Recently, Parkinson’s disease-associated α-synuclein (αS) has emerged as an important regulator for SNARE-dependent vesicle fusion. However, it is controversial if excessive accumulation of αS, even in the absence of aggregation, impairs neurotransmission. Here we use a single vesicle fusion assay with ms time resolution capable of dissecting the impact of αS on each step of membrane fusion. Unlike the previous results from various in vitro , cellular, and in vivo studies, we find that non-aggregated αS promotes vesicle merger even at exorbitant concentrations. The enhancement has been seen as much as 13 fold. Delving into the kinetics of the intermediate states for vesicle fusion reveals that αS stimulates vesicle docking without altering the dynamics of bilayer merger (lipid mixing). However, minute amounts of soluble aggregated species abolish SNARE-dependent bilayer merger completely. Thus, the results show that excessive accumulation of non-aggregated αS may not be toxic for neurotransmitter release.

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.

Botulinum toxins (BoNT) A and E block neurotransmitter release by specifically cleaving the C- terminal ends of SNAP-25, a plasma membrane SNARE protein. Here, we find that SNAP-25A and E, the cleavage products of BoNT A and E respectively, terminate membrane fusion via completely different mechanisms. Combined studies of single molecule FRET and single vesicle fusion assays reveal that SNAP-25E is incapable of supporting SNARE pairing and thus, vesicle docking. In contrast, SNAP-25A facilitates robust SNARE pairing and vesicle docking with somewhat reduced SNARE zippering, which leads to severe impairment of fusion pore opening. The EPR results show that the discrepancy between SNAP-25A and E might stem from the extent of the dynamic destabilization of the t-SNARE core at the N-terminal half which plays a pivotal role in nucleating SNARE complex formation. Thus, the results provide insights into the structure/dynamics-based mechanism by which BoNT A and E impair membrane fusion.

CRISPRs and CRISPR-associated (Cas) proteins provide a microbial adaptive immunity against phage infection. Cas2 is a universal Cas protein found in all types of CRISPR-Cas systems, and its role is implicated in the new spacer acquisition into CRISPR loci. In type I-C CRISPR-Cas systems, Cas2 proteins are metal-dependent double-stranded DNA (dsDNA) nucleases, and a pHdependent conformational transition has been proposed as prerequisite for the nuclease function. Here, we report the crystal structure of Xanthomonas albilineans Cas2 (XaCas2), and provide experimental evidence of a pHdependent conformational change during functional activation. XaCas2 crystallized at an acidic pH represented a catalytically inactive conformational state, and exhibited dsDNA nuclease activity only under neutral and basic conditions. In the crystal structure, significant conformational heterogeneity was evident in the putative hinge regions, suggesting that XaCas2 engages in hinge-bending conformational switching. The presence of a Trp residue in the hinge region enabled investigation of hinge dynamics by fluorescence spectroscopy. The pH dependence of the fluorescence intensity overlapped precisely with that of nuclease activity. Mutational analysis further confirmed that the conformational activation of the nuclease function was in play. Together, our results reveal strong correlations among the conformational states, catalytic activity, and hinge dynamics of XaCas2, and suggest that Cas2 may play a dual role as a dsDNA nuclease or as a central adaptor protein within the CRISPR-adaptation complex.

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