Structural Determinants for Allostery and Functional Switching in the E. coli Biotin Regulatory System

Abstract

The Escherichia coli protein, BirA, functions as an allosteric transcriptional repressor and a metabolic enzyme. BirA catalyzes synthesis of its intermediate, bio-5'-AMP, from biotin and ATP. In its repressor function, binding of bio-5'-AMP promotes homodimerization that is a prerequisite to DNA binding at the biotin biosynthetic operon. As an enzyme, BirA forms a heterodimer with the biotin carboxyl carrier protein and transfers biotin to activate the first committed step of fatty acid synthesis. Based on structural data the unliganded repressor is characterized by four flexible loops that are ordered in the ligand-bound homodimers. Two of these loops participate in intermediate binding. The biotin binding loop (BBL) orders over biotin and participates directly at the dimer interface. The adenylate binding loop (ABL), located distal to the interface orders over the adenine ring. Single site mutations in a hydrophobic cluster of the ABL are investigated for small ligand binding and dimerization employing isothermal titration calorimetry and sedimentation equilibrium techniques. Results indicate that coupling between adenylate binding and dimerization is compromised in each mutant. Further, the ligand-induced disorder-to-order transition in the ABL is shown to be integral to the allosteric response. The ABL and BBL are also examined for their role in the structural mechanism of intermediate-induced protein:DNA interaction. Based on a structural model of the repressor:DNA complex, electrostatic interactions are predicted between the flexible loops and the DNA backbone. The DNA binding properties of mutants in the loops are assessed by the DNase I footprinting technique. Contrary to predictions, results indicate no direct participatation of the loops in the protein:DNA interaction. Finally, the same loops are inspected for their role in mutually exclusive, isoenergetic protein:protein interactions. Combined with mutant proteins previously examined in homodimerization, additional mutants in the flexible loops are investigated in alternate protein:protein interactions. The hetero- and homo-dimerization processes are monitored by inhibition DNase I footprinting titrations, a kinetic assay, and sedimentation equilibrium measurements. Results indicate an overlap in structural determinants important for both interactions. Further, "hotspots" have evolved that are exclusive to one but not the other protein:protein interaction that is associated with each function of BirA.