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J Biol Chem. 2015 May 29;290(22):13654-66. doi: 10.1074/jbc.M115.651208. Epub 2015 Apr 1.

Crucial roles of single residues in binding affinity, specificity, and promiscuity in the cellulosomal cohesin-dockerin interface.

Author information

1
From the Department of Biological Chemistry, The Weizmann Institute of Science, 7610001 Rehovot, Israel.
2
Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Hadassah Medical School, The Hebrew University, 9112102 Jerusalem, Israel, and.
3
Department of Molecular Microbiology and Biotechnology, Tel Aviv University, 6997801 Ramat Aviv, Israel.
4
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.

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.

KEYWORDS:

Clostridium cellulolyticum; Clostridium thermocellum; carbohydrate-binding protein; cellulosome; cohesin; computer modeling; dockerin; iELISA; protein complex; substrate specificity

PMID:
25833947
PMCID:
PMC4447945
DOI:
10.1074/jbc.M115.651208
[Indexed for MEDLINE]
Free PMC Article

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