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J. Biol. Chem., Vol. 279, Issue 41, 42881-42888, October 8, 2004

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Pinpoint Mapping of Recognition Residues on the Cohesin Surface by Progressive Homologue Swapping^*

David Nakar, Tal Handelsman, Yuval Shoham, Henri-Pierre Fierobe¶, Jean-Pierre Belaich¶||, Ely Morag**, Raphael Lamed, and Edward A. Bayer

From the Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel, the Department of Biotechnology and Food Engineering, and Institute of Catalysis Science and Technology, Technion, Israel Institute of Technology, Haifa 32000, Israel, the ^¶Bioénergétique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique, IBSM, 13402 Marseille, France, the ^||Université de Provence, 3 Place Victor Hugo, 13331 Marseille, France, the ^**Zephyr ProteomiX, Kiryat-Shemona 11013, Israel, and the Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv 69978, Israel

The high affinity cohesin-dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The connection between affinity and species specificity was studied by exploring the recognition properties of two structurally related cohesin species of divergent specificity. The cohesins were examined by progressive rounds of swapping, in which corresponding homologous stretches were interchanged. The specificity of binding of the resultant chimeric cohesins was determined by enzyme-linked affinity assay and complementary protein microarray. In succeeding rounds, swapped segments were systematically contracted, according to the binding behavior of previously generated chimeras. In the fourth and final round we discerned three residues, reputedly involved in interspecies binding specificity. By replacing only these three residues, we were able to convert the specificity of the resultant mutated cohesin, which bound preferentially to the rival dockerin with 20% capacity of the wild-type interaction. These residues represent but 3 of the 16 contact residues that participate in the cohesin-dockerin interaction. This approach allowed us to differentiate, in a structure-independent fashion, between residues critical for interspecies recognition and binding residues per se.

Received for publication, July 1, 2004 , and in revised form, July 23, 2004.

^* This research was supported by the Israel Science Foundation (Grant Nos. 394/03, 771/01, and 446/01), the US-Israel Binational Agricultural Research and Development Fund (BARD Research Grant No. 3106-99C), and by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. Additional support was provided by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation (Munich, Germany). 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplementary Material.

To whom correspondence should be addressed: Dept. of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100 Israel. Tel.: 972-8-934-2373; Fax: 972-8-946-8256; E-mail: ed.bayer{at}weizmann.ac.il.

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