1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Ga chaperone and GEF Ric-8A

  1. Daniel Muñoz-Reyes
  2. Levi J McClelland
  3. Sandra Arroyo-Urea
  4. Sonia Sánchez-Yepes
  5. Juan Sabín
  6. Sara Pérez-Suárez
  7. Margarita Menendez
  8. Alicia Mansilla
  9. Javier García-Nafría
  10. Stephen Sprang
  11. Maria Jose Sanchez-Barrena  Is a corresponding author
  1. Institute of Physical-Chemistry Blas Cabrera, Spain
  2. University of Montana, United States
  3. University of Zaragoza, Spain
  4. Hospital Universitario Ramón y Cajal, Spain
  5. Software 4 Science Developments, Spain
  6. Institute of Physical-Chemistry, Spain
  7. Institute of Physical Chemistry Blas Cabrera, Spain
https://doi.org/10.7554/eLife.86151
Share this article
Cite this article
  1. Daniel Muñoz-Reyes
  2. Levi J McClelland
  3. Sandra Arroyo-Urea
  4. Sonia Sánchez-Yepes
  5. Juan Sabín
  6. Sara Pérez-Suárez
  7. Margarita Menendez
  8. Alicia Mansilla
  9. Javier García-Nafría
  10. Stephen Sprang
  11. Maria Jose Sanchez-Barrena
(2023)
The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Ga chaperone and GEF Ric-8A
eLife 12:e86151.
https://doi.org/10.7554/eLife.86151
  2. Download BibTeX
  3. Download .RIS

Abstract

The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

Data availability

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, https://www.pdb.org/. PDB with codes: Structure 1 (8ALH), Structure 2 (8AHY), Structure 3 (8ALM).All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figure 2, Figure 3, Figure 4, Figure 5

Article and author information

Author details

  1. Daniel Muñoz-Reyes

    Department of Crystallography and Structural Biology, Institute of Physical-Chemistry Blas Cabrera, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.

  2. Levi J McClelland

    Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Sandra Arroyo-Urea

    Institute for Biocomputation and Physics of Complex Systems, University of Zaragoza, Zaragoza, Spain
    Competing interests
    The authors declare that no competing interests exist.

  4. Sonia Sánchez-Yepes

    Department of Neurobiology, Hospital Universitario Ramón y Cajal, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.

  5. Juan Sabín

    AFFINImeter Scientific & Development, Software 4 Science Developments, Santiago de Compostela, Spain
    Competing interests
    The authors declare that no competing interests exist.

  6. Sara Pérez-Suárez

    Department of Crystallography and Structural Biology, Institute of Physical-Chemistry Blas Cabrera, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.

  7. Margarita Menendez

    Department of Biological Physical-Chemisty, Institute of Physical-Chemistry, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.

  8. Alicia Mansilla

    Department of Neurobiology, Hospital Universitario Ramón y Cajal, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.

  9. Javier García-Nafría

    Institute for Biocomputation and Physics of Complex Systems, University of Zaragoza, Zaragoza, Spain
    Competing interests
    The authors declare that no competing interests exist.

  10. Stephen Sprang

    Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, United States
    Competing interests
    The authors declare that no competing interests exist.

  11. Maria Jose Sanchez-Barrena

    Department of Crystallography and Structural Biology, Institute of Physical Chemistry Blas Cabrera, Madrid, Spain
    For correspondence
    xmjose@iqfr.csic.es
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5986-1804

Funding

Spanish National Plan for Scientific and Technical Research and Innovation (PID2019-111737RB-I00)

  • Maria Jose Sanchez-Barrena

Spanish National Plan for Scientific and Technical Research and Innovation (PID2019-106608RB-I00)

  • Alicia Mansilla

Spanish National Plan for Scientific and Technical Research and Innovation (PDC2022-133775-I00)

  • Alicia Mansilla

Spanish National Plan for Scientific and Technical Research and Innovation (RTI2018-099985-B-I00)

  • Margarita Menendez

Spanish National Plan for Scientific and Technical Research and Innovation (PID2020-113359GA-I00)

  • Javier García-Nafría

Spanish National Plan for Scientific and Technical Research and Innovation (RYC-2017-22392)

  • Alicia Mansilla

Spanish National Plan for Scientific and Technical Research and Innovation (RYC2018-025731-I)

  • Javier García-Nafría

National Institutes of Health (P30GM140963)

  • Stephen Sprang

National Institutes of Health (R01GM105993)

  • Stephen Sprang

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

Copyright

© 2023, Muñoz-Reyes 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

  • 1,255
    views
  • 196
    downloads
  • 2
    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. Daniel Muñoz-Reyes
  2. Levi J McClelland
  3. Sandra Arroyo-Urea
  4. Sonia Sánchez-Yepes
  5. Juan Sabín
  6. Sara Pérez-Suárez
  7. Margarita Menendez
  8. Alicia Mansilla
  9. Javier García-Nafría
  10. Stephen Sprang
  11. Maria Jose Sanchez-Barrena
(2023)
The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Ga chaperone and GEF Ric-8A
eLife 12:e86151.
https://doi.org/10.7554/eLife.86151

Share this article

https://doi.org/10.7554/eLife.86151

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Kinetic regulation of kinesin’s two motor domains coordinates its stepping along microtubules

    Yamato Niitani, Kohei Matsuzaki ... Michio Tomishige
    Research Article

    The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.

    1. Biochemistry and Chemical Biology
    2. Computational and Systems Biology

    Allosteric modulation by the fatty acid site in the glycosylated SARS-CoV-2 spike

    A Sofia F Oliveira, Fiona L Kearns ... Adrian J Mulholland
    Short Report

    The spike protein is essential to the SARS-CoV-2 virus life cycle, facilitating virus entry and mediating viral-host membrane fusion. The spike contains a fatty acid (FA) binding site between every two neighbouring receptor-binding domains. This site is coupled to key regions in the protein, but the impact of glycans on these allosteric effects has not been investigated. Using dynamical nonequilibrium molecular dynamics (D-NEMD) simulations, we explore the allosteric effects of the FA site in the fully glycosylated spike of the SARS-CoV-2 ancestral variant. Our results identify the allosteric networks connecting the FA site to functionally important regions in the protein, including the receptor-binding motif, an antigenic supersite in the N-terminal domain, the fusion peptide region, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected link between different allosteric sites. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spike revealed that glycans do not qualitatively change the internal allosteric pathways but can facilitate the transmission of the structural changes within and between subunits.

    1. Biochemistry and Chemical Biology
    2. Genetics and Genomics

    High-resolution deep mutational scanning of the melanocortin-4 receptor enables target characterization for drug discovery

    Conor J Howard, Nathan S Abell ... Nathan B Lubock
    Research Article

    Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G-protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6600 single amino acid substitutions on MC4R’s function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gαs- and Gαq-signaling pathways, which could be used to design drugs that selectively bias MC4R’s activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.