Abstract
Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn(37) show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background.
Highlights
Cellulosomal cohesin-dockerin interactions show intraspecies promiscuity but interspecies specificity
Computational Alanine Scanning Identifies Interface Residue Hot Spots in Two Distinct Patches at the C. thermocellum Cohesin-Dockerin Interface—The structure of the C. thermocellum cohesin-dockerin interface contains a hydrophobic patch that is conserved in both C. thermocellum and C. cellulolyticum cohesin-dockerin interactions (Figs. 1C and 2A) as well as an extensive network of hydrogen bonds at the center of the interface (Fig. 1D and Fig. 3A)
Structure-based ⌬⌬G Prediction Protocols Partially Succeed in the Identification of Interface Hot Spots at the Cohesin-Dockerin Interface—For the mutations tested by indirect ELISA (iELISA) experiments for their effect on binding in this study, we found an overall good agreement of predicted and experimentally measured effects except for the mutations N37A and D39N (Table 1)
Summary
Cellulosomal cohesin-dockerin interactions show intraspecies promiscuity but interspecies specificity. We combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. In a gene-swapping experiment, Nakar et al [23] were able to switch the binding specificity of a cohesin from C. cellulolyticum to dockerin of C. thermocellum by the replacement of only three residues In their large scale assessment of correlated mutations, Halperin et al [24] harnessed the ample information about intraspecies binding promiscuity and interspecies binding specificity in the cohesindockerin interaction to identify interface residues of this interaction in a precise manner. We applied a combination of computational design and binding experiments to identify crucial features in the C. thermocellum cohesin that determine the structural and physical bases for binding affinity and specificity in the type I cohesindockerin interface. Our study identified two types of hot spot residues in C. thermocellum cohesin: affinity hot spots such as Leu in the conserved hydrophobic patch of the cohesin-dockerin interface contribute significantly to binding affinity, whereas the specificity hot spot Asn in the hydrogen bond network in the C. thermocellum cohesin-dockerin interface plays a crucial role in determining binding specificity
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