Ligand-linked structural changes in the Escherichia coli biotin repressor:The significance of surface loops for binding and allostery

The Escherichia coli repressor of biotin biosynthesis (BirA) is an alloster ic site-specific DNA-binding protein. BirA catalyzes synthesis of biotinyl- 5'-AMP from substrates biotin and ATP and the adenylate serves as the posit ive allosteric effector in binding of the repressor to the biotin operator sequence. Although a three-dimensional structure of the ape-repressor has b een determined by X-ray crystallographic techniques, no structures of any l igand-bound forms of the repressor are yet available. Results of previously published solution studies are consistent with the occurrence of conformat ional changes in the protein concomitant with ligand binding. In this work the hydroxyl radical footprinting technique has been used to probe changes in reactivity of the peptide backbone of BirA that accompany ligand binding . Results of these studies indicate that binding of biotin to the protein r esults in protection of regions of the central domain in the vicinity of th e active site and the C-terminal domain from chemical cleavage. Biotin-link ed changes in reactivity constitute a subset of those linked to adenylate b inding. Binding of both bio-5'-AMP and biotin operator DNA suppresses cleav age at additional sites in the amino and carboxy-terminal domains of the pr otein. Varying degrees of protection of the five surface loops on BirA from hydroxyl radical-mediated cleavage are observed in all complexes. These re sults implicate the C-terminal domain of BirA, for which no function has pr eviously been known, in small ligand and site-specific DNA binding and high light the significance of surface loops, some of which are disordered in th e apoBirA structure, for ligand binding and transmission of allosteric info rmation in the protein. (C) 1999 Academic Press.