PMID:12486044
| Citation |
Appleman, JA and Stewart, V (2003) Mutational analysis of a conserved signal-transducing element: the HAMP linker of the Escherichia coli nitrate sensor NarX. J. Bacteriol. 185:89-97 |
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| Abstract |
The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX(+) strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic alpha-helices have different roles in signal transduction. |
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| Keywords |
Adenylate Cyclase/chemistry; Adenylate Cyclase/genetics; Adenylate Cyclase/metabolism; Amino Acid Sequence; Bacterial Proteins; Cell Membrane/metabolism; Conserved Sequence; DNA Mutational Analysis; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Deletion; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Proteins/metabolism; Molecular Sequence Data; Mutation, Missense; Nitrates/metabolism; Phosphoric Monoester Hydrolases/chemistry; Phosphoric Monoester Hydrolases/genetics; Phosphoric Monoester Hydrolases/metabolism; Protein Kinases/chemistry; Protein Kinases/genetics; Protein Kinases/metabolism; Signal Transduction |
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