Functional Versatility of a Single Protein Surface in Two Protein:Protein Interactions

• Published in: Journal of molecular biology 2012-06, Vol.419 (3-4), p.223-233
• Main Authors: Adikaram, Poorni R., Beckett, Dorothy
• Format: Article
• Language: English
• Subjects: Acetyl-CoA Carboxylase - metabolism; Amino Acid Substitution; Binding Sites; Biotin - metabolism; bispecificity; Carbon-Nitrogen Ligases - chemistry; Carbon-Nitrogen Ligases - metabolism; DNA-Binding Proteins - metabolism; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; evolution; Models, Molecular; Protein Binding; Protein Conformation; Protein Multimerization; protein:protein interactions; Repressor Proteins - chemistry; Repressor Proteins - metabolism; surface loops; Transcription, Genetic
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2012.03.010

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. [Display omitted] ► A single surface on the protein BirA participates in two distinct protein:protein interactions that support its transcription regulatory and posttranslational biotin addition functions. ► Alanine replacement of loop residues on the surface reveals their significance for the two interactions. ► The data support a model for coevolution of surface loop sequences to preserve one essential function while acquiring a second.