The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A
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eLife assessment
This important study reports biochemical and structural experiments that were carried out to determine the molecular basis of calcium-sensitive regulation of the guanine exchange factor Ric8A by the neuronal calcium sensor 1 (NCS-1). Structural and biochemical evidence for the NCS-1/Ric8A interface is convincing, but evidence for the full-length interactions is incomplete due to the low resolution of cryo-EM maps. This work will have important implications for scientists interested in G-protein signaling and molecular interactions that contribute to synapse function.
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Abstract
The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca 2+ 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 2+ 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 2+ 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.
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eLife assessment
This important study reports biochemical and structural experiments that were carried out to determine the molecular basis of calcium-sensitive regulation of the guanine exchange factor Ric8A by the neuronal calcium sensor 1 (NCS-1). Structural and biochemical evidence for the NCS-1/Ric8A interface is convincing, but evidence for the full-length interactions is incomplete due to the low resolution of cryo-EM maps. This work will have important implications for scientists interested in G-protein signaling and molecular interactions that contribute to synapse function.
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Reviewer #1 (Public Review):
The strongest aspect of the study is the identification of the probable Ric-8A/NCS-1 interface through the crystal structures of NCS-1 complexed with candidate peptide mimetics from Ric-8A. However, since the structures involve peptides, it is critical to validate this interface with mutational analysis of the full-length or truncated Ric-8A. Furthermore, the evidence for the complex structure based on cryo-EM reconstruction is weak. The low resolution does not allow for reliable modeling of the complex. Two analyses may support the authors' main conclusions: a) validation of the interface with mutational analysis of Ric-8A, and b) new optimized sample/grid preparation for cryo-EM data collection.
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Reviewer #2 (Public Review):
This manuscript by Muñoz-Reyes et al. presents studies on the molecular mechanisms by which NCS-1 regulates Ric-8A and its interaction with Ga. They have investigated how calcium ions and phosphorylation of Ric-8A affect these interactions. They found that NCS-1 induces a conformational change in Ric-8A that prevents its phosphorylation and subsequent interaction with Ga, and this can be reversed by increasing calcium ion concentration. Using structural biology methods, they determined the interaction surfaces between NCS-1 and Ric-8A. These mechanistic analyses are needed in the field and beneficial to helping us understand specificity in the regulation of G protein signaling.
Overall, this manuscript presents an abundance of data that supports the authors' conclusions. The introduction is thorough and …
Reviewer #2 (Public Review):
This manuscript by Muñoz-Reyes et al. presents studies on the molecular mechanisms by which NCS-1 regulates Ric-8A and its interaction with Ga. They have investigated how calcium ions and phosphorylation of Ric-8A affect these interactions. They found that NCS-1 induces a conformational change in Ric-8A that prevents its phosphorylation and subsequent interaction with Ga, and this can be reversed by increasing calcium ion concentration. Using structural biology methods, they determined the interaction surfaces between NCS-1 and Ric-8A. These mechanistic analyses are needed in the field and beneficial to helping us understand specificity in the regulation of G protein signaling.
Overall, this manuscript presents an abundance of data that supports the authors' conclusions. The introduction is thorough and well-written. The structure figures are beautiful and clear - well done. Most of the biochemical and biophysical experiments are convincing. In some cases, further elaborations and explanations of data interpretation are needed. The crystallographic data is solid. However, I have major concerns with the cryo-EM data presented due to its low quality and the conclusions drawn from it.
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Reviewer #3 (Public Review):
The current manuscript investigates the molecular basis of calcium-sensitive regulation of the guanine exchange factor Ric8A by the neuronal calcium sensor 1 (NCS-1). The authors provide insight into a number of aspects of this interaction, including high-resolution structures of the NCS1-Ric8A binding interface (using peptides based on Ric8A), low-resolution cryo-EM data that hints at a structural rearrangement, and a biochemical investigation of the influence of calcium binding, sodium binding, and phosphorylation on this interaction. Altogether, this manuscript provides a comprehensive set of experiments that provide insight into this important interaction. In particular, the identification of ions bound to NCS-1 using crystallography and binding assays is very nicely done and convincing. The cryo-EM data …
Reviewer #3 (Public Review):
The current manuscript investigates the molecular basis of calcium-sensitive regulation of the guanine exchange factor Ric8A by the neuronal calcium sensor 1 (NCS-1). The authors provide insight into a number of aspects of this interaction, including high-resolution structures of the NCS1-Ric8A binding interface (using peptides based on Ric8A), low-resolution cryo-EM data that hints at a structural rearrangement, and a biochemical investigation of the influence of calcium binding, sodium binding, and phosphorylation on this interaction. Altogether, this manuscript provides a comprehensive set of experiments that provide insight into this important interaction. In particular, the identification of ions bound to NCS-1 using crystallography and binding assays is very nicely done and convincing. The cryo-EM data is at low resolution and provides only weak support for the proposed mechanism.
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