HAMP Domain Rotation and Tilting Movements Associated with Signal Transduction in the PhoQ Sensor Kinase

Amino acid changes in PhoQ HAMP domain that confer increased activity under repressive conditions. (A) The schematic at the top is a linear representation of PhoQ HAMP domain. α1 (green) and α2 (blue) represent the two α-helices of PhoQ HAMP domain joined by a connector region. Residue numbers relative to full-length PhoQ are indicated in the first row of the chart. The second row shows the PhoQ HAMP domain wild-type sequence. Presented in the bottom row are the amino acid substitutions identified for each position. In red are the amino acid mutations that result in a highly derepressed PhoQ protein. (B) Alkaline phosphatase activity of PhoQ HAMP mutants that show more than 10-fold activity over the wild type under repressive conditions. A reporter fusion between PhoP-dependent acid phosphatase (PhoN) and PhoA was used to measure activation. Cultures were grown in N minimal medium supplemented with 10 mM MgCl₂. All graphed values are the means and standard deviations and are representative of at least three independent trials. (C) Model of the PhoQ HAMP domain based on Aer2 HAMP1. One monomer is shown in color with α1 in green and α2 in blue; the other monomer is grey. Residues where highly activating mutations were identified are in yellow, and their residue numbers are shown.

Correlation between the size of the side chain at position 228 and activity in PhoQ. (A) ClustalO primary sequence alignment of the HAMP domains from Af1503 (GenBank no. O28769), PhoQ (P23837, D0ZV89, and Q9I4F8), CpxA (P0AE82), EnvZ (P0AEJ4), NarX (P0AFA2), Tsr (P02942), Tar (P07017), Aer2 (Q9I6V6), and Aer (P50466). ST, S. Typhimurium; Ec, E. coli; Pa, P. aeruginosa. The residues represented in blue are at the equivalent position as A291 in Af1503. In red are the two glutamate residues conserved in PhoQ. (B) Alkaline phosphatase activity of PhoQ mutants with amino acid substitutions at position 228. Cultures were grown in N minimal medium supplemented with 10 mM MgCl₂ (high) or 10 µM MgCl₂ (low). All values are means and standard deviations and are representative of at least three independent trials.

Model of PhoQ full cytoplasmic domain showing a possible interaction between the bottom of the PhoQ HAMP domain and a loop connecting the dimerization domain to the catalytic domain. (A) I-TASSER model of PhoQ (residues 217 to 487) using as a model the repressed (color) and activated (grey) CpxA chains as the template. Residues E232 (red), E261 (red), H277 (yellow), and R336 (blue) are represented as sticks. (B) Cross section of the model showing a possible salt bridge between residues E232 (red) and R336 (blue), shown as sticks. (C) Alkaline phosphatase activity of PhoQ targeted mutants grown in N minimal medium under repressive conditions (10 mM MgCl₂). One-way analysis of variance (ANOVA) was performed. ns, not significant; **, P ≤ 0.005; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Kinetic activities of PhoQ^wt and PhoQ^E232K. (A) PhoQ autophosphorylation. S. Typhimurium PhoQ-enriched membranes were incubated with [γ-³³P]ATP. Reactions were stopped at the indicated time points, and the products were separated by SDS-PAGE and exposed overnight to a phosphorimager screen. The fraction of phosphorylated PhoQ was determined for each time point. The data were analyzed using the nonlinear regression model “one phase decay.” (B) Phosphotransfer activity. S. Typhimurium PhoQ-enriched membranes were incubated with purified PhoP in the presence of [γ-³³P]ATP. Reactions were stopped at different time points, and the products were separated by SDS-PAGE and exposed overnight to a phosphorimager screen. (C) Dephosphorylation of phosphorylated PhoP (PhoP~P). Purified PhoP~P was incubated with membranes containing overexpressed PhoQ^wt or PhoQ^E232K. Reactions were stopped at different time points, and the products were separated by SDS-PAGE and exposed overnight to a phosphorimager screen. The data were analyzed using the nonlinear regression model “one phase decay.” All values are means and standard deviations and are representative of at least three independent trials.