Thermodynamic and structural investigation of bispecificity in protein-protein interactions.

The ability of a single protein to interact with multiple protein partners is central to many biological processes. However, the physical-chemical and structural basis of the multispecificity is not understood. In Escherichia coli, the protein BirA can self-associate to a homodimer or form a heterodimer with the biotin carboxyl carrier protein of the biotin-dependent carboxylase, acetyl coenzyme A carboxylase. The first interaction results in binding of BirA to the biotin operator sequence to repress transcription initiation at the biotin biosynthetic operon and the second is a prerequisite to posttranslational biotin addition to the carrier protein for use in metabolism. A single surface on BirA is used for both interactions and previous studies indicate that, despite the structural differences between the alternative partners, the two dimerization reactions are isoenergetic. In this work, the underlying thermodynamic driving forces and the sequence determinants of the two interactions were investigated in order to elucidate the energetic and structural underpinnings of the dual specificity. Combined measurements of the temperature and salt dependencies of heterodimerization indicate a modest unfavorable enthalpy and no dependence on salt concentration. By contrast, homodimerization is characterized by a very large unfavorable enthalpy and a modest dependence on salt concentration. Measurements of the function of BirA variants with single amino acid replacements in the alternative dimerization reactions indicate that although considerable overlap in structural determinants for both interactions exists, hotspots specific for one but not the other were detected.