Biochemical, biophysical and structural approach to determine the molecular basis of the calcium-sensitive regulation of the Gα chaperone and guanine exchange factor Ric-8A by the neuronal calcium sensor NCS-1.

The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. The protein-protein interaction interface constitutes a pharmacological target under brain pathological conditions. Previous structural studies 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 was not well understood. Here, we have conducted a multimodal approach to show that NCS-1 and Ric-8A constitute a hub that integrates Ca2+, phosphorylation and G-protein signaling. The emergent picture indicates that at Ca2+ resting state, Ric-8A activity is under NCS-1 control and the Ca2+ sensor traps Ric-8A in a conformational state that hinders phosphorylation and Ga recognition. However, a specific Ca2+ signal triggers the disassembly of the NCS-1/Ric-8A complex, which in turn allows phosphorylation of Ric-8A, formation of the Ric-8A/Gα complex and activation of Gα nucleotide exchange. Strikingly, we found that NCS-1 binds Na+ in its regulatory Ca2+ site, decreasing the affinity of NCS-1 for Ca2+. Furthermore, we show that different Ca2+ signals promote the recognition of Ric-8A and dopamine D2 receptor. 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. Daniel Muñoz-Reyes, Levi J McClelland, Sandra Arroyo-Urea, Sonia Sánchez-Yepes, Juan Sabín, Sara Pérez-Suárez, Margarita Menendez, Alicia Mansilla, Javier García-Nafría, Stephen Sprang, Maria José Sánchez-Barrena. eLife (2023) 12:e86151 (doi: 10.7554/eLife.86151) 

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