Functional versatility of a single protein surface in two protein:protein interactions.

The ability of the Escherichia coli protein BirA to function as both a metabolic enzyme and a transcription repressor relies on the use of a single surface for two distinct protein:protein interactions. BirA forms a heterodimer with the biotin acceptor protein of acetyl-coenzyme A carboxylase and catalyzes posttranslational biotinylation. Alternatively, it forms a homodimer that binds sequence-specifically to DNA to repress transcription initiation at the biotin biosynthetic operon. Several surface loops on BirA, two of which exhibit sequence conservation in all biotin protein ligases and the remainder of which are highly variable, are located at the two interfaces. The function of these loops in both homodimerization and biotin transfer was investigated by characterizing alanine-substituted variants at 18 positions of one constant and three variable loops. Sedimentation equilibrium measurements reveal that 11 of the substitutions, which are distributed throughout conserved and variable loops, significantly alter homodimerization energetics. By contrast, steady-state and single-turnover kinetic measurements indicate that biotin transfer to biotin carboxyl carrier protein is impacted by seven substitutions, the majority of which are in the constant loop. Furthermore, constant loop residues that function in biotin transfer also support homodimerization. The results reveal clues about the evolution of a single protein surface for use in two distinct functions.

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