Single-molecule FRET–derived model of the synaptotagmin 1–SNARE fusion complex

Synaptotagmin-1 functions as a Ca 2+ sensor in neurotransmitter release through its two C 2 domains (the C 2 A and C 2 B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble Nethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C 2 AB) was shown to bind simultaneously to two membranes via the Ca 2+ -binding loops at the top of both domains and basic residues at the bottom of the C 2 B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC 2 AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC 2 AB oligomers bound to each membrane via the Ca 2+ -binding loops, with no contact of the bottom of the C 2 B domain with the membranes. We now show that lnC 2 AB containing impurities indeed aggregates in solution, but properly purified lnC 2 AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC 2 AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C 2 B domain contacts the membrane in a sizeable population of molecules of both membranebound C 2 AB and membrane-bound lnC 2 AB. NMR data on nanodiscs show that a fraction of C 2 AB molecules bind to membranes with antiparallel orientations of the C 2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C 2 AB and lnC 2 AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.membrane bending | Ca2+ sensing | exocytosis | synaptic transmission N eurotransmitter release is a key event in interneuronal communication that is acutely triggered by Ca 2+ influx into a presynaptic terminal (1). The synaptic vesicle protein synaptotagmin-1 acts as a Ca 2+ sensor in fast release through the two C 2 domains that form most of its cytoplasmic region (the C 2 A and C 2 B domains) (2-5). This function is coupled to membrane fusion through the neuronal soluble N-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) proteins (6, 7), which bring the synaptic vesicle and plasma membranes together by forming SNARE complexes (2, 3). Synaptotagmin-1 function also depends on a tight interplay with complexins (8-10) and other key proteins of the release machinery (11, 12). The synaptotagmin-1 C 2 domains bind three or two Ca 2+ ions through loops at the top of β-sandwich structures (13-15) (Fig. 1A). These top loops also mediate Ca 2+ -dependent phospholipid binding (15-17), which is crucial for synaptotagmin-1 function (5, 18). Moreover, the synaptotagmin-1 cytoplasmic region clusters chromaffin granules and liposomes (19), and a fragment containing its two C 2 domains was shown by cryo-EM to bind simultaneously to two membranes, bringing them i...

Section: Resultssupporting

confidence: 61%

Prevalent mechanism of membrane bridging by synaptotagmin-1

Seven¹,

Brewer²,

Shi

et al. 2013

“…To obtain a distribution of fluorophore positions within an accessible volume, 100 independent simulated annealing dynamics runs were performed in the context of a fixed DNA duplex using CNS (45) as previously described (45,46). The average dye position was taken as the average position of the spiro atom (C24) in the lactam form of the dye and fixed rigidly to each of the sites in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Nucleoprotein architectures regulating the directionality of viral integration and excision

Seah

Warren

Tong

et al. 2014

The virally encoded site-specific recombinase Int collaborates with its accessory DNA bending proteins IHF, Xis, and Fis to assemble two distinct, very large, nucleoprotein complexes that carry out either integrative or excisive recombination along regulated and essentially unidirectional pathways. The core of each complex consists of a tetramer of Integrase protein (Int), which is a heterobivalent DNA binding protein that binds and bridges a core-type DNA site (where strand cleavage and ligation are executed), and a distal arm-type site, that is brought within range by one or more DNA bending proteins. The recent determination of the patterns of these Int bridges has made it possible to think realistically about the global architecture of the recombinogenic complexes. Here, we combined the previously determined Int bridging patterns with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein Holiday junction recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination. Our confidence in the basic features of these architectures is based on the redundancy and self-consistency of the underlying data from two very different experimental approaches to establish bridging interactions, a set of strategic intracomplex distances from FRET experiments, and the model's ability to explain key aspects of the integrative and excisive recombination pathways, such as topological changes, the mechanism of capturing attB, and the features of asymmetry and flexibility within the complexes.regulation of directionality | topology | recombinogenic architectures | molecular machines H igh-precision DNA transactions responsible for a variety of fundamental processes are typically promoted and modulated by large multiprotein machines that use cooperative interactions and involve DNA bending and/or wrapping. One well-studied example is the tightly regulated and highly directional site-specific recombination by which bacteriophage λ inserts and excises its DNA into and out of the Escherichia coli host chromosome, using the phage attP and the bacterial attB DNA sequences for integration and the resulting junction sequences, attL and attR, for excision. These reactions are catalyzed by the phage-encoded Integrase protein (Int), the founding member of the tyrosine recombinase family of site-specific recombinases (1). In addition to mediating integration and excision of viral genomes, members of this family function in a variety of other cellular processes including chromosome segregation, gene regulation, and conjugative transposition (2).Int has three well-characterized domains: an N-terminal DNA-binding domain (NTD), a central core-binding domain (CB), and a C-terminal catalytic domain (CAT). The CB and CAT domains (referred to here as the CTD) are together responsible for binding to the core-type DNA sequences where strand exchange and ligation t...

“…-independent binding of SYT to neuronal soluble N-ethylmaleimide-sensitive factor activating protein receptor on the plasma membrane (t-SNARE) [syntaxin/ synaptosomal-associated protein 25 (SNAP25)], most likely via its interaction with SNAP25 (17)(18)(19)(20)(21). This interaction is believed to position SYT on the prefusion SNARE complexes to trigger rapid exocytosis in response to Ca 2+ (20,22).…”

Section: +mentioning

confidence: 99%

Calcium sensitive ring-like oligomers formed by synaptotagmin

Bello¹,

Auclair²,

Wang³

et al. 2014

159

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear.Here we report an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18-43 nm, corresponding to 11-26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded. S ynaptotagmin-1 (SYT) is the calcium (Ca 2+ ) sensor that triggers synchronous release of neurotransmitters for synaptic transmission (1-4). It is a transmembrane protein, localized to the synaptic vesicles (1, 5), with tandem cytosolic C2 domains (C2A and C2B) that bind phospholipids in both a Ca 2+ -independent and a Ca 2+ -dependent manner (1, 6). The membrane-distal C2B domain interacts with acidic lipids such as phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to mediate efficient docking of the synaptic vesicles (7-10) before the influx of Ca 2+ ions. Recent studies (7,8,11,12) have located this calciumindependent membrane-binding site to a polybasic patch on the C2B domain (site I in Fig. 1A). The binding of Ca 2+ to the calcium-coordination pocket of the C2B domain (site II in Fig. 1A) triggers the insertion of flanking portions of site II into the plasma membrane, and this Ca 2+ -triggered membrane insertion is absolutely required for neurotransmitter release (13-16).The C2B domain also mediates the Ca 2+ -independent binding of SYT to neuronal soluble N-ethylmaleimide-sensitive factor activating protein receptor on the plasma membrane (t-SNARE) [syntaxin/ synaptosomal-associated protein 25 (SNAP25)], most likely via its interaction with SNAP25 (17-21). This interaction is believed to position SYT on the prefusion SNARE complexes to trigger rapid exocytosis in response to Ca 2+ (20,22). How the insertion of site II into the membrane bilayer is coupled to the completion of the SNARE assembly to release neurotransmitter is still unclear, although some key points have been established. In the prefusion state, the SNARE complexes...