FIGURE 5.

Binding promiscuity of the C. thermocellum cohesin N37A mutant. The N37A mutation allows promiscuous binding of the C. thermocellum (Ct) cohesin both to its cognate C. thermocellum dockerin and non-cognate C. cellulolyticum dockerin. A and B, experimental validation of non-cognate binding. A, a protein cellulose microarray assay reveals that although as expected C. thermocellum dockerin binds only to C. thermocellum cohesins (upper panel) and C. cellulolyticum (Cc) dockerin binds only to C. cellulolyticum cohesins (lower panel) the promiscuous C. thermocellum cohesin N37A mutant binds both to its cognate C. thermocellum dockerin and to the non-cognate C. cellulolyticum dockerin (red boxes). Each row of dots represents a 2-fold dilution; the bottom row contains a CBM-Xyn location marker. B, confirmation of cellulose binding microarray results by iELISA. The C. thermocellum N37A cohesin mutant can indeed bind to both its cognate (C. thermocellum) and non-cognate (C. cellulolyticum) dockerins. C. thermocellum N37A cohesin mutant-covered plates were used in this assay. C and D, structural models of the non-cognate and cognate interactions with C. cellulolyticum dockerin. C, model of the non-cognate interaction of C. cellulolyticum dockerin with the C. thermocellum N37A cohesin mutant (generated using RosettaDock; see “Experimental Procedures”). D, C. cellulolyticum dockerin interacting with its cognate wild-type C. cellulolyticum cohesin (Protein Data Bank code 2VN6 (16)). While in the C. cellulolyticum cohesin-dockerin interaction the C. cellulolyticum dockerin motif residue Phe⁴⁸ points toward the cohesin (D), the model of non-cognate interaction suggests that Phe⁴⁸ points into a hydrophobic pocket formed by C. cellulolyticum dockerin residue Ile⁵² and C. thermocellum cohesin residue Leu¹²⁹, displacing dockerin residue Lys⁵⁵ to the solvent (C). Phe⁴⁸ is shown as spheres, and residues predicted to interact with Phe⁴⁸ in the non-cognate interaction are depicted as sticks and labeled and colored in red and blue for cohesin and dockerin, respectively. See Fig. 2 legend for more details about the iELISA assay in B.