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J. Biol. Chem., Vol. 279, Issue 47, 49338-49345, November 19, 2004

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Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking^*

Luis Giron-Monzon, Laura Manelyte, Robert Ahrends, Dieter Kirsch, Bernhard Spengler, and Peter Friedhoff¶

From the Institut für Biochemie (FB 08), Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany and Institut für Anorganische und Analytische Chemie (FB 08), Justus-Liebig Universität Giessen, D-35392 Giessen, Germany

Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH·MutL complex and to explain the ATP dependence of this interaction.

Received for publication, August 13, 2004 , and in revised form, September 7, 2004.

^* This work was supported by the Deutsche Forschungsgemeinschaft (Fr-1495/3-1) and the Deutsche Akademische Austauschdienst (International Quality Network "Biochemistry of Nucleic Acids"). 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.

^¶ To whom correspondence should be addressed: Institut für Biochemie (FB 08), Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. Tel.: 49-641-99-35407; Fax: 49-641-99-35409; E-mail: friedhoff{at}chemie.bio.uni-giessen.de.

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