Allosteric mechanism of signal transduction in the two-component system histidine kinase PhoQ

  1. Bruk Mensa  Is a corresponding author
  2. Nicholas F Polizzi
  3. Kathleen S Molnar
  4. Andrew M Natale
  5. Thomas Lemmin
  6. William F DeGrado  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Codexis Inc., United States
  3. Università della Svizzera Italiana, Switzerland

Abstract

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ's sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutations distant from the sensor or catalytic site strongly influence PhoQ's ligand-sensitivity as well as the magnitude and direction of the signal. Data from 35 mutants are explained by a semi-empirical three-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively 'kinase-on' conformation, while the HAMP domain favors the 'off' state; when coupled, they create a bistable system responsive to physiological concentrations of Mg2+. Mutations alter signaling by locally modulating domain intrinsic equilibrium constants and interdomain couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source data files have been provided for Figure 8. All source code for modeling work is provided as source code files 1-5.

Article and author information

Author details

  1. Bruk Mensa

    Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States
    For correspondence
    bruk.mensa@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8777-5946
  2. Nicholas F Polizzi

    Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Kathleen S Molnar

    Codexis Inc., Redwood City, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Andrew M Natale

    Biophysics PhD program, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Thomas Lemmin

    Euler Institute, Università della Svizzera Italiana, Lugano, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  6. William F DeGrado

    Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States
    For correspondence
    Bill.DeGrado@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institutes of Health (K99-GM138753)

  • Bruk Mensa
  • Nicholas F Polizzi
  • Andrew M Natale
  • Thomas Lemmin
  • William F DeGrado

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Mensa et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,177
    views
  • 358
    downloads
  • 18
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Bruk Mensa
  2. Nicholas F Polizzi
  3. Kathleen S Molnar
  4. Andrew M Natale
  5. Thomas Lemmin
  6. William F DeGrado
(2021)
Allosteric mechanism of signal transduction in the two-component system histidine kinase PhoQ
eLife 10:e73336.
https://doi.org/10.7554/eLife.73336

Share this article

https://doi.org/10.7554/eLife.73336

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Mai Nguyen, Elda Bauda ... Cecile Morlot
    Research Article

    Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.

    1. Biochemistry and Chemical Biology
    2. Computational and Systems Biology
    Shinichi Kawaguchi, Xin Xu ... Toshie Kai
    Research Article

    Protein–protein interactions are fundamental to understanding the molecular functions and regulation of proteins. Despite the availability of extensive databases, many interactions remain uncharacterized due to the labor-intensive nature of experimental validation. In this study, we utilized the AlphaFold2 program to predict interactions among proteins localized in the nuage, a germline-specific non-membrane organelle essential for piRNA biogenesis in Drosophila. We screened 20 nuage proteins for 1:1 interactions and predicted dimer structures. Among these, five represented novel interaction candidates. Three pairs, including Spn-E_Squ, were verified by co-immunoprecipitation. Disruption of the salt bridges at the Spn-E_Squ interface confirmed their functional importance, underscoring the predictive model’s accuracy. We extended our analysis to include interactions between three representative nuage components—Vas, Squ, and Tej—and approximately 430 oogenesis-related proteins. Co-immunoprecipitation verified interactions for three pairs: Mei-W68_Squ, CSN3_Squ, and Pka-C1_Tej. Furthermore, we screened the majority of Drosophila proteins (~12,000) for potential interaction with the Piwi protein, a central player in the piRNA pathway, identifying 164 pairs as potential binding partners. This in silico approach not only efficiently identifies potential interaction partners but also significantly bridges the gap by facilitating the integration of bioinformatics and experimental biology.