Sequence-Function Relationships in Allostery Mediated by Disorder-To-Order Transitions

Abstract

Allostery functions in many biological systems including signal transduction, transcription regulation, and metabolism. Although disorder-to-order transitions contribute to numerous allosteric processes, the relationship of the functional allosteric response to the sequences that undergo these transitions is not known. The Escherichia coli biotin repressor, BirA, is an allosteric transcription regulatory protein that undergoes ligand-induced loop folding upon corepressor, bio-5′-AMP, binding. Ligand binding and dimerization are coupled processes with bio-5′-AMP binding enhancing the dimerization free energy by −4.0 kcal/mole. Previous work has demonstrated that adenylate binding is coupled to folding of a loop in which any single alanine replacement disrupts effector binding, loop folding and functional coupling. In this work, coupling between residues that contribute to the disorder-to-order transition was investigated by studying variant proteins with several combinations of alanine substitutions. Combined kinetic and equilibrium measurements reveal non-additive effects of multiple amino acid substitutions for all BirA functions. The results also indicate that specific combinations of alanine substitutions lead to reversion of the allosteric response toward that observed for the wild type protein. In combination the results suggest that, analogous to protein folding, full function in disorder-to-order transitions requires appropriate packing of the relevant side chains.