A NMR Study of the Cellulosome Dockerin:Cohesin Dual Binding Mode
The recalcitrant nature of the carbon-rich plant cell wall is largely owed to its array of structural polysaccharides, which are efficiently hydrolyzed by the anaerobic bacterium Clostridium thermocellum via a multi-enzyme complex termed the cellulosome. Cellulosome assembly requires various carbohydrate-active enzymes grafting onto a scaffoldin subunit through a high-affinity interaction between the enzyme-borne type-I dockerin modules and scaffoldin-borne type-I cohesin modules. Type-I dockerins contain duplicated F-hand motifs that comprise a calcium binding loop and an adjacent helix, in which the sequential duplication is manifested into a near-perfect internal two-fold symmetry of the dockerin structure. X-ray crystallographic studies have revealed that cohesin binding can occur through either of its F-helices and a critical Ser/Thr pair, although substituting the second Ser/Thr sequence with alanine residues was required to observe the alternative conformation. Thus, there has yet to be a direct structural observation of the type-I dockerin-cohesin dual binding mode in solution. The type-I dockerin from Cel48S and the seventh type-I cohesin from the scaffoldin of C. thermocellum were recombinantly expressed in E. coli as uniformly 13C/15N-labeled and unlabeled forms, respectively. Alanine substitutions were introduced to the first and second set of critical Ser/Thr pairs of Cel48S dockerin via site-directed mutagenesis and were similarly expressed to serve as controls for the individual binding modes. Wild-type and mutant Cel48S dockerin and type-I cohesin modules as separate entities and as 1:1 complexes were purified via Ni2+-affinity and size exclusion chromatography. 1H-15N HSQC NMR analyses indicated that although the bound wild-type Cel48S dockerin spectrum comprised fewer resonances than those of the dockerin variants fixated into a single orientation, the visible resonances were intermediate to those of the mutants. These observations are consistent with an interaction undergoing chemical exchange on the intermediate to fast NMR time scale. Weaker signal intensity would also be consistent with the Cel48S dockerin construct existing in two orientations. Thus, these findings collectively indicate that Cel48S dockerin interacts with its cognate cohesin module in more than one orientation. The dockerin dual binding mode would overcome steric hindrance associated with neighbouring enzymatic subunits assembling onto the cellulosomal scaffoldin subunit.