Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli.
Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.
Kenney
L J
LJ
Department of Molecular Biology, Princeton University, NJ 08544-1014, USA.
Bauer
M D
MD
Silhavy
T J
TJ
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism; D014882:Water-Electrolyte Balance
D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D010755:Organophosphates; D010446:Peptide Fragments; D010750:Phosphoproteins; C011632:acetyl phosphate; D014357:Trypsin