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bioRxiv · 10.1101/2022.12.09.519724

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

Munoz-Reyes, D.·McClelland, L. J.·Arroyo-Urea, S.·Sanchez-Yepes, S.·Sabin, J.·Perez-Suarez, S.·Menendez, M.·Mansilla, A.·Garcia-Nafria, J.·Sprang, S. R.·Sanchez-Barrena, M. J.
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Abstract

The Neuronal Calcium Sensor 1 and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to G have revealed how Ric-8A phosphorylation promotes G 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 G 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 G. Our data show that the binding of NCS-1 and G to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a profound 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 G when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here that define the NCS-1/Ric-8A interface will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

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Munoz-Reyes, D., McClelland, L. J., Arroyo-Urea, S., Sanchez-Yepes, S., Sabin, J., Perez-Suarez, S., Menendez, M., Mansilla, A., Garcia-Nafria, J., Sprang, S. R., Sanchez-Barrena, M. J.. 2022-12-10. The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A. https://doi.org/10.1101/2022.12.09.519724

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