cellulose; cellulosome; cohesin; dockerin; protein engineering; protein structure; protein-protein interaction.

Figure 1.

Architecture of A. cellulolyticus cellulosome. The scheme is color-coded to highlight the different Coh–Doc specificities. A, Doc-containing enzymes are incorporated into the ScaA scaffoldin through interaction with the seven ScaA Cohs (light green). ScaB plays the role of an adaptor protein that mediates between the ScaA Doc (yellow) and the Cohs of the anchoring scaffoldin (red) ScaC. The entire complex is attached to the cell surface via the SLH module of ScaC (orange). ScaA contains also a CBM (blue) and a GH9 (light brown) catalytic module. B, an additional mechanism of cellulosome attachment; ScaA is bound to the type-II Cohs of ScaD (yellow), which can also accept a single enzyme via its third type-I Coh (light green). The SLH module of ScaD serves to anchor the alternative complex to the cell surface.

Figure 2.

Structures of the A. cellulolyticus cohesin–dockerin complexes. A, structure of AcCohScaA6–DocCel5 M1 with the Doc color-ramped from N terminus (blue) to C terminus (red) and the Coh in gold. Ser-51 and Leu-52, which dominate Coh recognition, and engineered residues Ile-15 and Asn-16, to force a single binding mode, are labeled and shown in a stick configuration. Ca²⁺ ions are depicted as purple spheres. B, structure of AcCohScaA6–DocCel5 M2 with the Doc color-ramped from N terminus (blue) to C terminus (red) and the Coh in burgundy. Ser-15 and Ile-16 that dominate Coh recognition and engineered residues Ile-51 and Asn-52, to force a single binding mode, are again labeled and shown as stick representations. C, overlay of the two binding modes showing the high degree of overall similarity reflecting the internal 2-fold symmetry of the Doc module. The transparent gray disk marks the plane defined by the 8-3-6-5 β-sheet, where the β-strands form a distinctive Doc-interacting plateau. A also depicts a representation of the molecular surface contour of the Coh and Doc, respectively. Ca²⁺ ions are depicted as green spheres.

Figure 3.

Cohesin-dockerin interface of AcCohScaA6–DocCel5 M1 and AcCohScaA6–DocCel5 M2. Shown are the structures of AcCohScaA6–DocCel5 M1 (gold cohesin) and AcCohScaA6–DocCel5 M2 (burgundy cohesin) complexes with a detailed view of the Coh–Doc interface showing the main polar interactions (A and C) and main hydrophobic contacts (B and D). In all panels, the most important residues involved in Coh–Doc recognition are depicted in stick configuration, with a dark background label for the Doc residues and a light background label for the Coh residues, using the AcDocCel5 and AcCohScaA6 numbering. Solid black lines, hydrogen-bond interactions. The Docs are shown color-ramped from N terminus (blue) to C terminus (red). Ca²⁺ ions are depicted as green spheres. In all panels, the transparent gray disk marks the plane defined by the 8-3-6-5 β-sheet, where the β-strands form a distinctive Doc-interacting plateau.

Figure 4.

Symmetric nature of A. cellulolyticus dockerins exemplified by structures of different specificities. From left to right, AcDocCel5 (brown), AcDocScaB (green), and AcXDocScaA (blue) structures overlaid with a 180° rotated version of themselves, showing conservation of key Coh-interacting residues. This suggests that the A. cellulolyticus cellulosome is assembled exclusively via dual-binding mode Coh–Doc interactions, therefore having a highly dynamic architecture.

Figure 5.

Multiple-sequence alignment of AcDocCel5 and AcDocScaB in a C terminus (helix 3)–dominated Coh–Doc interface. Based on the AcCohScaB6-DocCel5 M1 complex (PDB code 5NRK; top) and AcCohScaC3-DocScaB (PDB code 4UYP; bottom), a schematic representation of the secondary structure is shown. The residues involved in the molecular interactions with the respective Coh partner are highlighted as follows: blue arrow for polar contacts and yellow circles for hydrophobic contacts. Residues involved in the coordination with the first and second calcium ions are marked with a blue star, and those implicated in binding the third calcium (AcDocCel5) are shown with a purple star. Below the alignment, the ClustalO consensus symbols represent the position conservation status. The primary sequence background is colored according to the ALSCRIPT Calcons convention, implemented in ALINE (22): red, identical residues; orange to blue, lowering color-ramped scale of conservation.

Figure 6.

Overlay of AcDocCel5 and AcDocScaB. Structures of AcDocCel5 (brown) and AcDocScaB (light green) are overlaid, showing the size difference in the gap between the Coh contacting helices. AcDocCel5 residues Phe-18 and Phe-54 are highlighted.