The response regulator OmpR oligomerizes via beta-sheets to form head-to-head dimers.
In Escherichia coli, the EnvZ/OmpR two-component regulatory system regulates expression of the porin genes ompF and ompC in response to changes in osmolarity. It has recently become apparent that OmpR functions as a global regulator, by regulating the expression of many genes in addition to the porin genes. OmpR consists of two domains; phosphorylation of the N-terminal receiver domain increases DNA binding affinity of the C-terminal domain and vice versa. Many response regulators including PhoB and FixJ dimerize upon phosphorylation. Here, we demonstrate that OmpR dimerization is stimulated by phosphorylation or by DNA binding. The dimerization interface revealed here was unanticipated and had previously not been predicted. Using the accepted head-to-tail tandem-binding model as a guide, we set out to examine the intermolecular interactions between OmpR dimers bound to DNA by protein-protein cross-linking methods. Surprisingly, amino acid positions that we expected to form cross-linked dimers did not. Conversely, positions predicted not to form dimers did. Because of these results, we designed a series of 23 cysteine-substituted OmpR mutants that were used to investigate dimer interfaces formed via the beta-sheet region. This four-stranded beta-sheet is a unique feature of the OmpR group of winged helix-turn-helix proteins. Many of the cysteine-substituted mutants are dominant to wild-type OmpR, are phosphorylated by acetyl phosphate as well as the cognate kinase EnvZ, and the cross-linked proteins are capable of binding to DNA. Our results are consistent with a model in which OmpR binds to DNA in a head-to-head orientation, in contrast to the previously proposed asymmetric head-to-tail model. They also raise the possibility that OmpR may be capable of adopting more than one orientation as it binds to a vast array of genes to activate or repress transcription.
Maris
Ann E
AE
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569, USA.
Walthers
Don
D
Mattison
Kirsten
K
Byers
Nicole
N
Kenney
Linda J
LJ
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins