Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor

  1. Steven De Gieter
  2. Casey Gallagher
  3. Eveline Wijckmans
  4. Diletta Pasini
  5. Chris Ulens  Is a corresponding author
  6. Rouslan G Efremov  Is a corresponding author
  1. VIB-VUB Center for Structural Biology, Belgium
  2. KU Leuven, Belgium

Abstract

Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high potential as drug targets, Cys-loop receptors from humans and closely related organisms have been thoroughly investigated, whereas molecular mechanisms of neurotransmission in invertebrates are less understood. When compared with vertebrates, the invertebrate genomes underwent a drastic expansion in the number of the nACh-like genes associated with receptors of unknown function. Understanding this diversity contributes to better insight into the evolution and possible functional divergence of these receptors. In this work, we studied orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Sequence analysis points towards its remote relation to characterized nACh receptors. We solved the cryo-EM structure of the lophotrochozoan nACh-like receptor in which a CHAPS molecule is tightly bound to the orthosteric site. We show that the binding of CHAPS leads to extending of the loop C at the orthosteric site and a quaternary twist between extracellular and transmembrane domains. Both the ligand binding site and the channel pore reveal unique features. These include a conserved Trp residue in loop B of the ligand binding site which is flipped into an apparent self-liganded state in the apo structure. The ion pore of Alpo4 is tightly constricted by a ring of methionines near the extracellular entryway of the channel pore. Our data provide a structural basis for a functional understanding of Alpo4 and hints towards new strategies for designing specific channel modulators.

Data availability

The cryo-EM density maps and atomic models generated in this study have been deposited in the PDB and EMDB database under accession codes: 8BYI / EMDB-16326 (Alpo4CHAPS), 8BXF / EMDB- 16317 (Alpo4APO), 8BX5 / EMDB-16308 (Alpo4LMNG_APO), 8BXB / EMDB-16314 (Alpo4ACH), 8BKE / EMDB-16316 (Alpo4COMB), 8BKD / EMDB-16315 (Alpo4SER).

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Steven De Gieter

    VIB-VUB Center for Structural Biology, Brussels, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  2. Casey Gallagher

    Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  3. Eveline Wijckmans

    Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  4. Diletta Pasini

    Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium
    Competing interests
    The authors declare that no competing interests exist.
  5. Chris Ulens

    Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium
    For correspondence
    chris.ulens@kuleuven.be
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8202-5281
  6. Rouslan G Efremov

    VIB-VUB Center for Structural Biology, Brussels, Belgium
    For correspondence
    rouslan.efremov@vub.vib.be
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7516-8658

Funding

Fonds Wetenschappelijk Onderzoek (G0H5916N)

  • Rouslan G Efremov

Fonds Wetenschappelijk Onderzoek (G054617N)

  • Rouslan G Efremov

Fonds Wetenschappelijk Onderzoek (G0H5916N)

  • Steven De Gieter

Fonds Wetenschappelijk Onderzoek (G0C1319N)

  • Chris Ulens

Fonds Wetenschappelijk Onderzoek (G087921N)

  • Chris Ulens

KU Leuven (C3/19/023)

  • Chris Ulens

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

Copyright

© 2023, De Gieter 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.

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. Steven De Gieter
  2. Casey Gallagher
  3. Eveline Wijckmans
  4. Diletta Pasini
  5. Chris Ulens
  6. Rouslan G Efremov
(2023)
Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor
eLife 12:e86029.
https://doi.org/10.7554/eLife.86029

Share this article

https://doi.org/10.7554/eLife.86029

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Joar Esteban Pinto Torres, Mathieu Claes ... Yann G-J Sterckx
    Research Article

    African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.

    1. Structural Biology and Molecular Biophysics
    Manming Xu, Sarath Chandra Dantu ... Shozeb Haider
    Research Article

    The relationship between protein dynamics and function is essential for understanding biological processes and developing effective therapeutics. Functional sites within proteins are critical for activities such as substrate binding, catalysis, and structural changes. Existing computational methods for the predictions of functional residues are trained on sequence, structural, and experimental data, but they do not explicitly model the influence of evolution on protein dynamics. This overlooked contribution is essential as it is known that evolution can fine-tune protein dynamics through compensatory mutations either to improve the proteins’ performance or diversify its function while maintaining the same structural scaffold. To model this critical contribution, we introduce DyNoPy, a computational method that combines residue coevolution analysis with molecular dynamics simulations, revealing hidden correlations between functional sites. DyNoPy constructs a graph model of residue–residue interactions, identifies communities of key residue groups, and annotates critical sites based on their roles. By leveraging the concept of coevolved dynamical couplings—residue pairs with critical dynamical interactions that have been preserved during evolution—DyNoPy offers a powerful method for predicting and analysing protein evolution and dynamics. We demonstrate the effectiveness of DyNoPy on SHV-1 and PDC-3, chromosomally encoded β-lactamases linked to antibiotic resistance, highlighting its potential to inform drug design and address pressing healthcare challenges.