Abstract
The neuronal calcium sensor 1 (NCS-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 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 Ca2+ 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 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.
| Original language | English |
|---|---|
| Journal | eLife |
| Volume | 12 |
| DOIs | |
| State | Published - Nov 29 2023 |
Funding
MJS-B would like to thank ALBA (XALOC beamline) and ESRF synchrotrons for access and support of the staff, the mass spectrometry service from Institute 'Blas Cabrera' and Prof. Armando Albert and Prof. Alberto Ferrús for critical revision of the manuscript. The proteomic analysis was performed in the proteomics facility of 'Centro Nacional de Biotecnología' (CSIC). SEC-MALS experiments were performed at the Spectroscopy and NMR Unit (CNIO) with the assistance of Clara M Santiveri and Ramón Campos-Olivas. This work was funded by grants from Spanish Ministry of Science and Innovation PID2019-111737RB-I00 (to MJS-B), PID2019-106608RB-I00 and PDC2022-133775-I00 (to AM), RTI2018-099985-B-I00 (to MM) and PID2020-113359GA-I00 (to JG-N). MM was supported also by CIBER of Respiratory Diseases (CIBERES) from ISCIII. AM and JG-N were supported by 'Ramón y Cajal' contracts from the Spanish Ministry of Science and Innovation (RYC-2017-22392 and RYC2018-025731-I, respectively). SA-U was funded by a PhD fellowship of the 'Diputación General de Aragón'. SP-S was supported by a contract from 'Programa de Empleo Juvenil de la Comunidad de Madrid' PEJ-2020-AI/BMD-18666. SRS would like to acknowledge NIH grant P30GM140963 for support of the Center for Biomolecular Structure and Dynamics Integrated Structural Biology Core at the University of Montana, a Pilot Project grant to LJM, and R01GM105993 to SRS.
| Funders | Funder number |
|---|---|
| PEJ-2020-AI/BMD-18666 | |
| R01GM105993, P30GM140963 | |
| CSIC - National Center for Biotechnology | |
| Instituto de Salud Carlos III | RYC2018-025731-I, RYC-2017-22392 |
| RTI2018-099985-B-I00, PID2020-113359GA-I00, PID2019-111737RB-I00, PID2019-106608RB-I00, PDC2022-133775-I00 | |
Keywords
- E. coli
- biochemistry
- calcium signaling
- chemical biology
- guanine nucleotide exchange
- molecular biophysics
- molecular chaperone
- phosphorylation
- protein-protein interaction
- structural biology
- synapse function regulation