Current views of multidrug (MD) recognition focus on large drug-binding cavities with flexible elements. However, MD recognition in BmrR is supported by a small, rigid drug-binding pocket. Here, a detailed description of MD binding by the noncanonical BmrR protein is offered through the combined use of X-ray and solution studies. Low shape complementarity, suboptimal packing, and efficient burial of a diverse set of ligands is facilitated by an aromatic docking platform formed by a set of conformationally fixed aromatic residues, hydrophobic pincer pair that locks the different drug structures on the adaptable platform surface, and a trio of acidic residues that enables cation selectivity without much regard to ligand structure. Within the binding pocket is a set of BmrR-derived H-bonding donor and acceptors that solvate a wide range of ligand polar substituent arrangements in a manner analogous to aqueous solvent. Energetic analyses of MD binding by BmrR are consistent with structural data. A common binding orientation for the different BmrR ligands is in line with promiscuous allosteric regulation.

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Folding coupled to binding is ubiquitous in biology. Nevertheless, the relationship of sequence to function for protein segments that undergo coupled binding and folding remains to be determined. Specifically, it is not known if the well-established rules that govern protein folding and stability are relevant to ligand-linked folding transitions. Upon small ligand biotinoyl-5 0 -AMP (bio-5 0 -AMP) binding the Escherichia coli protein BirA undergoes a disorder-to-order transition that results in formation of a network of packed hydrophobic side chains. Ligand binding is also allosterically coupled to protein association, with bio-5 0 -AMP binding enhancing the dimerization free energy by 24.0 kcal/mol. Previous studies indicated that single alanine replacements in a three residue hydrophobic cluster that contributes to the larger network disrupt cluster formation, ligand binding, and allosteric activation of protein association. In this work, combined equilibrium and kinetic measurements of BirA variants with alanine substitutions in the entire hydrophobic network reveal large functional perturbations resulting from any single substitution and highly non-additive effects of multiple substitutions. These substitutions also disrupt ligand-linked folding. The combined results suggest that, analogous to protein folding, functional disorder-to-order linked to binding requires optimal packing of the relevant hydrophobic side chains that contribute to the transition. The potential for many combinations of residues to satisfy this requirement implies that, although functionally important, segments of homologous proteins that undergo folding linked to binding can exhibit sequence divergence.

Solvent reorganization can contribute significantly to the energetics of protein:protein interactions. However, our knowledge of the magnitude of the energetic contribution is limited, in part, by a dearth of quantitative experimental measurements. The biotin repressor forms a homodimer as a prerequisite to DNA binding to repress transcription initiation. At 20°C the dimerization reaction, which is thermodynamically coupled to binding of a small ligand, bio-5'-AMP, is characterized by a Gibbs free energy of -7 kcal/mole. This modest net dimerization free energy reflects underlying very large opposing enthalpic and entropic driving forces of +41±3 and -48±3 kcal/mole. The thermodynamics have been interpreted as indicating coupling of solvent release to dimerization. In this work this interpretation has been investigated by measuring the effect of replacing H2O with D2O on the dimerization thermodynamics. Sedimentation equilibrium measurements performed at 20°C reveal a solvent isotope effect of -1.5 kcal/mole on the Gibbs free energy of dimerization. Analysis of the temperature dependence of the reaction in D2O indicates enthalpic and entropic contributions of +28 and -37 kcal/mole, respectively, considerably smaller than the values measured in H2O. These large solvent isotope perturbations to the thermodynamics are consistent with a significant contribution of solvent release to the dimerization reaction.

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