Swapping functional specificity of a MADS box protein: residues required for Arg80 regulation of arginine metabolism.
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.
Pubmed ID: 12138185 RIS Download
Amino Acid Sequence | Arginine | DNA-Binding Proteins | Genetic Complementation Test | Humans | MADS Domain Proteins | Models, Molecular | Molecular Sequence Data | Mutation | Protein Binding | Protein Structure, Secondary | Protein Structure, Tertiary | Saccharomyces cerevisiae | Saccharomyces cerevisiae Proteins | Sequence Alignment | Transcription Factors | Two-Hybrid System Techniques