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Research Article

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

Susana Matamouros, Kyle R. Hager, Samuel I. Miller
John Mekalanos, Editor
Susana Matamouros
aDepartment of Microbiology, University of Washington, Seattle, Washington, USA
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Kyle R. Hager
aDepartment of Microbiology, University of Washington, Seattle, Washington, USA
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Samuel I. Miller
aDepartment of Microbiology, University of Washington, Seattle, Washington, USA
bDepartment of Genome Sciences, University of Washington, Seattle, Washington, USA
cDepartment of Medicine, University of Washington, Seattle, Washington, USA
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John Mekalanos
Harvard Medical School
Roles: Editor
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DOI: 10.1128/mBio.00616-15
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  • FIG 1 
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    FIG 1 

    Disulfide cross-linking shows PhoQ HAMP domain present in distinct packing conformations in the presence and absence of the activating signal, antimicrobial peptide C18G (AMP). (A) Alkaline phosphatase activity of the PhoQ HAMP cysteine mutants. Cultures were grown in N minimal medium supplemented with 1 mM MgCl2 in the presence (blue) and absence (green) of the antimicrobial peptide C18G. All graphed values are the means and standard deviations and are representative of at least three independent trials. (B) Percent PhoQ dimer was calculated and plotted for each PhoQ cysteine mutant in the absence and in the presence of the activating signal, C18G. Graphed values are the means and standard deviations and are representative of at least three independent trials. A statistical t test was performed (*, P ≤ 0.05). (C) Cross section of the superposition of the PhoQ HAMP models based on Aer2 HAMP1 (green) and Aer2 HAMP1L44H (blue).

  • FIG 2 
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    FIG 2 

    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.

  • FIG 3 
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    FIG 3 

    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.

  • FIG 4 
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    FIG 4 

    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.

  • FIG 5 
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    FIG 5 

    Kinetic activities of PhoQwt and PhoQE232K. (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 PhoQwt or PhoQE232K. 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.

  • FIG 6 
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    FIG 6 

    Model of PhoQ activation. In the absence of an activating signal (left), the PhoQ periplasmic domain is tethered to the membrane via divalent cation bridges between the acidic patch of the PhoQ periplasmic domain (red sticks) and the negatively charged phospholipid heads via the presence of metal ions (green spheres), and the cytoplasmic CA domain is in a repressed state with dominant phosphatase activity. Interactions between the CA and the DHp domains as well as between the HAMP domain (present in an a-d conformation) and a loop connecting the CA to the DHp help stabilize the repressed state. Upon sensing of an activating signal (right), a change in conformation in the PhoQ periplasmic domain occurs which is propagated through the transmembrane region, resulting in loosening and rotation of the PhoQ HAMP domain, possibly to a conformation closer to a complementary x-da packing. The interactions between the HAMP, DHp, and CA domains that kept PhoQ in an inactive state are disrupted, freeing the CA to adopt a kinase-competent conformation.

Tables

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  • TABLE 1 

    β-β carbon distance for each residue calculated for each of the models

    ResidueaDistance (Å)
    Af1053Af1053A291FAer2 HAMP1Aer2 HAMP1L44H
    I2218.41166.4
    L224*10.211.3810.9
    A225*13.315.113.211.3
    V2288.89.38.38.3
    L24711.610.712.515.6
    L2507.16.57.910.6
    L254*8.910.110.611.1
    L257*4.67.56.95.9
    S260*913.311.69.9
    C score0.220.30.410.35
    • ↵a Asterisks denote residues for which statistically significant changes were observed by disulfide cross-link assay upon PhoQ activation.

Supplemental Material

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  • Figure S1 

    Circular dichroism (CD) of purified HAMPwt (●), HAMPE232K (■), and HAMPR236H (▲). Download Figure S1, PDF file, 0.1 MB.

    Copyright © 2015 Matamouros et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S2 

    Helical wheel representation of a four-helix bundle in an x-da packing conformation (left) and in an a-d packing conformation (right). The corresponding PhoQ residues are indicated. Download Figure S2, PDF file, 0.4 MB.

    Copyright © 2015 Matamouros et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S3 

    Model of PhoQ HAMP domain based on Aer2 HAMP1L44H. One monomer is shown in color with α1 in green and α2 in blue, and the other monomer is in grey. Download Figure S3, PDF file, 0.2 MB.

    Copyright © 2015 Matamouros et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S4 

    Comparison of phosphatase activity between activating (10 µM MgCl2) and repressive (10 mM MgCl2) growth conditions. Download Figure S4, PDF file, 0.1 MB.

    Copyright © 2015 Matamouros et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Table S1 

    Fold activation of the different PhoQ alleles compared to the wild type grown under PhoQ-repressive conditions. Table S1, PDF file, 0.1 MB.

    Copyright © 2015 Matamouros et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S5 

    ClustalO primary sequence alignment of several PhoQ homologs. Download Figure S5, PDF file, 0.1 MB.

    Copyright © 2015 Matamouros et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Additional Files

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  • Supplemental Material
  • Supplementary Data

    Supplementary Data

    • Figure sf1, PDF - Figure sf1, PDF
    • Figure sf2, PDF - Figure sf2, PDF
    • Figure sf3, PDF - Figure sf3, PDF
    • Figure sf4, PDF - Figure sf4, PDF
    • Figure sf5, PDF - Figure sf5, PDF
    • Table st1, PDF - Table st1, PDF
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HAMP Domain Rotation and Tilting Movements Associated with Signal Transduction in the PhoQ Sensor Kinase
Susana Matamouros, Kyle R. Hager, Samuel I. Miller
mBio May 2015, 6 (3) e00616-15; DOI: 10.1128/mBio.00616-15

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HAMP Domain Rotation and Tilting Movements Associated with Signal Transduction in the PhoQ Sensor Kinase
Susana Matamouros, Kyle R. Hager, Samuel I. Miller
mBio May 2015, 6 (3) e00616-15; DOI: 10.1128/mBio.00616-15
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