Daniel Muñoz-Reyes, Department of Crystallography and Structural Biology, Institute of Physical-Chemistry 'Blas Cabrera', CSIC, Madrid, Spain.
Levi J. McClelland, Center for Biomolecular Structure and Dynamics, and Division of Biological Sciences, University of Montana, Missoula, United States.
Sandra Arroyo-Urea, Institute for Biocomputation and Physics of Complex Systems (BIFI) and Laboratorio de Microscopías Avanzadas (LMA), University of Zaragoza, Zaragoza, Spain.
Sonia Sánchez-Yepes, Department of Neurobiology, Instituto Ramón y Cajal de Investigación Sanitaria, Hospital Universitario Ramón y Cajal, Madrid, Spain.
Juan Sabín, AFFINImeter Scientific & Development team, Software 4 Science Developments, Santiago de Compostela, Spain.
Sara Pérez-Suárez, Department of Crystallography and Structural Biology, Institute of Physical-Chemistry 'Blas Cabrera', CSIC, Madrid, Spain.
Margarita Menendez, Department of Biological Physical-Chemisty, Institute of Physical-Chemistry 'Blas Cabrera', CSIC, Madrid, Spain.
Alicia Mansilla, Department of Neurobiology, Instituto Ramón y Cajal de Investigación Sanitaria, Hospital Universitario Ramón y Cajal, Madrid, Spain.
Javier García-Nafría, Institute for Biocomputation and Physics of Complex Systems (BIFI) and Laboratorio de Microscopías Avanzadas (LMA), University of Zaragoza, Zaragoza, Spain.
Stephen SprangFollow
Maria Jose Sanchez-Barrena, Department of Crystallography and Structural Biology, Institute of Physical-Chemistry 'Blas Cabrera', CSIC, Madrid, Spain.

Document Type

Article

Publication Title

eLife

Publication Date

11-29-2023

Volume

12

Disciplines

Biology | Life Sciences

Abstract

The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα 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 Gα 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 Ca signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα 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 guanine nucleotide exchange factor (GEF) activity toward Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca 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.

Keywords

E. coli, biochemistry, calcium signaling, chemical biology, guanine nucleotide exchange, molecular biophysics, molecular chaperone, phosphorylation, protein-protein interaction, structural biology, synapse function regulation

DOI

10.7554/eLife.86151

Rights

© 2023 Muñoz-Reyes et al.

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

Download

Included in

Biology Commons

Share

COinS