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

Abstract

The ability of the Escherichia coli protein, BirA, to function as both a metabolic enzyme and a transcription repressor relies on use of a single surface for two distinct protein:protein interactions. BirA forms a heterodimer with the biotin acceptor protein of acetyl-CoA carboxylase and catalyzes post-translational 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 BCCP is impacted by 7 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.

Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation

“…17 An alanine substitution at residue G142 completely abolishes allosteric coupling. 11 The structure of the holoBirAG142A variant indicates that the disorder-to-order transitions in the 140−146 and 193−199 loops are perturbed, consistent with a functional role for these transitions in activation of dimerization. 18 Furthermore, 33 Å from the alanine substitution, the disorderto-order transition in the effector binding site ABL is also disrupted.…”

“…Despite its location far from the dimerization interface, the I280A substitution also perturbs the dimerization free energy by 3.5 kcal/mol. 11 The wild-type holoBirA structure indicates that the I280A substitution disrupts a hydrophobic interaction with the W309 side chain (Figure 8). The relationship between this local disruption and the more extensive loss of folding upon binding predicted from the thermodynamics has yet to be determined.…”

Within-population variability in a moth sex pheromone blend: genetic basis and behavioural consequences

“…R. Soc. B 281: 20133054 systems [61,62], although none have yet been described for desaturases. This could explain the finding that the HL heterozygote in the backcross unexpectedly produces more unsaturated compounds than does the LL homozygote: HL desaturase activity would be higher than LL because its desaturase expression is repressed to a lesser degree.…”

Protein:Protein Interactions in Control of a Transcriptional Switch

“…2) and results of previous studies of alanine-substituted BirA variants indicate sensitivity of homodimerization to amino acid changes in any of these five interface loops [17,18]. By contrast, the heterodimeric interaction of holoBirA with apoBCCP is sensitive primarily to substitutions in the loops composed of residues 116–124 and 170–176 [18,19]. …”

“…The variants are characterized by homodimerization equilibrium constants ranging from millimolar to nanomolar, corresponding to Gibbs free energies ranging from −3 to −10 kcal/mol [18]. This range of self-association properties can be exploited to investigate the relationship of dimerization energetics to repression complex assembly and the functional switch.…”