A phosphorylation site mutant of OmpR reveals different binding conformations at ompF and ompC

• Published in: Journal of molecular biology 2002-01, Vol.315 (4), p.497-511
• Main Authors: Mattison, Kirsten, Oropeza, Ricardo, Byers, Nicole, Kenney, Linda J
• Format: Article
• Language: English
• Subjects: Amides - metabolism; Amino Acid Substitution - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins; Base Sequence; DNA Footprinting; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; EnvZ protein; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression Regulation, Bacterial; Genes, Dominant - genetics; Models, Genetic; Models, Molecular; Molecular Sequence Data; Multienzyme Complexes - metabolism; Mutation - genetics; ompC gene; OmpF protein; OmpR protein; Organophosphates - metabolism; Phenotype; Phosphoric Acids - metabolism; Phosphorylation; Porins - genetics; Promoter Regions, Genetic - genetics; Protein Binding; Protein Kinases - metabolism; Protein Structure, Tertiary; Response Elements - genetics; Trans-Activators - chemistry; Trans-Activators - genetics; Trans-Activators - metabolism; Transcription, Genetic
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2001.5222

In Escherichia coli, the two-component regulatory system that controls the expression of outer membrane porins in response to environmental osmolarity consists of the sensor kinase EnvZ and the response regulator OmpR. Phosphorylated OmpR activates expression of the OmpF porin at low osmolarity, and at high osmolarity represses ompF transcription and activates expression of OmpC. We have characterized a substitution in the amino-terminal phosphorylation domain of OmpR, T83I, its phenotype is OmpF(-) OmpC(-). The mutant protein is not phosphorylated by small molecule phosphodonors such as acetyl phosphate and phosphoramidate, but it is phosphorylated by the cognate kinase EnvZ. Interestingly, the active site T83I substitution alters the DNA binding properties of the carboxyl-terminal effector domain. DNase I protection assays indicate that DNA binding by the mutant protein is similar to wild-type OmpR at the ompF promoter, but at ompC, the pattern of protection is different from OmpR. Our results indicate that all three of the OmpR binding sites at the ompC promoter must be filled in order to activate gene expression. Furthermore, it appears that OmpR-phosphate must adopt different conformations when bound at ompF and ompC. A model is presented to account for the reciprocal regulation of OmpF and OmpC porin expression.