Structural And Functional Studies Of The C₂-PH Module Of CAPS-1 In Vesicle Exocytosis

Vesicular secretion plays an important role in intercellular communication.The assembly of the SNARE complex（soluble N-ethylmaleimide-sensitive factor attachment protein receptor,SNARE）provides energy for vesicle exocytosis.SNARE complex assembly is strictly regulated,and CAPSs(Ca²⁺-dependent activator proteins for secretion)are important regulatory proteins involved in SNARE complex assembly.Loss of CAPS in nematodes or mice results in a severe reduction in vesicular secretion.CAPS contains four domains:the N-terminal C₂ domain,,a PH domain that binds PI（4,5）P2-membrane,a DAMH domain involved in SNARE interaction and the DCV domain bound to the vesicle membrane.Munc13 is another important regulatory proteins involved in SNARE complex assembly,which binds to the PI（4,5）P2 via its C₂B domain,and participates in the regulation of vesicle secretion.PI（4,5）P2 is an important phospholipid molecule on the presynaptic membrane,the binding of CAPS to PI（4,5）P2 is crucial for the proper localization of CAPS and the anchoring of vesicles.Although the PH domain of CAPS-1 can bind PI（4,5）P2,further studies have revealed that CAPS-1 can still bind to PI（4,5）P2 even when the PH domain was removed,indicating the existence of other PI（4,5）P2 binding sites on CAPS-1.However,it is not clear which domains are involved in the binding between CAPS-1 and PI（4,5）P2.Interestingly,another work found that a natural CAPS splice isoform,which comprises the C₂ and PH domains but lacks the DAMH and DCV-binding domains,is able to rescue exocytosis in chromaffin cells and neurons deficient in CAPS.This suggests that the C₂ and PH domains of CAPS play important functions in the early stages of mouse development,but the specific mechanism is unclear.In this study,we used X-ray crystallography,protein-vesicle co-floating techniques,fluorescence resonance energy transfer techniques,total internal reflection microscopy,and electrophysiology to systematically investigate the mechanisms underlying the function of the C₂-PH functional unit composed of the CAPS-1 C₂ and PH domains in vesicular secretion,and obtained the following results:（1）The crystal structure of the rat CAPS-1 C₂-PH module was solved（PDB:7VS3）,and through crystal structure analysis,it was found that the C₂ and PH domains of CAPS-1are tightly bound by hydrophobic interactions,and destroying the hydrophobic interaction between C₂ and PH domains would reduce the locomotion speed of nematodes and reduce the secretion of nematode neurotransmitters;（2）A new PI（4,5）P2 binding site was identified on the CAPS-1 C₂ domain,which synergized with the PI（4,5）P2 binding site on the PH domain,so that the C₂-PH module obtained stronger PI（4,5）P2 binding ability than the PH domain alone,and destroyed the PI（4,5）P2 binding site on the C₂-PH module would reduce the locomotion speed of nematodes and reduce the secretion of nematode neurotransmitters;（3）The PI（4,5）P2 binding capacity of the Munc13-1 C₂B domain and C₂-PH module were compared,and it was found that the PI（4,5）P2 binding order of the two proteins was regulated by the concentration of calcium:the CAPS-1 C₂-PH module bound PI（4,5）P2 in the absence of calcium while the C₂B domain of Munc13-1 binds to PI（4,5）P2 when the calcium concentration increases;（4）It was found that the C₂-PH module and the DAMH domain interacte with the MUN domain of Munc13-1,CAPS-1 could recruit Munc13-1 to the cell membrane in the resting state,according to this the C₂-PH-Munc13-1 chimera was designed,and it was found that the chimera protein could compensate for the decrease in vesicle secretion caused by CAPS-1 deletion in PC12 cells.In summary,our study elucidates the structure and function of CAPS-1 C₂-PH module in detail.Compared the different characteristics of CAPS-1 C₂-PH module and Munc13-1C₂B domain binding PI（4,5）P2,and evealed the mechanism of Munc13-1 and CAPS-1 in vesicle secretion.This lays the foundation for a better understanding of the vesicle release process.