Critical Role of a Glycine Residue in an Allosteric Switch

Abstract

Thermodynamic coupling in proteins is ubiquitous but mechanistically poorly understood, particularly for systems in which the coupling occurs over large distances. In the E. coli biotin repressor the coupling between homodimerization and allosteric effector, bio-5′-AMP, is −4.0 kcal/mol. The two coupled processes occur at sites separated by 33A. Structural and thermodynamic studies indicate that the coupling is accompanied by disorder-to-order transitions at the two distant functional sites. Perturbations to the transition at the ligand binding site via alanine substitutions alter both ligand binding and coupled dimerization. Alanine substitutions in four loops in the dimerization surface yield a range of energetic consequences for BirA dimerization. For one of these variants, BirAG142A, the free energies of dimerization and corepressor binding are consistent with complete abolition of coupling. Structural studies of the variant indicate that the loss of coupling is accompanied by disruption of the disorder-to-order transitions at both functional surfaces. In this work the structural basis of coupling between the dimerization and ligand binding surfaces was further investigated by measuring the consequences of alanine substitutions distributed in three of the dimerization loops on corepressor binding. Isothermal titration calorimetry measurements indicate that, in contrast to BirAG142A, the variants, several of which dimerize very weakly, all bind to bio-5′-AMP with energetics indistinguishable from wild-type BirA. The results indicate that a single glycine residue serves as a switch for allosteric regulation of BirA.