1. Structural Biology and Molecular Biophysics
  2. Microbiology and Infectious Disease
Download icon

Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action

  1. Felipe Trajtenberg
  2. Juan A Imelio
  3. Matías R Machado
  4. Nicole Larrieux
  5. Marcelo A Marti
  6. Gonzalo Obal
  7. Ariel E Mechaly
  8. Alejandro Buschiazzo  Is a corresponding author
  1. Institut Pasteur de Montevideo, Uruguay
  2. Universidad de Buenos Aires, Argentina
Research Article
  • Cited 29
  • Views 1,606
  • Annotations
Cite this article as: eLife 2016;5:e21422 doi: 10.7554/eLife.21422

Abstract

Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring.

Article and author information

Author details

  1. Felipe Trajtenberg

    Laboratory of Molecular and Structural Microbiology, Institut Pasteur de Montevideo, Montevideo, Uruguay
    Competing interests
    The authors declare that no competing interests exist.
  2. Juan A Imelio

    Laboratory of Molecular and Structural Microbiology, Institut Pasteur de Montevideo, Montevideo, Uruguay
    Competing interests
    The authors declare that no competing interests exist.
  3. Matías R Machado

    Biomolecular Simulations, Institut Pasteur de Montevideo, Montevideo, Uruguay
    Competing interests
    The authors declare that no competing interests exist.
  4. Nicole Larrieux

    Laboratory of Molecular and Structural Microbiology, Institut Pasteur de Montevideo, Montevideo, Uruguay
    Competing interests
    The authors declare that no competing interests exist.
  5. Marcelo A Marti

    Dto. Química Biológica e IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
    Competing interests
    The authors declare that no competing interests exist.
  6. Gonzalo Obal

    Protein Biophysics Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay
    Competing interests
    The authors declare that no competing interests exist.
  7. Ariel E Mechaly

    Laboratory of Molecular and Structural Microbiology, Institut Pasteur de Montevideo, Montevideo, Uruguay
    Competing interests
    The authors declare that no competing interests exist.
  8. Alejandro Buschiazzo

    Laboratory of Molecular and Structural Microbiology, Institut Pasteur de Montevideo, Montevideo, Uruguay
    For correspondence
    alebus@pasteur.edu.uy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2509-6526

Funding

Agencia Nacional de Investigación e Innovación (FCE2009_1_2679)

  • Felipe Trajtenberg
  • Alejandro Buschiazzo

Agence Nationale de la Recherche (PCV06_138918)

  • Alejandro Buschiazzo

FOCEM (COF 03/11)

  • Alejandro Buschiazzo

Centro de Biologia Estructural del Mercosur

  • Alejandro Buschiazzo

Agencia Nacional de Investigación e Innovación (FCE2007_219)

  • Felipe Trajtenberg
  • Alejandro Buschiazzo

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

Reviewing Editor

  1. Michael Laub, Massachusetts Institute of Technology, United States

Publication history

  1. Received: September 11, 2016
  2. Accepted: December 9, 2016
  3. Accepted Manuscript published: December 12, 2016 (version 1)
  4. Version of Record published: January 12, 2017 (version 2)
  5. Version of Record updated: September 5, 2017 (version 3)

Copyright

© 2016, Trajtenberg 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

  • 1,606
    Page views
  • 434
    Downloads
  • 29
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

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)

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

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

Further reading

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Lukas Ded et al.
    Research Article

    Out of millions of ejaculated sperm, only a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here, using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that all spermatozoa past the UTJ possess intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Sandip Basak et al.
    Research Article

    Serotonin receptors (5-HT3AR) play a crucial role in regulating gut movement, and are the principal target of setrons, a class of high-affinity competitive antagonists, used in the management of nausea and vomiting associated with radiation and chemotherapies. Structural insights into setron-binding poses and their inhibitory mechanisms are just beginning to emerge. Here, we present high-resolution cryo-EM structures of full-length 5-HT3AR in complex with palonosetron, ondansetron, and alosetron. Molecular dynamic simulations of these structures embedded in a fully-hydrated lipid environment assessed the stability of ligand-binding poses and drug-target interactions over time. Together with simulation results of apo- and serotonin-bound 5-HT3AR, the study reveals a distinct interaction fingerprint between the various setrons and binding-pocket residues that may underlie their diverse affinities. In addition, varying degrees of conformational change in the setron-5-HT3AR structures, throughout the channel and particularly along the channel activation pathway, suggests a novel mechanism of competitive inhibition.