Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states

  1. Hans Moldenhauer
  2. Ignacio Díaz-Franulic
  3. Horacio Poblete
  4. David Naranjo  Is a corresponding author
  1. Universidad de Valparaíso, Chile
  2. Universidad de Talca, Chile

Abstract

In-silico and in-vitro studies have made progress in understanding protein-protein complexes formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

Data availability

Data used for Figures 2 to 7 is available in dryad.org

The following data sets were generated

Article and author information

Author details

  1. Hans Moldenhauer

    Instituto de Neurociencia, Universidad de Valparaíso, Valpararaíso, Chile
    Competing interests
    The authors declare that no competing interests exist.
  2. Ignacio Díaz-Franulic

    Instituto de Neurociencia, Universidad de Valparaíso, Valparaíso, Chile
    Competing interests
    The authors declare that no competing interests exist.
  3. Horacio Poblete

    Center for Bioinformatics and Molecular Simulations, Universidad de Talca, Talca, Chile
    Competing interests
    The authors declare that no competing interests exist.
  4. David Naranjo

    Instituto de Neurociencia, Universidad de Valparaíso, Valparaíso, Chile
    For correspondence
    david.naranjo@uv.cl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3482-5126

Funding

Fondo Nacional de Desarrollo Científico y Tecnológico (3160321)

  • Hans Moldenhauer

Fondo Nacional de Desarrollo Científico y Tecnológico (3170599)

  • Ignacio Díaz-Franulic

Fondo Nacional de Desarrollo Científico y Tecnológico (1171155)

  • Horacio Poblete

Ministerio de Economía, Fomento y Turismo (MiNICAD)

  • Horacio Poblete

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

Reviewing Editor

  1. Leon D Islas, Universidad Nacional Autónoma de México, Mexico

Publication history

  1. Received: February 18, 2019
  2. Accepted: July 4, 2019
  3. Accepted Manuscript published: July 4, 2019 (version 1)
  4. Version of Record published: July 26, 2019 (version 2)

Copyright

© 2019, Moldenhauer 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

  • 1,230
    Page views
  • 159
    Downloads
  • 6
    Citations

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

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. Hans Moldenhauer
  2. Ignacio Díaz-Franulic
  3. Horacio Poblete
  4. David Naranjo
(2019)
Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states
eLife 8:e46170.
https://doi.org/10.7554/eLife.46170

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Rajesh Sharma et al.
    Research Article

    Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cGMP signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here we present a crystal structure of PKG Iβ in which the auto-inhibitory sequence and the cyclic nucleotide binding domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ auto-inhibitory sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I cyclic nucleotide binding domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wild type cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yitong Li et al.
    Research Article

    Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryo-EM structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multi-partite contacts at structured cores to activate the methylesterase. B56-interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56-interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.