title: Crucial roles of single residues in binding affinity, specificity, and promiscuity in the cellulosomal cohesin-dockerin interface.
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.
authors.affiliation: From the Department of Biological Chemistry, The Weizmann Institute of Science, 7610001 Rehovot, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Slutzki
authors.affiliation: Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Hadassah Medical School, The Hebrew University, 9112102 Jerusalem, Israel, and.
authors.forename: Dan
authors.initials: D
authors.lastname: Reshef
authors.affiliation: From the Department of Biological Chemistry, The Weizmann Institute of Science, 7610001 Rehovot, Israel.
authors.forename: Yoav
authors.initials: Y
authors.lastname: Barak
authors.affiliation: From the Department of Biological Chemistry, The Weizmann Institute of Science, 7610001 Rehovot, Israel.
authors.forename: Rachel
authors.initials: R
authors.lastname: Haimovitz
authors.affiliation: Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Hadassah Medical School, The Hebrew University, 9112102 Jerusalem, Israel, and.
authors.forename: Shahar
authors.initials: S
authors.lastname: Rotem-Bamberger
authors.affiliation: Department of Molecular Microbiology and Biotechnology, Tel Aviv University, 6997801 Ramat Aviv, Israel.
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.affiliation: From the Department of Biological Chemistry, The Weizmann Institute of Science, 7610001 Rehovot, Israel.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
authors.affiliation: Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Hadassah Medical School, The Hebrew University, 9112102 Jerusalem, Israel, and oraf@ekmd.huji.ac.il.
authors.forename: Ora
authors.initials: O
authors.lastname: Schueler-Furman
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002241:Carbohydrates / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry*; D018797:Cell Cycle Proteins / Q000737:chemistry*; D002482:Cellulose / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry*; D046973:Ruminiclostridium cellulolyticum / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000378:metabolism; D019295:Computational Biology; D004797:Enzyme-Linked Immunosorbent Assay; D020128:Inhibitory Concentration 50; D009154:Mutation; D040081:Protein Array Analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012984:Software; D013045:Species Specificity; D013379:Substrate Specificity; D013816:Thermodynamics; D000097722:Cohesins
chemical_list: D001426:Bacterial Proteins; D002241:Carbohydrates; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002482:Cellulose; D002868:Chromosomal Proteins, Non-Histone; D000097722:Cohesins
keywords: Clostridium cellulolyticum; Clostridium thermocellum; carbohydrate-binding protein; cellulosome; cohesin; computer modeling; dockerin; iELISA; protein complex; substrate specificity
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