Biomedical subjects

E D Streaker

Publications and source records attributed to E D Streaker.

4 recordsLinked to original sources

PubMed2000

Multiple disordered loops function in corepressor-induced dimerization of the biotin repressor.

Cooperative association of the Escherichia coli biotin repressor with the biotin operator is allosterically activated by binding of the corepressor, bio-5'-AMP. The corepressor function of the adenylate is due, in part, to its ability to induce repressor dimerization. Since a high-resolution structure of only the apo or unliganded repressor is currently available, the location of the dimerization interface on the protein structure is not known. Here, five mutants in the corepressor-binding domain of the repressor have been analyzed with respect to their DNA-binding and self-assembly properties. Results of these studies reveal that four of the mutant proteins exhibit defects in DNA binding. These same proteins are compromised in self-assembly. Furthermore, in the three-dimensional structure of the apo protein the mutations all lie in partially disordered surface loops, one of which is known to participate directly in corepressor binding. These results suggest that multiple disordered surface loops function in the corepressor-induced dimerization required for sequence-specific DNA binding by the biotin repressor.

Adenosine Monophosphate↗

PubMed1999

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 allosteric site-specific DNA-binding protein. BirA catalyzes synthesis of biotinyl-5'-AMP from substrates biotin and ATP and the adenylate serves as the positive allosteric effector in binding of the repressor to the biotin operator sequence. Although a three-dimensional structure of the apo-repressor has been determined by X-ray crystallographic techniques, no structures of any ligand-bound forms of the repressor are yet available. Results of previously published solution studies are consistent with the occurrence of conformational 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 results in protection of regions of the central domain in the vicinity of the active site and the C-terminal domain from chemical cleavage. Biotin-linked changes in reactivity constitute a subset of those linked to adenylate binding. Binding of both bio-5'-AMP and biotin operator DNA suppresses cleavage at additional sites in the amino and carboxy-terminal domains of the protein. Varying degrees of protection of the five surface loops on BirA from hydroxyl radical-mediated cleavage are observed in all complexes. These results implicate the C-terminal domain of BirA, for which no function has previously been known, in small ligand and site-specific DNA binding and highlight the significance of surface loops, some of which are disordered in the apoBirA structure, for ligand binding and transmission of allosteric information in the protein.

Adenosine Monophosphate↗

PubMed1998

A map of the biotin repressor-biotin operator interface: binding of a winged helix-turn-helix protein dimer to a forty base-pair site.

The Escherichia coli biotin repressor is a member of the "winged helix-turn-helix" class of site-specific DNA binding proteins. The protein binds as a dimer to the 40 bp biotin operator sequence. Although the structure of the aporepressor has been solved by X-ray crystallographic techniques, no structure of the holorepressor-DNA complex is yet available. In order to characterize the structural features of the biotin repressor-biotin operator interface we have applied a number of solution techniques including DNase I, hydroxyl radical and dimethyl sulfate footprinting and the circular permutation or "bending" assay. Results of these combined studies indicate that each repressor monomer forms a bipartite interface with each half-site of the biotin operator sequence. The results imply that, in addition to the helix-turn-helix module of each monomer, a second structural element participates in the protein-DNA interface. The two bipartite protein-DNA interfaces appear, moreover, to primarily involve the two 12 bp termini of the operator site. Results of combined DNase I footprinting and circular permutation analysis indicate, furthermore, that the central 16 bp region that links the two termini becomes distorted concomitant with binding of holoBirA.

Base Sequence↗

PubMed1998

Coupling of site-specific DNA binding to protein dimerization in assembly of the biotin repressor-biotin operator complex.

The Escherichia coli repressor of biotin biosynthesis, BirA, binds site-specifically to the biotin operator, a 40 base pair imperfect inverted palindrome. Two repressor monomers have been shown to bind to the two operator half-sites. Analysis of results of quantitative DNase I footprint titrations performed on the wild-type biotin operator template indicate that binding is well described by a cooperative mechanism. The data obtained from these studies were, however, insufficient to independently resolve all of the energetic parameters associated with cooperative binding of the two repressor monomers to the operator site. In this work, to further dissect the energetics of assembly of the biotin repressor-biotin operator complex, measurements of binding of BirA to four bioO variants designed to reduce the valency of repressor binding from 2 to 1 have been performed. Results of these measurements indicate, as was found with the wild-type biotin operator template, that two repressor monomers bind simultaneously to the two half-sites of all variant operators. Protein dimerization and DNA binding are thus obligatorily coupled in the biotin repressor system. Furthermore, the results suggest that, in the context of a cooperative binding mechanism, the cooperative free energy associated with the biotin repressor-biotin operator interaction is significantly more favorable than the previously estimated -2 kcal/mol.

Bacterial Proteins↗