Structural And Functional Study Of CAPS-1 DAMH Domain In Regulating SNARE Complex Formation And Exocytosis

The synaptic exocytosis is crucial for neurodevelopment,synapse formation,and synaptic plasticity,which includes five stages:trafficking,tethering,docking,priming,and fusion of the synaptic vesicles（SVs）and dense core vesicles（DCVs）.These processes in presynaptic active zone provide an important basis for the Ca²⁺-triggered fusion.Priming of exocytosis is widely believed to involve the assembly of trans-soluble N-ethylmaleimide-sensitive factor attachment protein receptor（SNARE）complex,which is composed of synaptobrevin-2/VAMP-2（Syb2）,syntaxin-1（Syx1）,and SNAP-25（SN25）.Except for the core apparatus,the SNARE complex,the release of neurotransmitters further requires the precise regulation of other presynaptic active zone proteins,e.g.,Munc13 and Ca²⁺-dependent activator proteins for secretion（CAPS）.As a conserved multi-domain protein family,CAPS is composed of four important functional domains:the C₂ domain and the pleckstrin homologous（PH）domain at the N-terminus,and the DAMH domain and the DCV binding domain（DCVBD）at the C-terminus.CAPS participates in the regulation of exocytosis through the DAMH domain promoting the SNARE complex assembly.CAPS and Munc13 are believed to play crucial roles in SNARE-mediated exocytosis.Moreover,the molecular mechanism of Munc13-regulated exocytosis has been extensively elucidated.However,the three-dimensional structure of CAPS,and the underlying mechanism of how it regulates the SNARE-mediated membrane fusion remain unclear.In this study,We used X-ray crystallography,GST pull-down,fluorescence resonance energy transfer（FRET）,liposome co-flotation,and in vitro recombinant liposome fusion assay to i)report a crystal structure of the CAPS-1 DAMH domain at2.9-（?）resolution;ii)demonstrate the interaction between CAPS-1 and Munc13-1;iii)identify the key sites（E890/H891/E894,W898/D901/L902,and E905）in DAMH domain for specific binding to SNARE complex;iv)reveal a dual role of CAPS-1 in SNARE complex formation.On one hand,CAPS-1 inhibits SNARE complex formation dependent on binding of the DAMH domain to the MUN domain of Munc13-1.The binding hinders the ability of Munc13-1 to catalyze opening of Syx1.On the other hand,CAPS-1 plays a chaperone role to facilitate SNARE complex formation dependent on the interaction of the DAMH domain with the Syx1/SN25complex.The interaction stabilizes the open conformation of Syx1.Our results suggest that CAPS-1 facilitates SNARE complex formation via the DAMH domain in a manner dependent on sequential and cooperative interaction with Munc13-1 and SNARE proteins.In conclusion,we propose a model by which CAPS-1 regulates SNARE-mediated synaptic exocytosis.