Evidence for a dual binding mode of dockerin modules to cohesins

*Rede de Química e Tecnologia/Centro de Química Fina e Biotecnologia (REQUIMTE/CQFB), Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal;
^†Centro Interdisciplinar de Investigação em Sanidade Animal Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal;
^‡Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, The Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom;
^§Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel; and
^¶Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom

Communicated by Roy H. Doi, University of California, Davis, CA, December 20, 2006 (received for review October 24, 2006)

Abstract

The assembly of proteins that display complementary activities into macromolecular complexes is critical to cellular function. One such enzyme complex, of environmental significance, is the plant cell wall degrading apparatus of anaerobic bacteria, termed the cellulosome. The complex assembles through the interaction of enzyme-derived “type I dockerin” modules with the multiple “cohesin” modules of the scaffolding protein. Clostridium thermocellum type I dockerin modules contain a duplicated 22-residue sequence that comprises helix-1 and helix-3, respectively. The crystal structure of a C. thermocellum type I cohesin-dockerin complex showed that cohesin recognition was predominantly through helix-3 of the dockerin. The sequence duplication is reflected in near-perfect 2-fold structural symmetry, suggesting that both repeats could interact with cohesins by a common mechanism in wild-type (WT) proteins. Here, a helix-3 disrupted mutant dockerin is used to visualize the reverse binding in which the dockerin mutant is indeed rotated 180° relative to the WT dockerin such that helix-1 now dominates recognition of its protein partner. The dual binding mode is predicted to impart significant plasticity into the orientation of the catalytic subunits within this supramolecular assembly, which reflects the challenges presented by the degradation of a heterogeneous, recalcitrant, insoluble substrate by a tethered macromolecular complex.

Footnotes

^‖To whom correspondence may be addressed. E-mail: mromao{at}dq.fct.unl.pt or cafontes{at}fmv.utl.pt
Author contributions: A.L.C., F.M.V.D., and T.N. contributed equally to this work; C.M.G.A.F. designed research; A.L.C., F.M.V.D., T.N., J.A.M.P., M.R.P., and N.S. performed research; T.N. contributed new reagents/analytic tools; E.A.B., G.J.D., L.M.A.F., M.J.R., C.M.G.A.F., and H.J.G. analyzed data; and A.L.C., E.A.B., G.J.D., M.J.R., C.M.G.A.F., and H.J.G. wrote the paper.
The authors declare no conflict of interest.
Data deposition: Coordinates and observed structure factor amplitudes for the Coh-Doc S45A-T46A mutant complex, to 2 Å resolution, have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID code 2CCL).
This article contains supporting information online at www.pnas.org/cgi/content/full/0611173104/DC1.
Abbreviations:

Coh,

cohesin;

Doc,

dockerin;

ITC,

isothermal titration calorimetry.