Complexin-1 regulated assembly of single neuronal SNARE complex revealed by single-molecule optical tweezers

Abstract

The dynamic assembly of the Synaptic-soluble N-ethylmaleimide-sensitive factor Attachment REceptor (SNARE) complex is crucial to understand membrane fusion. Traditional ensemble study meets the challenge to dissect the dynamic assembly of the protein complex. Here, we apply minute force on a tethered protein complex through dual-trap optical tweezers and study the folding dynamics of SNARE complex under mechanical force regulated by complexin-1 (CpxI). We reconstruct the clamp and facilitate functions of CpxI in vitro and identify different interplay mechanism of CpxI fragment binding on the SNARE complex. Specially, while the N-terminal domain (NTD) plays a dominant role of the facilitate function, CTD is mainly related to clamping. And the mixture of 1-83aa and CTD of CpxI can efficiently reconstitute the inhibitory signal identical to that the full-length CpxI functions. Our observation identifies the important chaperone role of the CpxI molecule in the dynamic assembly of SNARE complex under mechanical tension, and elucidates the specific function of each fragment of CpxI molecules in the chaperone process.

The stability of the primed pool of synaptic vesicles and the clamping of spontaneous neurotransmitter release relies on the integrity of the C-terminal half of the SNARE domain of Syntaxin-1A

“…This was further confirmed by single-molecule optical tweezer studies which determined the sequential assembly of the SNARE complex through diverse staged assisted by these two subdomains (Gao et al, 2012). Additionally, it has been determined that accessory proteins, such as Munc18-1 and Complexin, chaperone the stability of the N-terminus and facilitate or clamp the assembly of the C-terminus in the partially-zippered SNARE domain, which without their regulation is fast and spontaneous (Gao et al, 2012;Hao et al, 2023;Ma et al, 2015).…”

“…However, STX1 mutations of outward residues were inconclusive and were always accompanied by hydrophobic layer mutations (Jiao et al, 2018), which affect the assembly kinetics and energetics of the SNARE complex (Ma et al, 2015). Single molecule optical-tweezer studies have focused on the impact of regulatory molecules on the stability of assembly such as Munc18-1 (Ma et al, 2015;Jiao et al, 2018) and complexin (Hao et al, 2023), or on the intrinsic stability of the hydrophobic layers in the step-wise assembly of the SNARE complex (Gao et al, 2012;Ma et al, 2015;Zhang et al, 2017). Although the conserved hydrophobic layers in the SNARE domains of STX1A and STX2 (Figure 1) suggest unchanged zippering and intrinsic stability of the complex, further studies addressing the contribution of surface residues on the stability of the alpha-helical structure of the SNARE domain of STX1 (Li et al, 2022) or the stability of the SNARE complex should be conducted.…”

“…For that purpose, we used a comprehensive structure-function approach and compared the efficacy of STX1 to the closely-related isoform STX2 (Sherry et al, 2006), which is implicated in the secretion of various peripheral secretory cells (Abonyo et al, 2004;Dolai et al, 2018;Hutt et al, 2005). When expressed in STX1-null hippocampal mouse neurons (Vardar et al, 2016), STX2 synapses showed impaired vesicle priming, unclamping of spontaneous release, as well as inefficient and slowed Ca 2+evoked fusion, consistent with an impaired SNARE complex function (Hao et al, 2023;Jiao et al, 2018;Lai et al, 2017;Stepien et al, 2022;Voleti et al, 2020). Therefore, we proceeded with a structure-function chimeric analysis between STX1 and STX2, to isolate the precise involvement of the SNARE domain of STX1 in the regulatory mechanisms of release.…”

“…Although studies using STX1/STX3 chimeric constructs revealed that the SNARE motif of STX1 may carry structural features that regulate spontaneous release (Vardar et al, 2022), research on the SNARE domain of STX1 has been limited to the studies of its cognate interaction partners such as Munc18-1, Synaptotagmin-1 (SYT1) and Complexin (Hao et al, 2023;Jiao et al, 2018;Ma et al, 2015;Zhou et al, 2015Zhou et al, , 2017.…”

Key determinants of the dual clamp/activator function of Complexin

“…Notably, a comparable slowing in exocytosis timing was not observed in the absence of the entire CTD of CpxII (i. e. by deleting the amino acid positions 101-134, Makke et al, 2018), suggesting that this SytI- CpxII interaction site is specifically required for lifting the clamp signal imposed by Cpx’s downstream amphipathic helix. While CpxII aligns with its central helix in an antiparallel orientation on the SNARE complex, downstream protein regions of CpxII may provide sufficient structural flexibility for the far C-terminus to fold back in a parallel orientation on membrane-proximal layers of the partially assembled SNARE complex (Bowen et al, 2005; Makke et al, 2018, Hao et al, 2023). Under these conditions, the glutamate cluster of Cpx’s CTD may come into close proximity to the position of crucial amino acids of SNAP-25 (i.e.…”