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Molecular and Cellular Biology, August 2002, p. 5741-5752, Vol. 22, No. 16
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.16.5741-5752.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Swapping Functional Specificity of a MADS Box Protein: Residues Required for Arg80 Regulation of Arginine Metabolism

Institut de Recherches Microbiologiques J. M. Wiame and Laboratoire de Microbiologie de l'Université Libre de Bruxelles, 1070 Brussels, Belgium,¹ Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854-8020²

Received 4 April 2002/ Returned for modification 7 May 2002/ Accepted 23 May 2002

Arg80 and Mcm1, two members of the MADS box family of DNA-binding proteins, regulate the metabolism of arginine in association with Arg81, the arginine sensor. In spite of the high degree of sequence conservation between the MADS box domains of the Arg80 and Mcm1 proteins (56 of 81 amino acids), these domains are not interchangeable. To determine which amino acids define the specificity of Arg80, we swapped the amino acids in each secondary-structure element of the Arg80 MADS box domain with the corresponding amino acids of Mcm1 and assayed the ability of these chimeras to regulate arginine-metabolic genes in place of the wild-type Arg80. Also performed was the converse experiment in which each variant residue in the Mcm1 MADS box domain was swapped with the corresponding residue of Arg80 in the context of an Arg80-Mcm1 fusion protein. We show that multiple regions of Arg80 are important for its function. Interestingly, the residues which have important roles in determining the specificity of Arg80 are not those which could contact the DNA but are residues that are likely to be involved in protein interactions. Many of these residues are clustered on one side of the protein, which could serve as an interface for interaction with Arg81 or Mcm1. This interface is distinct from the region used by the Mcm1 and human serum response factor MADS box proteins to interact with their cofactors. It is possible that this alternative interface is used by other MADS box proteins to interact with their cofactors.

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