Protein: Protein Interactions in Control of the Escherichia Coli Biotin Protein Ligase Functional Switch

Abstract

Although many proteins are known to undergo functional switches in response to cellular signals, there are very few cases for which the detailed mechanism of control of the switch is known. The Escherichia coli biotin protein ligase, BirA, is a bifunctional protein that functions in biotin homeostasis. In its metabolic role BirA catalyzes post-translational biotin addition to the BCCP subunit of acetyl-CoA carboxylase and as a transcription repressor it homodimerizes and binds to the biotin operator sequence of the biotin biosynthetic operon. In performing its alternative functions BirA utilizes a single surface that is characterized by several loops to form two mutually exclusive protein-protein interactions. Previous results indicate the importance of these loop sequences for the energetics of homodimerization and the rates of association with BCCP. In this work DNaseI footprint titrations were employed to investigate the influence of the two dimerization interactions on the energetics of transcription repression complex assembly and the switch between the two functions. Direct footprint titrations reveal that homodimerization energetics dictate the energetics of repression complex assembly. Inhibition footprint titrations reveal a direct correlation between inhibition of repression complex assembly and the rate of heterodimer association. This correlation firmly establishes kinetics as the controlling factor in regulating the BirA functional switch.