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J. Biol. Chem., Vol. 283, Issue 26, 18422-18430, June 27, 2008

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The Clostridium cellulolyticum Dockerin Displays a Dual Binding Mode for Its Cohesin Partner^*

Benedita A. Pinheiro¹, Mark R. Proctor¹, Carlos Martinez-Fleites^¶1, José A. M. Prates, Victoria A. Money^¶, Gideon J. Davies^¶2, Edward A. Bayer^||, Carlos M. G. A. FontesM³, Henri-Pierre Fierobe^**, and Harry J. Gilbert⁴

From the Centro Interdisciplinar de Investigação em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal, Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, United Kingdom, ^¶Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, YO10 5YW, United Kingdom, ^**Laboratoire de Chimie Bactérienne (UPR9043), CNRS, IBSM, 13402 Marseille, France, and ^||Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

The plant cell wall degrading apparatus of anaerobic bacteria includes a large multienzyme complex termed the "cellulosome." The complex assembles through the interaction of enzyme-derived dockerin modules with the multiple cohesin modules of the noncatalytic scaffolding protein. Here we report the crystal structure of the Clostridium cellulolyticum cohesin-dockerin complex in two distinct orientations. The data show that the dockerin displays structural symmetry reflected by the presence of two essentially identical cohesin binding surfaces. In one binding mode, visualized through the A16S/L17T dockerin mutant, the C-terminal helix makes extensive interactions with its cohesin partner. In the other binding mode observed through the A47S/F48T dockerin variant, the dockerin is reoriented by 180° and interacts with the cohesin primarily through the N-terminal helix. Apolar interactions dominate cohesin-dockerin recognition that is centered around a hydrophobic pocket on the surface of the cohesin, formed by Leu-87 and Leu-89, which is occupied, in the two binding modes, by the dockerin residues Phe-19 and Leu-50, respectively. Despite the structural similarity between the C. cellulolyticum and Clostridium thermocellum cohesins and dockerins, there is no cross-specificity between the protein partners from the two organisms. The crystal structure of the C. cellulolyticum complex shows that organism-specific recognition between the protomers is dictated by apolar interactions primarily between only two residues, Leu-17 in the dockerin and the cohesin amino acid Ala-129. The biological significance of the plasticity in dockerin-cohesin recognition, observed here in C. cellulolyticum and reported previously in C. thermocellum, is discussed.

Received for publication, February 25, 2008 , and in revised form, April 25, 2008.

The atomic coordinates and structure factors (codes 2vn5 and 2vn6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Biotechnology and Biological Sciences Research Council, Fundação para a Ciência e a Tecnologia (POCTI/BIA-PRO/59118/2004), and the individual grant SFRH/BD/25439/2005 (to B. A. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² A Royal Society-Wolfson research merit award holder.

³ To whom correspondence may be addressed. E-mail: cafontes{at}fmv.utl.pt.

⁴ To whom correspondence may be addressed. E-mail: h.j.gilbert{at}ncl.ac.uk.

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