ANRV378-BI78-31 ARI 5 May 2009 14:58

Single-Molecule Studies of the Neuronal SNARE Fusion Machinery

Axel T. Brunger,1 Keith Weninger,2 Mark Bowen,3 and Steven Chu4

1The Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science, Stanford University, California 94305; email: [email protected] 2Department of Physics, North Carolina State University, Raleigh, North Carolina 27695-8202; email: [email protected] 3Department of Physiology and Biophysics, Stony Brook University Medical Center, Stony Brook, New York, 11794-8661; email: [email protected] 4Department of Energy, Washington, District of Columbia 20585; email: [email protected]

Annu. Rev. Biochem. 2009. 78:903–28 Key Words The Annual Review of Biochemistry is online at FRET, membrane fusion, neurotransmission, synaptic vesicle biochem.annualreviews.org

This article’s doi: Abstract 10.1146/annurev.biochem.77.070306.103621 SNAREs are essential components of the machinery for Ca2+-triggered

by 72.255.12.226 on 06/04/09. For personal use only. Copyright c 2009 by Annual Reviews. fusion of synaptic vesicles with the plasma membrane, resulting in neu- All rights reserved rotransmitter release into the synaptic cleft. Although much is known 0066-4154/09/0707-0903$20.00 about their biophysical and structural properties and their interactions with accessory proteins such as the Ca2+ sensor synaptotagmin, their

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org precise role in membrane fusion remains an enigma. Ensemble stud- ies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecu- lar insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applica- tions of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological mem- branes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.

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ten vesicles are stably docked at the active zone Contents awaiting an action potential (1–4). Exocytosis is triggered within approximately 0.2 ms of INTRODUCTION ...... 904 the Ca2+ influx that follows arrival of an action SINGLE-MOLECULE potential (5, 6). Although extremely rapid, the APPROACHES TO STUDY neurotransmitter release probability has a sig- MEMBRANE PROTEIN nificant heterogeneity in single synaptic release INTERACTIONS sites in hippocampal neurons (7). At most, one AND FUSION ...... 904 synaptic vesicle per synapse undergoes exocy- SNARES ...... 907 tosis upon depolarization in the central nervous SINGLE-MOLECULE STUDIES system (8). Thus, regulation of neurotransmit- OF THE SYNTAXIN·SNAP-25 ter release occurs at the level of synaptic vesicle BINARY COMPLEX ...... 908 release probability. There is also a background SINGLE-MOLECULE STUDIES rate of fusion of about one per minute per OF THE SYNTAXIN- synapse in the absence of action potentials. SYNAPTOBREVIN BINARY Synaptic vesicle fusion involves a highly con- COMPLEX ...... 910 served family of proteins termed SNAREs (sol- SINGLE-MOLECULE STUDIES uble N-ethyl maleimide sensitive factor attach- OF THE TERNARY SNARE ment protein receptors) (9–11). SNAREs are COMPLEX: PARALLEL OR directly linked to Ca2+ triggering of exocyto- ANTIPARALLEL? ...... 911 sis in conjunction with a Ca2+ sensor, such as EVIDENCE FOR A TRANS STATE synaptotagmin (12–14). Genetic rescue experi- OF THE TERNARY SNARE ments with mutants of synaptotagmin have now COMPLEX? ...... 912 firmly established that synaptotagmin is the MODELS OF MEMBRANE Ca2+ sensor for the synchronous component FUSION ...... 912 of synaptic exocytosis (15), but the mechanism RECONSTITUTION OF of action of the synaptotagmin·SNARE·mem- SNARE-MEDIATED brane fusion machinery remains a matter of MEMBRANE FUSION ...... 913 intense research (14, 16–23). Numerous other NUMBER OF SNARE COMPLEXES auxiliary proteins have been found to be essen- INVOLVED IN SYNAPTIC tial for Ca2+-dependent neurotransmitter re-

by 72.255.12.226 on 06/04/09. For personal use only. VESICLE FUSION ...... 916 lease, such as complexin, Munc18, and Munc13. SYNAPTOTAGMIN ...... 917 Thus, SNAREs form only one part, albeit the COMPLEXIN ...... 918 central part, of the complex system of synap- MUNC18 ...... 919 tic neurotransmission. In this review, we fo- MUNC13 ...... 920

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org cus on single-molecule studies of the neuronal SUMMARY AND FUTURE SNAREs, i.e., syntaxin, synaptobrevin, SNAP- ISSUES ...... 920 25, some of their binding partners and com- plexes, and their role in synaptic vesicle docking and fusion. INTRODUCTION Synaptic neurotransmitter release involves the Ca2+-triggered fusion of synaptic vesicles with SINGLE-MOLECULE SNARE: soluble the plasma membrane in the presynaptic ter- APPROACHES TO STUDY N-ethyl maleimide minal, releasing the neurotransmitter into the MEMBRANE PROTEIN sensitive factor synaptic cleft. Synaptic vesicles are recruited to INTERACTIONS AND FUSION attachment protein receptors the active zone in the presynaptic membrane One of the key advantages of single-molecule but do not readily fuse. Instead, an average of experiments is that they allow one to study the

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behavior of a single or countable number of Quantitative interpretation of FRET effi- molecules, or molecular complexes, and their ciencies in terms of absolute distances is not action on an individual molecule or particle straightforward. It requires careful measure- smFRET: single- such as a synaptic vesicle. This is especially use- ment of fluorophore dynamics to correct the molecule fluorescence ful for systems that show significant variability anisotropy and quantum yield terms in the resonance energy of individual molecular “trajectories” for bio- Forster¨ radius (29b). Interpreting quantitative transfer logical or chemical processes, such as protein smFRET values in terms of macromolecu- FRET: fluorescence folding, protein synthesis, or protein-assisted lar structure is a developing field (29c–29f). resonance energy membrane fusion. The ability to observe in- An ideal fluorophore attachment site has free, transfer dividual events therefore removes the require- isotropic rotation so there is a high degree of ment of synchronizing events in ensemble ex- uncertainty when correlating fluorophore sep- periments that monitor the average behavior of aration to protein structure. Recently, molec- many individual molecules or particles (often on ular dynamics simulations of protein-attached the order of Avogadro’s number), and it allows dyes show some promise in obtaining a con- one to perform statistical analysis of a popula- version function between FRET efficiency and tion of individual trajectories that would not be absolute FRET distance (29g). possible in bulk owing to ensemble averaging. The sample is illuminated with laser light There are several recent reviews that discuss using total internal reflection in order to re- applications of single-molecule techniques to strict illumination to the region near the surface biological systems (24–28). (the electric field intensity is restricted to a sur- Here, we briefly discuss the principles face layer with decay length ∼100 nm) and thus and focus primarily on single-molecule flu- has reduced background fluorescence. Two or orescence approaches such as the particu- more lasers emitting at different wavelengths lar experiment shown in Figure 1a. Protein- are used to study colocalization and FRET protein interactions are monitored between the between the fluorescent dyes. A similar setup membrane-bound syntaxin·SNAP-25 complex was used to study single-vesicle interactions and synaptobrevin, which is introduced above between protein-containing liposomes and de- the supported bilayer (29). Fluorescent labels posited bilayers while simultaneously monitor- are covalently attached at different positions in ing content mixing (30). the individual proteins. These dyes are planar An alternative to the supported bilayer ge- aromatic ring structures that range in size from ometry is to tether “acceptor” liposomes to an by 72.255.12.226 on 06/04/09. For personal use only. around 0.5 to 1.2 kDa and may be charged as inert surface and then to monitor the inter- well. Because any covalent modification of a action of these liposomes with “donor” lipo- macromolecule with such a dye can affect the somes in solution. This geometry has been used energtic and kinetic properties of the system, for single-vesicle studies (31). Both geometries

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org it is important to study this effect. In our ex- have advantages and disadvantages. The sup- periments, we therefore tested for the potential ported bilayers mimic the geometry of synap- influence of the labels by repeating them with tic vesicles docked to a relatively flat presy- different labeling combinations. Using avail- naptic membrane, but they tend to produce able crystal structures, we selected sites that limited protein mobility for at least a portion were surface exposed in the particular macro- of the reconstituted proteins owing to interac- molecules that we studied. As such, we usually tions of the proteins with the underlying surface found that the effect of the labels is small on (30, 32). By contrast, the tethered liposome ap- qualitative properties such as colocalization of proach suffers from nonspecific binding of the dyes or presence or absence of FRET efficiency. free liposomes with the surface, and the pro- However, labeling sites near binding inter- tein density in the tethered liposomes is more faces can affect protein folding and interactions difficult to control than in supported bilayers (29a). (33).

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a Laser

40 Å Lipid bilayer Syntaxin

SNAP-25 + Synaptobrevin

Cover slip

Microscope objective

bcDonor Acceptor

Cy3 Cy5 (donor) (acceptor)

SNAP-25 SN2

Colocalized spot Syntaxin

SNAP-25 SN1 by 72.255.12.226 on 06/04/09. For personal use only.

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org d Donor 1000 Acceptor 800 600 400 200 0

0 20406080100 Time (seconds)

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Another very useful approach to study in- molecules indicated frequent switching be- dividual biological processes is single-molecule tween both conformations with a relaxation atomic force microscopy often combined with time of 0.8 ms (43), whereas an ensemble NMR optical trapping or fluorescence measurements study primarily showed a closed conformation (34–37). A particularly important application in (44). the context of membrane fusion is the determi- Synaptobrevin has a short unstructured N- nation of the force and formation enthalpy be- terminal region that is adjacent to the SNARE tween individual interacting proteins (38, 39). core domain. The entire cytoplasmic domain of isolated synaptobrevin is unfolded in solution (45, 46). SNARES Isolated SNAP-25 is an unfolded pro- For a comprehensive review of the chemi- tein, consisting of two SNARE core domains cal, biophysical, and structural properties of (termed SN1 and SN2, respectively) (47) and SNARE proteins see Reference 40. Both syn- a linker that includes four palmitoylated cys- taxin and synaptobrevin have a C-terminal teine residues. These palmitoylated cysteines transmembrane domain and an adjacent do- are probably important for membrane associ- main that is involved in interacting with ation and exocytosis, although mutation of all SNAREs; we will refer to these domains as four cysteines in chromaffin cells has surpris- SNARE core domains (Figure 2a). ingly mild effects on exocytosis and electro- Syntaxin has a folded N-terminal domain physiological parameters (48). consisting of a three-helix bundle that is con- The SNARE core domains exhibit a nected to the partially unfolded SNARE core plethora of configurational, conformational, domain by a short linker (41). Syntaxin switches and oligomeric states (40). These differ- between closed and open conformations. In the ent states allow SNAREs to interact with closed conformation, the N-terminal domain their matching SNARE partners or auxil- interacts with part of the SNARE core domain, iary proteins, sometimes in a mutually ex- preventing interactions with other SNAREs, clusive fashion. SNAREs undergo progressive whereas in the open conformation the syn- disorder-to-order transitions upon interactions taxin’s SNARE core domain is free to interact with binding partners, culminating with the with synaptobrevin and SNAP-25 (42). Fluo- fully folded ternary SNARE complex, con- rescence correlation spectroscopy of individual sisting of an elongated four-helix bundle (49) by 72.255.12.226 on 06/04/09. For personal use only.

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 1

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org A typical single-molecule fluorescence resonance energy transfer (smFRET) experiment. (a) Shown is the experimental setup for the smFRET experiments of the binary complex (29). Briefly, dual dye (donor/acceptor)-labeled binary complex (syntaxin·SNAP-25) was reconstituted into a supported bilayer. Evanescent wave illumination was performed through total internal reflection. Laser light was chosen at two wavelengths to monitor donor and acceptor fluorescence. Synaptobrevin or other factors were injected, and binding to binary complex was monitored by a change in FRET from the dual-labeled syntaxin·SNAP-25. A similar setup was used for docking and fusion experiments wherein synaptobrevin was reconstituted into liposomes that contained the soluble dye calcein that served as a content mixing indicator (30). (b) Donor ( green) and acceptor (red ) dye labeling positions in the dual-labeled SNAP-25 molecule that forms the binary complex with syntaxin. Shown is a model of the three-helix bundle complex consisting of the SNARE core domains of syntaxin (orange), SNAP-25 SN1 ( green), and SNAP-25 SN2 (red ). (c) Fields of view (50 × 100 μm) of donor (left) and acceptor (right) fluorescence arising from a dual-labeled SNAP-25 molecule in the syntaxin·SNAP-25 binary complex. (d ) Selected time trace of the donor and acceptor fluorescence arising from a colocalized spot (similar to the marked one in panel c).

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(Figure 2b). These SNARE folding and as- SINGLE-MOLECULE STUDIES sembly events are intimately coupled to their OF THE SYNTAXIN·SNAP-25 function in synaptic vesicle docking and fusion. BINARY COMPLEX Below, we discuss three of the assembly states The binary interaction between syntaxin and that have been studied with single-molecule SNAP-25 is generally considered to be the first methods. intermediate in the path to SNARE complex

a SNARE core domain N-terminal domain

Syntaxin C TM N

Binding to munc18

SNARE core domain (SN1) SNARE core domain (SN2)

SNAP-25 N C Palmitoylation sites

SNARE core domain

Synaptobrevin C TM N

b Synaptobrevin SNAP-25 Syntaxin

C

N by 72.255.12.226 on 06/04/09. For personal use only.

c Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org

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formation. This complex has also been called single-molecule studies (25, 53, 54). The dy- the target (t)-SNARE or acceptor complex be- namic changes of the binary complex included cause it forms the binding site on the plasma both frame-by-frame variability in smFRET ef- membrane for synaptic-vesicle localized synap- ficiency as well as stochastic switching between tobrevin (50). The binding of synaptobrevin stable intermediate and high smFRET states to the binary syntaxin·SNAP-25 complex thus (Figure 3a). These large changes in smFRET serves to stably dock synaptic vesicles near the efficiency indicate conformational transitions active zone of the synapse. within the binary complex involving large Syntaxin and SNAP-25 readily form a sta- (>5 nm) movements. These results are consis- ble “dead-end” 2:1 complex in vitro, where tent with earlier studies using circular dichro- a second syntaxin SNARE domain takes the ism that indicated some induced structure upon usual position of the synaptobrevin helix in binary complex formation but much less than the SNARE complex (47, 50). The 2:1 state what one would expect if it were to form a helix forms a stable four-helix bundle (51, 52). The bundle (47). prevalence of this 2:1 species during solution An approximate model of the configurations assembly of SNARE proteins made it impos- of the 1:1 binary complex is shown in Figure 3b sible to study the 1:1 binary complex by en- (lower panels). Prior to synaptobrevin binding, semble methods. Using an extremely low con- an equilibrium exists between a configuration centration of syntaxin (about 100 molecules consisting of the SNARE domains of syntaxin in an area of 50 × 50 μm) embedded in a and SNAP-25 (SX-SN1-SN2) and two con- supported bilayer, it was possible for the figurations involving the SNARE domain of first time to study the structure and dynam- syntaxin and either one of the two SNAP-25 ics of the neuronal binary complex in its SNARE domains with the other SNAP-25 do- 1:1 state with single-molecule methods (29) main dissociated (SX-SN1 and SX-SN2). This (Figure 1b). model assumes that the syntaxin N-terminal The conformation of the 1:1 binary com- domain is in the open conformation (41) be- plex is more variable than one would expect cause if it were in the closed conformation, it if it formed a stable three-helix bundle. With would prevent interactions between syntaxin a labeling site pair in syntaxin and SN1, and and SNAP-25, at least in the N-terminal por- a dual labeling site pair in SNAP-25, dynamic tion of the SNARE domains. changes in smFRET efficiency levels were ob- Upon addition of synaptobrevin, the equi- by 72.255.12.226 on 06/04/09. For personal use only. served. The discovery of fluctuating molecular librium shifts toward the three-helix bun- structural states is a common feature of many dle (SX-SN1-SN2) configuration (Figure 3b). ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 2 Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org The neuronal SNARE complex. (a) Primary structure diagram for syntaxin (red ), SNAP-25 ( green), and synaptobrevin (blue). The experiments referenced in this review refer to the following isoforms: syntaxin 1A, synoptobrevin II, and SNAP-25A which we simply refer to as syntaxin, synaptobrevin, and SNAP-25. The SNARE core domains are defined through the 16 layers as found in the crystal structure of the neuronal SNARE complex (49). (b) X-ray crystal structure of the core of the neuronal SNARE complex consisting of synaptobrevin (blue), SNAP-25 ( green), and syntaxin (red ) [Protein Data Bank (PDB) 1SFC] (49). This structure represents the fully folded postfusion state of the complex, also referred to as the cis state. The N- and C-terminal sides of the core complex are indicated. (c) Model of the trans state of two SNARE complexes that dock a liposome to a supported bilayer in vitro. This model was obtained by modifying the membrane-proximal end of the crystal structure of the neuronal SNARE complex to allow the transmembrane domains to enter into the juxtaposed membranes. The transmembrane domains were assumed to be helical (156). The connecting regions between the transmembrane domains and the core complex are likely flexible. Two SNARE complexes are shown; the exact number is unknown, but 1–2 SNARE complexes suffice to dock liposomes (30). Abbreviation: TM, transmembrane domain.

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a 1000 Donor Acceptor 500 Intensity

0 0 5 10 15 20 25 30 Time (seconds)

b + Synaptobrevin 0.020 0.030 0.015 0.020 0.010 0.010 0.005005 Probability Probability 0.000000 0.000 00.0.0 1.0 0.0 1.0 FRET FRET

SN1•SN2•syntaxin SN1•SN2•syntaxin• SN2N2•syntaxinsyntaxin syntaxinsyntaxi synaptobrevin

SN1 SN2

Figure 3 Single-molecule fluorescence resonanace energy transfer (smFRET) studies of the binary complex. (a) Selected time trace of the donor and acceptor fluorescence of the binary complex (syntaxin·SNAP-25, see by 72.255.12.226 on 06/04/09. For personal use only. Figure 1) arising from a colocalized spot (similar to the marked one in panel 1c) (29). Note the switching between two different FRET states as indicated by the correlated changes in donor and acceptor fluorescence. (b) FRET distributions of donor and acceptor dyes on the binary complex before (left panel ) and after addition of synaptobrevin (right panel ). Note that the intermediate FRET states have disappeared after addition of synaptobrevin (29). Below the FRET distributions, models of the binary complex

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org conformations are shown. Before the addition of synaptobrevin, the binary complex exhibits three configurations: only the SN2 SNAP-25 domain bound to syntaxin (SX-SN2), only the SN1 domain of SNAP-25 bound to syntaxin (SX-SN1), or both SNAP-25 SNARE domains bound to the syntaxin SNARE domain (SX-SN1-SN2). Upon addition of synaptobrevin (right) or accessory proteins, these configurations collapse into the SX-SN1-SN2 configuration.

This effect occurs on a fast scale, orders of SINGLE-MOLECULE STUDIES magnitude faster than the rate constants for OF THE SYNTAXIN- synaptobrevin binding to the dead-end 2:1 bi- SYNAPTOBREVIN BINARY nary complex. Addition of synaptobrevin to the COMPLEX 1:1 binary complex also completely eliminated A weak interaction exists between syntaxin dynamic variability in smFRET efficiency lev- and synaptobrevin as corroborated by a small els and stochastic switching (29). increase in circular dichroism helicity and

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proton NMR chemical shifts upon binding the assembly of the SNARE complex, so many (45, 46). The observed structural changes are biophysical and structural studies were carried much smaller than those for the formation out with recombinant proteins in the absence of of the syntaxin·SNAP-25 binary complex. the transmembrane domains and palmitoylated Still, single-molecule experiments showed that cysteines. These truncated SNARE constructs docking and fusion of liposomes to deposited readily form a variety of helical bundles of vary- bilayers can be accomplished with just synap- ing composition and configuration (40, 45, 47). tobrevin and syntaxin in different membranes, Ternary SNARE complex formation in- despite the absence of SNAP-25 (30, 55) as was duces major disorder-to-order transitions in hinted previously by ensemble experiments SNARE core domains (45), in addition to those (56). Furthermore, single-molecule atomic observed in the binary complexes discussed force microscopy studies have revealed that the above. The assembled ternary SNARE complex activation free energy for the formation of the consists of a parallel four-helix bundle (49, 62) syntaxin-synaptobrevin complex is comparable (Figure 2b). The core of the four-helix bundle to that of leucine zippers (38, 39). In compar- of the SNARE complex consists of 16 primarily ison, an ensemble study using a surface force hydrophobic layers formed by interacting side apparatus produced an estimate for the for- chains from each of the four α-helices. At the mation enthalpy of the trans ternary SNARE center of the core complex, a conserved ionic complex between two separate membranes layer is present, consisting of an arginine and

(35 kB T) (57). Bulk liposome and single- three glutamine residues contributed from each molecule force measurements revealed that the of the four α-helices. This ionic layer is sealed ternary SNARE complex is much more stable off against solvent by adjacent hydrophobic lay- than the syntaxin-synaptobrevin complex (38, ers, but it contains a buried water molecule 58). Furthermore, in order to obtain a high (62). Structures of the neuronal, endosomal, FRET efficiency signal between syntaxin and and yeast SNARE complexes are very similar, synaptobrevin labeling sites, SNAP-25 had indicating a high degree of evolutionary con- to be added, suggesting that the syntaxin- servation (49, 62–65). synaptobrevin complex is not a helical bundle. smFRET experiments revealed a surprising Interestingly, studies of SNAP-25 knockout characteristic of SNARE complex assembly: A mice revealed a phenotype where vesicle dock- mixture of parallel as well as antiparallel config- ing and stimulus-independent (spontaneous) urations was found between the SNARE core by 72.255.12.226 on 06/04/09. For personal use only. fusion persisted, although Ca2+-triggered domains of syntaxin and synaptobrevin and to release was abolished (59, 60). Thus, an a lesser degree between those of syntaxin and explanation of these in vivo experiments could SNAP-25; we confirmed that this result is not be that even in the absence of SNAP-25 and an artifact by the introduction of covalent dye

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org its homologs, the weak interaction between labels by using different labeling pair combi- synaptobrevin and syntaxin can promote nations to probe for parallel and antiparallel synaptic vesicle docking and fusion, although configurations (66). The subpopulation with no Ca2+ triggering is possible. the parallel four-helix bundle configuration was greatly enriched by an additional purification step in the presence of denaturant, indicating SINGLE-MOLECULE STUDIES OF that the parallel configuration is the energet- THE TERNARY SNARE COMPLEX: ically most favorable state. This explains why PARALLEL OR ANTIPARALLEL? only the parallel configuration was found in The ternary SNARE complex, consisting of the crystal structures of the SNARE complex syntaxin, synaptobrevin, and SNAP-25, can be because extensive purification, including the readily isolated from neuronal cell extracts (61). temporary use of denaturants, was performed The membrane anchors are not required for for crystallization. Interconversion between the

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parallel and antiparallel configurations was not The zipper model suggests that SNAREs observed on the hour timescale. The discov- exist in a partially assembled state prior to the ery of mixtures of parallel and antiparallel con- arrival of the Ca2+ signal (76–79) (Figure 2c). figurations now explains previously puzzling In this state, the SNAREs would still be results of smaller than expected mean FRET susceptible to cleavage by a subset of clostridial between parallel reporting sites in ensem- neurotoxins (80–82). Furthermore, folding- ble studies of SNARE complexes (67) and specific SNARE antibodies affect some but also those of nonzero mean FRET in ensem- not all components of the electrophysiological ble studies of syntaxin C-terminally fused to response (77). However, these experiments blue fluorescent protein and synaptobrevin N- did not show a direct interaction between the terminally fused to green fluorescent protein in membrane distal regions of the ternary SNARE 20S complexes (68). complex while the transmembrane domains To investigate if such mixtures exist in the are in opposite membranes. An important first membrane environment of docked liposomes, step in this direction is an smFRET study smFRET efficiency studies were carried out where SNAREs promoted liposome docking to determine the configuration of the SNARE to supported bilayers and high FRET was complexes involved in docking liposomes to de- observed between labels at the membrane distal posited bilayers (30). The antiparallel popu- ends of syntaxin and synaptobrevin (table 1 in lation was approximately one-fifth the size of Reference 30). It is unlikely that the observed the parallel population. Thus, liposome dock- FRET signal occurred after liposome fusion ing to a supported bilayer favors the assembly because fusion was a rare event in these of SNAREs into the parallel configuration as experiments, although this question deserves seen in the crystal structure (Figure 2c). further study.

EVIDENCE FOR A TRANS STATE OF THE TERNARY SNARE MODELS OF MEMBRANE FUSION COMPLEX? Models of membrane fusion have been largely Numerous biochemical, structural, and genetic restricted to a phenomenological description of studies have lent support to the zipper model, elastic materials representing monolayers (for a which posits that SNARE complex assembly review, see Reference 83). These models make begins in trans (i.e., residing on opposite mem- the assumptions that the elastic moduli of the by 72.255.12.226 on 06/04/09. For personal use only. branes), with separate SNAREs on the donor monolayers are uniform and that their surfaces and acceptor membranes, and ends with forma- vary smoothly. A commonly used model has tion of a cis complex (i.e., residing on the same emerged from these largely theoretical stud- membrane). Directional folding of SNAREs ies: Fusion starts with a stalk state and proceeds

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org into a highly stable parallel four-helix bundle through one or more hemifusion diaphragm in- is then thought to drive membrane juxtaposi- termediate states, leading to the formation of a tion and, eventually, fusion (69–73) (Figure 2c). fusion pore (Figure 4, pathway 1). A hemifu- Single-molecule atomic force microscopy has sion diaphragm state is characterized by outer shown that the assembly of the ternary SNARE leaflet mixing with no inner leaflet mixing (84, complex, in principle, provides sufficient en- 85). However, the only intermediate state that ergy to drive membrane juxtaposition and fu- has actually been observed experimentally is the sion (39). In addition to juxtaposing mem- stalk state (86). Furthermore, this dogma of a branes through SNARE complex formation, stalk-diaphragm-fusion pathway has been chal- the SNARE transmembrane domains may also lenged by computer simulations that demon- participate in the later stages of fusion (74), e.g., strate the possibility of direct transitions from by stabilizing or destabilizing fusion intermedi- stalk to fusion pore using Monte Carlo (87) ate states (31, 75). and coarse-grain ensemble molecular dynamics

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Hemifusion diaphragm synaptobrevin were reconstituted into sepa- rate liposomes, and lipid mixing was observed on a minute timescale. In the literature, the 1 1 PEG: polyethylene lipid-mixing events are interpreted as “fusion,” glycol 1,2 2 even though these experiments typically do not probe content mixing. We therefore refer to the events observed in these experiments as “lipid Docking Stalk Fusion mixing/fusion” because the experimental data Figure 4 cannot resolve this ambiguity. We refer to this Alternative pathways for liposome fusion. Computer assay and its subsequent variants as “bulk lipo- simulations of liposome-membrane fusion (88). some assay.” Pathway 1 shows the canonical progression from an The bulk liposome assay was an important unfused starting state through a stalk intermediate and a hemifused diaphragm intermediate to the fully advance as it demonstrated that SNAREs are fused state. Pathway 2 shows an alternative reaction capable of docking and lipid mixing/fusion of pathway observed in ensemble molecular dynamics liposomes in vitro. However, it suffers from sev- simulations (88): rapid fusion from the stalk eral deficiencies: Only lipid mixing is observed, intermediate to the fully fused state. All renderings the protein-to-lipid ratio can be too high, and are of snapshots from observed reaction trajectories; lipids are colored to distinguish the outer (red and no individual fusion events can be observed. It is green) and inner ( gold and blue) leaflets of each essential to measure both lipid mixing and con- vesicle. Explicit water was present in all simulations tent mixing to obtain a detailed kinetic model but not rendered. of the fusion process (84, 85), to distinguish fu- sion events from liposome instability and leak- algorithms (88) (Figure 4, pathway 2). The ing (58), to account for lipid mixing arising relative importance of these two pathways de- from lipid “flip-flop” without fusion (69), and to pends on the lipid composition; ∼90% of the account for possible spontaneous lipid dye fusion events proceed via pathway 2 for 2:1 transfer between adjacent membranes (91). palmitoyloleoyl phosphatidylcholine: palmi- Historically, only lipid mixing was probed in toyloleoyl phosphatidylethanolamine (POPE) bulk liposome experiments except in one case lipid mixtures, whereas most fusion events pro- where duplex formation of oliognucleotides was ceed via pathway 1 for liposomes containing used to assay content mixing (92). pure POPE (89). In experimental support of The protein density in the early bulk lipo- by 72.255.12.226 on 06/04/09. For personal use only. this alternative pathway, polyethylene glycol some assays is now considered generally too (PEG)-mediated liposome fusion could only be high to be physiologically relevant [e.g., 750 modeled with direct transitions between stalk synaptobrevins per 45-nm vesicles (90) com- and fusion pore in addition to the pathway pared to the physiological density of roughly via a hemifusion diaphragm intermediate (85).

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org 70 synaptobrevins per 50-nm synaptic vesicle Touncover these underlying intermediates and (93)]. In a more recent experiment, physiolog- transitions, it was essential to simultaneously ical average protein-to-lipid ratios were used monitor lipid mixing, content mixing, and leak- (94). SNARE complex formation was assayed iness of the fusing liposomes (58). with C-terminal FRET labels, and lipid mix- ing was observed with liposome dye dequench- RECONSTITUTION OF ing. Both signals were highly correlated, and SNARE-MEDIATED application of botulinum neurotoxin (serotype MEMBRANE FUSION E) or tetanus neurotoxin disrupted both pro- Rothman and coworkers (90) developed the cesses. No content mixing indicator was used, first in vitro assay to study SNARE-mediated so fusion was inferred indirectly by an increase vesicle docking and fusion. Coexpressed of liposome size as observed by electron mi- acceptor complex (syntaxin·SNAP-25) and croscopy. However, the ensemble timescale of

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the observed lipid-mixing/fusion events was The importance of membrane anchors was still on the minute timescale. Furthermore, studied by replacement of the SNARE trans- even though the average concentration of re- membrane domains with covalently attached constituted proteins was physiological in these lipids (102, 103). Replacing either syntaxin or and some more recent studies, there is still a synaptobrevin transmembrane domains with concern because there can be a large variation covalent phospholipid anchors prevented lipid of reconstituted protein density in individual li- mixing/fusion but still allowed docking of li- posomes depending on the reconstitution pro- posomes. The membrane-proximal region of tocol (33), resulting in a subpopulation of un- synaptobrevin could be modified by helix- stable liposomes, which could give raise to false breaking proline residues with little effect on fusion positives and consequently results that the assay, and insertion of a flexible linker had do not agree with physiological data. a moderate effect, with increasing influence for The lipid-mixing/fusion events observed in longer linkers (104). the bulk liposome experiments are often infre- A recent bulk liposome study of SNARE- quent or slow, resulting in rounds of fusion that mediated vacuolar fusion showed that it re- occur over a minute timescale, orders of mag- quires physiological lipid compositions and nitude slower than individual fusion events that protein-to-lipid ratios to obtain a reconstituted occur in synaptic neurotransmission. For com- system that faithfully reproduces the critical parison, in goldfish bipolar neurons, activation dependence on other factors in addition to of Ca2+ current drives secretion at a rate of SNAREs for vacuolar fusion (105). Further- 10,000 synaptic vesicles per second (95), and more, careful controls were performed to rule in calyx of Held nerve terminals three to five out instability or leakiness of the liposomes. It readily releasable synaptic vesicles fuse within is disappointing that such carefully designed <1 ms at each active zone upon Ca2+ triggering bulk liposome experiments are rare for neu- (96). ronal SNAREs. Despite the deficiencies of the bulk liposome Single-molecule microscopy and spec- assay, a number of results have been obtained troscopy can overcome some of the key that are consistent with structural knowledge deficiencies of the bulk liposome assay because of SNAREs and their complexes. For exam- individual events can be observed rather than ple, an increase in the number of rounds of an ensemble average. Using multiple dye lipid mixing/fusion within a time interval re- reporters, content and lipid mixing and protein by 72.255.12.226 on 06/04/09. For personal use only. sulted from removal of the N-terminal domain localization can be monitored simultaneously. of syntaxin (73). Lipid mixing/fusion was sen- In principle, smFRET studies should also allow sitive to particular SNARE pairings (97–99), the study of single protein-protein interactions presumably caused by the kinetic differences of conditional on the occurrence of a fusion

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org the assembly of these pairings despite very sim- reaction, although there are many challenges ilar thermal stabilities (100, 101). Finally, a dra- to overcome to perform this experiment. matic acceleration of the lipid-mixing/fusion With synaptobrevin reconstituted in liposomes rate was obtained by the addition of a fragment and syntaxin·SNAP-25 in deposited bilayers, of synapotobrevin that reduces the prevalence efficient SNARE-dependent docking was ob- of the dead-end 2:1 syntaxin·SNAP-25 com- served (30). Infrequent fusion events were also plex (50). This work also showed that coexpres- observed using the content mixing reporter cal- sion of syntaxin and SNAP-25, as opposed to cein. The fusion events appeared to be triggered formation of the binary complex by addition by the start of the laser illumination. The most of SNAP-25 to membrane-reconstituted syn- likely explanation of this effect is that the illumi- taxin, is not essential for the efficiency of the nation of the calcein dye caused heating, which lipid-mixing/fusion rates, which is contrary to provided the energy to initiate fusion. Another what had been suggested in earlier works (90). explanation might be photobleaching-induced

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radical formation. Clearly, illumination- Time (seconds) induced heating or radical formation is not 5.9 6.06.1 6.2 6.3 6.4 restricted to single-molecule experiments, but could also occur in ensemble experiments. It is Synaptobrevin possible that the use of a supported bilayer may have affected the kinetics of the system, requir- ing heat to trigger fusion. Further ensemble Content and single-molecule studies are required to investigate these effects because they might Figure 5 affect the observed energetics and kinetics. Individual fusion event observed by single-particle studies. Liposomes The timescale of these individual fusion containing the content dye calcein were reconstituted with dye-labeled reactions, once initiated, was faster than the synaptobrevin molecules and then introduced above palmitoyloleoyl time resolution of the camera employed in phosphatidylcholine/palmitoyloleoyl phosphatidylserine bilayers with these experiments, indicating that fusion is reconstituted syntaxin in complex with SNAP-25 (30). The images represent a single 11 μmby11μm patch of membrane with docked liposomes observed in intrinsically faster than 100 ms (Figure 5). A two different spectral ranges to detect the content dye and synaptobrevin dye surprising result was found when SNAP-25 fluorescence. Two liposomes are docked to the bilayer in the field of view as was left out: Docking and thermally induced indicated by the synaptobrevin dyes. A single-fusion event occurs at6sas fusion still occurred. The previously men- indicated by the sudden appearance of a bright content dye signal. The increase tioned weak interaction between syntaxin and of content dye fluorescence is due to dequenching. Fusion proceeds faster than the time resolution of the camera used in this experiment; in other words, the synaptobrevin is thus sufficient for constitutive fusion reaction is faster than 100 ms. docking and fusion in vitro. Clearly, for a fully + functional Ca2 -sensitive system, SNAP-25 in SNAREs incapable of promoting lipid mix- is required because it interacts with synato- ing, which should serve as a cautionary tale for tagmin, and manipulations of SNAP-25 (e.g., everyone working with these proteins. by the action of clostridial neurotoxins) affect The experiments by Bowen et al. (30) and neurotransmission (13). Liu et al. (55) agree on three aspects. First, A similar liposome/bilayer topology was individual events [content mixing observed by used in the single-vesicle study by Liu et al. (55, Bowen et al. (30) or lipid mixing observed 106), although the bilayer preparation and lipid by Liu et al. (55)] are fast (faster than 100 compositions were quite different, the lipid and 25 ms, respectively). Second, Ca2+ has density was lower than that of Bowen et al. (30), no effect on this SNARE-only system. Third, by 72.255.12.226 on 06/04/09. For personal use only. and only lipid mixing was monitored. SNARE- SNAP-25 is not required for SNARE-mediated dependent docking of liposomes was observed docking and fusion. There are also differences at the start of the experiment. Lipid mixing between the two experiments. Only thermally was monitored by dequenching of fluorescence induced fusion events were observed by Bowen

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org from lipid dyes incorporated into the lipo- et al. (30), in contrast to the burst of lipid- somes. Fusion events were thus inferred from mixing events at the start of the experiment dequenching and subsequent decay of the lipid by Liu and coworkers (55). It should be noted, dyes owing to diffusion within the deposited bi- however, that the experimental setup by Bowen layer. After initiation of the experiment, 65% et al. (30) prevented measurement of events at of the docked vesicles exhibited lipid mixing the early stage because the system was initially within less than 25 ms after docking. When equilibrated to establish single-molecule con- the concentration of syntaxin·SNAP-25 was in- ditions and to avoid nonspecific liposome bind- creased about 100-fold, only few events were ing. It is therefore possible that initial events observed. This result was explained by the for- might have also occurred in the experiments by mation of large aggregates at these higher con- Bowen and coworkers (30), albeit unobservable. centrations of SNAREs as revealed by atomic In summary, these studies (30, 55) both produce force microscopy (55). Presumably, this results individual events on the millisecond timescale,

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while they differ in terms what is being ob- to differences in the experimental conditions. served: Liu et al. (55) observed lipid-mixing Reconstituted syntaxin·SNAP-25 binary com- events that occurred spontaneously, whereas plexes partitioned into a mobile and a fixed DOPS: dioleoyl phosphatidylserine thermally observed fusion events were observed fraction in deposited bilayers; the mobile frac- by Bowen et al. (30). tion was significantly reduced in the presence The notion that trans SNARE complexes of negatively charged lipids, such as palmitoy- alone are insufficient for fusion but require a loleoyl phosphatidylserine or PIP2 (32). Sim- trigger, such as the Ca2+ sensor synaptotag- ilarly, Bowen et al. (30) observed a significant min or thermal heating (in vitro), to promote fraction of immobile reconstituted syntaxin efficient fusion (30) is supported by bulk lipo- molecules in deposited bilayers that were ob- some experiments of SNARE-containing vesi- tained by liposome condensation on the quartz cles that are brought into contact by a low surface; some of the reduced mobility may be concentration of PEG (58). In this experimen- related to interactions between syntaxin trans- tal setup, the neuronal SNARE complex alone membrane domains involving upside-down did not trigger fusion as determined by a con- syntaxin molecules whose cytoplasmic domain tent mixing indicator. SNAREs did enhance interacts with the surface (30). PEG-triggered fusion by favoring formation of the stalk intermediate. These studies also revealed that high protein-to-lipid ratios for NUMBER OF SNARE COMPLEXES syntaxin (>1:500), and to a lesser degree for INVOLVED IN SYNAPTIC synaptobrevin (>1:100), cause liposomes to VESICLE FUSION loose integrity, calling into question bulk lipo- How many SNARE complexes are involved some studies carried out at high protein-to-lipid in docking and fusion of synaptic vesicles and ratios. whether these SNARE complexes interact with Single-vesicle studies revealed appar- each other are still unknown. There are some ent hemifusion states induced by neuronal estimates that generally suggest a low number SNAREs in the presence of 20% dioleoyl phos- of SNARE complexes involved in these pro- phatidylethanolamine but not in the presence cesses. One such study involved a permeabi- of 15% DOPS (dioleoyl phosphatidylserine) lized PC12 cell system (108). Upon injection (106). Apparent intermediate hemifusion states of the cytosolic domain of synaptobrevin, exo- were also induced by the yeast homologs of cytosis was inhibited. The increased inhibition by 72.255.12.226 on 06/04/09. For personal use only. neuronal SNAREs (31), even in the absence of of fusion with increasing synaptobrevin con- phosphatidylethanolamine. However, because centration was best fit to a function involving no protein and content mixing reporters were three SNARE complexes. A model based on used, it is not possible to directly correlate mutagenesis studies of syntaxin transmembrane

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org the state of the membrane (e.g., hemifusion segments suggested five to eight complexes in- diaphragm) with formation of fusion pores and volved in formation of a putative fusion pore protein conformational changes. (74). SNARE complexes obtained from brain Additional insights into SNARE-membrane extracts as well as from purified components interactions have been reported by using the appeared as star-shaped oligomers in negative- Langmuir-Blodgett trough to obtain single pla- stain electron micrographs (109). However, a nar phospholipid bilayers supported on PEG different atomic force microscopy experiment cushions (107). Reconstituted synaptobrevin revealed that as little as one SNARE complex exchanged between the supported bilayer and can dock a liposome to a target membrane (38). vesicles in solution (i.e., unbound) (107). How- A previous study using single-molecule fluores- ever, little protein transfer was observed in the cence microscopy came to a similar conclusion: single-molecule experiments by Bowen et al. As little as one complex per liposome is suffi- (30), although this difference could be due cient for docking to supported bilayers (30).

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SYNAPTOTAGMIN Sutton, S. Chu, & A.T. Brunger, submitted) are + also available. The relative positions between Synaptotagmin is the Ca2 sensor for the syn- the C2 domains are very different in these struc- chronous component of synaptic exocytosis tures and thus indicate a high flexibility of the (15). Synaptotagmin occurs in both neuronal linker connecting the two C2 domains. Single- and nonneuronal cells, and there are at least molecule studies of the C2A-C2B fragment of 16 different isoforms in different subcellular synaptotagmins I and III revealed highly dy- localizations in the brain (109a). For example, namic interactions between the two C2 do- synaptotagmins I and II are localized on synap- mains and indicated that interactions with a tic vesicles (110); these isoforms are often col- SNARE complex and/or liposomes stabilize lectively referred to as simply synaptotagmin. In one of the conformations (M. Vrljic, P. Strop, contrast, synaptotagmin III is primarily found J. Ernst, R.B. Sutton, S. Chu, & A.T. Brunger, in the plasma membrane. Synaptotagmins are submitted; U.B. Choi, P. Strop, M. Vrljic, S. composed of a short intravesicular (luminal) Chu, A.T.Brunger, & K. Weninger, submitted). N-terminal region, a single-membrane span- No high-resolution structures are available at ning domain, a lysine- and arginine-rich jux- this time of complexes between synaptotag- tamembrane region, and two homologous C2 min, SNAREs, phospholipids, or other binding domains, termed C2A and C2B. A fragment partners. consisting of both C2 domains interacts in a + A direct interaction between synaptotag- Ca2 -dependent manner with acidic lipids and + min and the syntaxin SNAP-25 binary com- in both a Ca2 -dependent and -independent · plex was revealed by smFRET experiments manner with SNAP-25 and syntaxin, the bi- (29). Synaptotagmin stabilizes the helix bun- nary complex, and the ternary SNARE com- + dle configuration of the binary complex to the plex (111–113). The Ca2 -dependent interac- same extent as synaptobrevin, even in the ab- tions with the SNARE complexes are very sence of Ca2+. smFRET between an acceptor- salt dependent; at 200 mM salt concentra- + labeled binary complex and donor-labeled tion, the Ca2 dependence of these interactions synaptotagmin confirmed the molecular inter- disappears (14). action. The observed stabilization of the binary It should be noted that resequencing of the t-SNARE complex might provide an expla- synaptotagmin I cDNA revealed an accidental nation for the increase in lipid mixing/fusion mutation (Gly374Asp) in the C2B domain of in bulk liposome assays upon incubation of

by 72.255.12.226 on 06/04/09. For personal use only. the original clone (114), resulting in misfolding, syntaxin SNAP-25 liposomes with the C2A- so in vitro studies prior to 2000 and papers that · C2B fragment of synaptotagmin I (20, 119). reference these early works are affected by this However, because synaptotagmin I/II and the mutation. binary complex are thought to primarily re- The X-ray crystal structure of the C2A do-

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org side on opposite membranes, this interaction main of synaptotagmin I revealed a β-sandwich + could only take place if the synaptic vesicle is fold with a cluster of three Ca2 -binding loops + already docked to the target membrane. Fur- at the apex of the fold (115). Upon Ca 2 bind- thermore, in one study, the lipid-mixing accel- ing, few structural changes occurred in the diva- erating property of the C2A-C2B fragment is lent cation-binding pocket of the C2A domain, eliminated when the syntaxin SNAP-25 com- apart from changes in the side chain rotamers · + plex is “activated” with a C-terminal fragment for the Ca2 -coordinating aspartate residues of synaptobrevin (22). Another report showed and a general decrease in flexibility of the do- no Ca2+ dependence of lipid mixing/fusion us- main (115, 116). Crystal structures of the C2A- ing full-length synaptotagmin (minus the lumi- C2B fragment of synaptotagmins I and III with- + nal domain) (120). Furthermore, bulk liposome out Ca2 (117, 118) and that of synaptotagmin + studies do not reproduce the observation that III with Ca2 (M. Vrljic, P. Strop, J. Ernst, R.B. disrupting Ca2+ binding to the C2B domain

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impairs neurotransmitter release more strongly complex and C2B is more consistent with the than with C2A (15, 121–124). Ca2+-binding sites oriented away from the Synaptotagmin preferentially binds to SNARE complex rather than in direct contact. POPS: 2+ palmitoyloleoyl curved membranes, suggesting possible inter- This would leave the Ca -binding sites acces- phosphatidylserine actions with highly curved fusion intermediates sible for phospholipid binding, allowing con- (125). However, the model drawn from this current binding of both SNAREs and mem- study does not explain the above-mentioned brane. This prediction has been confirmed higher sensitivity of mutations of the C2B by monitoring the partitioning of synapto- domain compared to the C2A domain to neuro- tagmin to POPS containing membranes ver- transmitter release. Furthemore, it is of interest sus SNARE-reconstituted DOPS membranes to note that purified synaptic vesicles fuse with (132): Synaptotagmin preferentially binds to binary complexes containing proteoliposomes the SNARE-containing membrane while its inaCa2+-independent manner (23) despite Ca2+-binding loops are inserted into the mem- the fact that the synaptic vesicles contain both brane. Clearly, these studies need to be extented synaptobrevin and synaptotagmin. Thus, the to investigate the interaction between synapto- observed synaptotagmin-accelerating effect tagmin and the trans SNARE complex as this in current in vitro experiments is not yet is probably the most relevant interaction in the representative of the mechanism of Ca2+ context of synaptic vesicle fusion. triggering in neurons. smFRET studies revealed structural insights for the interactions between a C2A-C2B frag- COMPLEXIN ment of synaptotagmin I and the ternary cis Complexin is a soluble protein, with a molec- SNARE complex, although the generally noisy ular weight of ∼15 kDa, that shows rapid and FRET efficiency data and the unknown fluo- high-affinity binding to the SNARE complex rophore dynamics precluded quantitative inter- (133, 134). Solution NMR studies of complexin pretation in terms of absolute distances (126). and its interactions with SNAREs revealed an Interactions were found between the C2B do- α-helical region involved in SNARE binding main of synaptotagmin I and the membrane- and an unstructured portion (135). Complexin proximal portion of the SNARE complex, but binds in an antiparallel α-helical conforma- only in the presence of Ca2+. Few high FRET tion to the groove between the synaptobrevin efficiency interactions were observed with the and syntaxin α-helices of the ternary SNARE by 72.255.12.226 on 06/04/09. For personal use only. C2A domain. Thus, the low number of FRET complex (136, 137). A report of complexin- events observed between C2A and the SNARE dependent oligomerization of SNARE com- labeling sites suggests that the C2A domain plexes (138) was probably an artifact caused by does not closely interact with the SNARE core disulfide bond formation (134). Knockout ex-

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org complex. periments in mice showed that complexin is The labeling sites on the SNARE com- essential for the Ca2+ dependency of synaptic plex showing high FRET efficiency with la- vesicle release with a phenotype related to that beling sites on the C2B domain were near the of synaptotagmin knockout mice (139). How- ionic layer and the membrane-proximal region, ever, because complexin has no Ca2+-binding which is in qualitative agreement with bio- sites, the observed phenotype must be related chemical studies that implied the membrane- to some interplay between the SNAREs, com- proximal region of the SNARE complex in plexin, and synaptotagmin (14). synaptotagmin binding (127–131). Because the Single-molecule experiments indicated a fluorescent probes were in a loop distal to novel interaction between complexin and the the Ca2+-binding sites, the appearance of membrane-bound syntaxin·SNAP-25 complex high FRET efficiency between the SNARE (29). Previously, no interaction had been

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observed between complexin and the individual liposomes was observed in single-vesicle SNAREs or the binary complex by using studies (142), although only a fraction of isothermal titration calorimetry in solution the liposomes showed this Ca2+ effect. The (134). In these experiments, the binary com- observed Ca2+ stimulation is puzzling because plex was likely in the 2:1 state, which might neither SNARE nor complexin contain Ca2+- have prevented complexin binding. Interest- binding sites. The distribution of reconstituted ingly, the N-terminal accessory helix of com- proteins in liposomes can vary significantly; plexin (residues 29–48) has an inhibitory ef- thus, some liposomes may have a much higher fect on neurotransmitter release, suggesting protein concentration, effectively destabilizing that this region serves as a placeholder for the vesicle. Furthermore, it should be noted the C-terminal portion of the synaptobrevin that this study used a relatively high protein- SNARE motif, thereby regulating assembly of to-lipid ratio (1:100 for syntaxin) that might the SNARE complex (140). This result sug- have made the liposomes leaky. Upon addition gested possible interactions between complexin of complexin, these liposomes could thus be and the binary complex. Single-molecule exper- further destabilized, making them prone to iments have now clearly established that there is nonspecific Ca2+-induced events in combina- a direct interaction between complexin and the tion with POPS-containing membranes. As the 1:1 binary complex and also have shown that authors suggest, it is possible that this type of this interaction dramatically stabilizes the he- synaptotagmin-independent Ca2+ triggering lix bundle configuration. Recently, this inter- could play a role in asynchronous release, but action has also been confirmed with two en- it clearly does not explain the requirement tirely different methods: liposome cofloatation for complexin for synchronous release where assays (141) and electron paramagnetic reso- synaptotagmin is also required. In this context, nance spectroscopy (142). synaptotagmin 1 knockout mice exhibited Kinetic binding studies with complexin and some Ca2+-triggered release in the presence of the cis ternary SNARE complex were carried an N-terminal fragment of complexin (140). out by stopped-flow fluorescence and isother- A possible role of complexin function in syn- mal titration calorimetry (134) as well as by chronous release is provided by experiments smFRET spectroscopy (29a, 126). The mea- using the bulk liposome assay involving both

sured dissociation constant (KD) was in the synaptotagmin and complexin: Complexin- nanomolar range with fast on and off rates. The impaired inner leaflet mixing and the addition by 72.255.12.226 on 06/04/09. For personal use only. single-molecule experiments revealed a tran- of synaptotagmin in the presence of Ca2+ sient nature of the complexin·SNARE inter- released this inhibition and led to lipid mixing action despite its relatively high affinity. Al- (144). Taken together, these studies indicate though the dissociation constants are similar, both activating and inhibitory activities of

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org there are signficant variations for the on and complexin (157, 158). off rates in the two smFRET studies (29a, 126), which are likely due to the different dye label- ing sites altering the kinetics of this interaction. MUNC18 FRET efficiency distributions involving a label Sec1/Munc18 (SM) proteins are in a small fam- attached to the unstructured C-terminal por- ily of cytoplasmic proteins that play an impor- tion of complexin were broader than those seen tant but poorly understood role in intracellu- for the structured SNARE-binding region of lar membrane fusion. Interactions between the complexin (126). Again, these studies need to neuronal SM protein Munc18 and syntaxin (42, be extended to interactions between complexin 145), the binary syntaxin·SNAP-25 SNARE and the trans SNARE complex. complex (146), and the ternary SNARE com- Complexin-dependent Ca2+-accelerated plex have been found (147). On the the lipid mixing between SNARE-containing basis of available structural and biophysical

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information, several possible interaction in- SUMMARY AND FUTURE ISSUES terfaces and conformations have been found: Single-molecule studies have revealed a num- ( ) a tight interaction between the closed a ber of new and sometimes provocative features form of syntaxin and Munc18, involving part of SNAREs and their accessory proteins. Mix- of the syntaxin SNARE motif and the N- tures of parallel and antiparallel SNARE com- terminal domain of syntaxin (42); as well as plexes were found in solution, but mostly par- ( ) interactions between the SNARE domains b allel complexes are involved in the docking of the ternary complex and the short N- of liposomes to supported bilayers. Transient terminal sequence of syntaxin (148). In ad- docking of liposomes can occur with only syn- dition, smFRET experiments demonstrated a taxin and synaptobrevin, i.e., without SNAP- direct interaction between the MUN domain 25. Very few (one to three) SNARE complexes of Munc18 and the membrane-anchored 1:1 are sufficient for stable docking of liposomes. binary complex accompanied by stabilization The SNARE-induced fusion mechanism is in- of the helix bundle configuration (29). De- trinsically fast (less than 100 ms), but infre- spite increasing information about Munc18- quent. The binary syntaxin·SNAP-25 complex SNARE interactions, the function of this is flexible with three different rapidly inter- protein in membrane fusion is still unclear converting configurational states that collapse (148, 149). into a single state upon binding of synaptotag- min (Figure 3b). The syntaxin·SNAP-25 com- MUNC13 plex can also be stabilized by the accessory fac- Munc13 is an ∼200-kDa protein that is es- tors (in decreasing order) synaptotagmin (with + sential for priming of synaptic vesicles to the and without Ca2 ), complexin, Munc13, and release-ready state (150) and that is also in- Munc18. We conclude that in the cellular en- volved in presynaptic plasticity (151–153). It vironment the binary complex will be primar- has been suggested that Munc13 catalyzes the ily in the three-helix bundle configuration as transition from the closed to the open state of there is a high likelihood that at least one syntaxin based on the observation that double- of the accessory proteins is near a particular knockout (Munc13 and syntaxin) Caenorhabdi- syntaxin·SNAP-25 complex. Because it is this tis elegans mutants are rescued by a constitu- configuration to which synaptobrevin can read- tively open syntaxin (154). The α-helical MUN ily bind, the formation of this configuration of

by 72.255.12.226 on 06/04/09. For personal use only. domain of Munc13 is sufficient for rescuing the syntaxin·SNAP-25 complex is likely not go- neurotransmitter release in hippocampal neu- ing to be a limiting step in neurotransmitter rons lacking Munc13s (155). It does not in- release. teract with syntaxin alone, but single-molecule SNARE-mediated fusion events are rela- experiments reported an interaction with the tively rare in single-molecule experiments (or Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org membrane-bound binary syntaxin·SNAP-25 slow in bulk liposome assays). Nevertheless, complex that has a similarly pronounced effect single-molecule experiments revealed an in- as complexin, leading to stabilization of the he- trinsically fast kinetics (<10 ms) for individ- lix bundle configuration (29). The Mun domain ual fusion events. Using the bulk liposome as- of munc13 is highly α-helical, so its strong ef- say, some acceleration of lipid mixing/fusion fect on diminishing the SX-SN1 and SX-SN2 has been observed upon addition of other configurations could be explained by stabiliza- factors, but it is desirable to have these ac- tion of the SX-SN1-SN2 complex by four-helix celerating effects also examined with single- bundle formation. These single-molecule re- molecule methodologies. Thus, the single- sults have been reproduced with an entirely molecule experiments need to be expanded different approach, using liposome cofloatation in order to mimic the pertinent properties of + assays (141). Ca2 -triggered synaptic vesicle fusion. Such a

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system would contribute to the understanding important questions about the molecular mech- of the molecular machinery of vesicle fusion anism of Ca2+-triggered neurotransmitter because the sequence of protein-protein, lipid- release. lipid, and protein-lipid interactions could be The probabilistic nature of synaptic vesicle studied during the fusion process. Other open release results in one (or zero) synaptic vesicles questions are as follows: fusing in response to an action potential out of a larger, readily releasable pool. Fusion proba-  Do antiparallel SNARE configurations bility is dynamically regulated by many factors exist in the physiological context and are and contributes to presynaptic plasticity. Thus, they regulated by chaperones? each synaptic vesicle undergoes a series of se-  Is the fusion pore of pure lipidic charac- quential interactions as they mature from dock- ter or does it involve SNAREs or other ing to a fusion-competent state. Reconstituting proteins? this type of dynamic and heterogeneous reac-  How many SNARE complexes are re- tion pathway is precisely where single-molecule quired for synaptic vesicle fusion? methods excel, and thus, these methods will Single-molecule methods will certainly play a be invaluable in untangling this complex major role in addressing these fundamentally system.

DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We thank Dr. Josep Rizo for critical reading of the manuscript. Support by the National Institutes of Health to A.T.B.and S.C. (1-RO1-MH63105), to K.W.(GM076039), and to M.B. (MH081923- 01), and a CASI award from the Burroughs Wellcome Fund to K.W. are gratefully acknowledged. S.C. was at Lawrence Berkeley National Laboratory and at the Departments of Physics and Molecular and Cell Biology, University of California, Berkeley, when this article was written. by 72.255.12.226 on 06/04/09. For personal use only. LITERATURE CITED 1. Heuser JE, Reese TE. 1977. The structure of the synapse. In Cellular Biology of Neurons, ed. ER Kandel, SR Geiger, VB Mountcastle, JM Brookhart, pp. 261–94. Bethesda, MD: Am. Physiol. Soc. 2. Schikorski T, Stevens CF. 1997. Quantitative ultrastructural analysis of hippocampal excitatory

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Annual Review of Biochemistry Contents

Preface ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppv Volume 78, 2009 Prefatory Articles Frontispiece E. Peter Geiduschek pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppxii Without a License, or Accidents Waiting to Happen E. Peter Geiduschek pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1 Frontispiece James C. Wang pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp30 A Journey in the World of DNA Rings and Beyond James C. Wang pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp31 Biochemistry and Disease Theme The Biochemistry of Disease: Desperately Seeking Syzygy John W. Kozarich pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp55 Biosynthesis of Phosphonic and Phosphinic Acid Natural Products William W. Metcalf and Wilfred A. van der Donk ppppppppppppppppppppppppppppppppppppppppp65 New Antivirals and Drug Resistance Peter M. Colman pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp95 Multidrug Resistance in Bacteria by 72.255.12.226 on 06/04/09. For personal use only. Hiroshi Nikaido pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp119 Conformational Pathology of the Serpins: Themes, Variations, and Therapeutic Strategies ppppppppppppppppppppppppppppppppppppppppppppppppppppppppp

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org Bibek Gooptu and David A. Lomas 147 Getting a Grip on Prions: Oligomers, Amyloids, and Pathological Membrane Interactions Byron Caughey, Gerald S. Baron, Bruce Chesebro, and Martin Jeffrey ppppppppppppppppp177 Ubiquitin-Mediated Protein Regulation RING Domain E3 Ubiquitin Ligases Raymond J. Deshaies and Claudio A.P. Joazeiro pppppppppppppppppppppppppppppppppppppppppp399 Regulation and Cellular Roles of Ubiquitin-Specific Deubiquitinating Enzymes Francisca E. Reyes-Turcu, Karen H. Ventii, and Keith D. Wilkinson pppppppppppppppppppp363

vi AR378-FM ARI 7 May 2009 15:43

Recognition and Processing of Ubiquitin-Protein Conjugates by the Proteasome Daniel Finley pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp477 Degradation of Activated Protein Kinases by Ubiquitination Zhimin Lu and Tony Hunter ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp435 The Role of Ubiquitin in NF-κB Regulatory Pathways Brian Skaug, Xiaomo Jiang, and Zhijian J. Chen pppppppppppppppppppppppppppppppppppppppp769 Biological and Chemical Approaches to Diseases of Proteostasis Deficiency Evan T. Powers, Richard I. Morimoto, Andrew Dillin, Jeffery W. Kelly, and William E. Balch pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp959

Gene Expression RNA Polymerase Active Center: The Molecular Engine of Transcription Evgeny Nudler ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp335 Genome-Wide Views of Chromatin Structure Oliver J. Rando and Howard Y. Chang pppppppppppppppppppppppppppppppppppppppppppppppppppp245 The Biology of Chromatin Remodeling Complexes Cedric R. Clapier and Bradley R. Cairns ppppppppppppppppppppppppppppppppppppppppppppppppppp273 The Structural and Functional Diversity of Metabolite-Binding Riboswitches Adam Roth and Ronald R. Breaker ppppppppppppppppppppppppppppppppppppppppppppppppppppppppp305 Lipid and Membrane Biogenesis

by 72.255.12.226 on 06/04/09. For personal use only. Genetic and Biochemical Analysis of Non-Vesicular Lipid Traffic Dennis R. Voelker pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp827 Cholesterol 24-Hydroxylase: An Enzyme of Cholesterol Turnover in the Brain

Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org David W. Russell, Rebekkah W. Halford, Denise M.O. Ramirez, Rahul Shah, and Tiina Kotti pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1017 Lipid-Dependent Membrane Protein Topogenesis William Dowhan and Mikhail Bogdanov pppppppppppppppppppppppppppppppppppppppppppppppppp515 Single-Molecule Studies of the Neuronal SNARE Fusion Machinery Axel T. Brunger, Keith Weninger, Mark Bowen, and Steven Chu ppppppppppppppppppppppp903 Mechanisms of Endocytosis Gary J. Doherty and Harvey T. McMahon pppppppppppppppppppppppppppppppppppppppppppppppp857

Contents vii AR378-FM ARI 7 May 2009 15:43

Recent Advances in Biochemistry Motors, Switches, and Contacts in the Replisome Samir M. Hamdan and Charles C. Richardson ppppppppppppppppppppppppppppppppppppppppppp205 Large-Scale Structural Biology of the Human Proteome Aled Edwards pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp541 Collagen Structure and Stability Matthew D. Shoulders and Ronald T. Raines pppppppppppppppppppppppppppppppppppppppppppppp929 The Structural and Biochemical Foundations of Thiamin Biosynthesis Christopher T. Jurgenson, Tadhg P. Begley, and Steven E. Ealick pppppppppppppppppppppppp569 Proton-Coupled Electron Transfer in Biology: Results from Synergistic Studies in Natural and Model Systems Steven Y. Reece and Daniel G. Nocera ppppppppppppppppppppppppppppppppppppppppppppppppppppp673 Mechanism of Mo-Dependent Nitrogenase Lance C. Seefeldt, Brian M. Hoffman, and Dennis R. Dean ppppppppppppppppppppppppppppp701 Inorganic Polyphosphate: Essential for Growth and Survival Narayana N. Rao, Mar´ıa R. G´omez-Garc´ıa, and Arthur Kornberg ppppppppppppppppppppp605

Essentials for ATP Synthesis by F1F0 ATP Synthases Christoph von Ballmoos, Alexander Wiedenmann, and Peter Dimroth pppppppppppppppppp649 The Chemical Biology of Protein Phosphorylation Mary Katherine Tarrant and Philip A. Cole pppppppppppppppppppppppppppppppppppppppppppppp797 Sphingosine 1-Phosphate Receptor Signaling Hugh Rosen, Pedro J. Gonzalez-Cabrera, M. Germana Sanna, and Steven Brown pppp743 The Advent of Near-Atomic Resolution in Single-Particle Electron

by 72.255.12.226 on 06/04/09. For personal use only. Microscopy Yifan Cheng and Thomas Walz ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp723 Super-Resolution Fluorescence Microscopy Bo Huang, Mark Bates, and Xiaowei Zhuang ppppppppppppppppppppppppppppppppppppppppppppp993 Annu. Rev. Biochem. 2009.78:903-928. Downloaded from arjournals.annualreviews.org

Indexes

Cumulative Index of Contributing Authors, Volumes 74–78 pppppppppppppppppppppppppp1041 Cumulative Index of Chapter Titles, Volumes 74–78 ppppppppppppppppppppppppppppppppppp1045

Errata

An online log of corrections to Annual Review of Biochemistry articles may be found at http://biochem.annualreviews.org/errata.shtml

viii Contents