880 VOLUME 14 NUMBER 10 OCTOBER 2007 NATURE STRUCTURAL & MOLECULAR BIOLOGY

SNARE proteins are central components of a conserved machinery that governs most types of intracellular membrane fusion in organisms ranging from yeast to mammals1. Studies of the neuronal SNAREs that control neurotransmitter release, namely the synaptic vesicle protein synaptobrevin (also known as VAMP) and the plasma membrane proteins syntaxin and SNAP-25, led to a general model in which vesicle SNAREs (v-SNAREs) and SNAREs from the target membrane (t-SNAREs) form tight complexes bridging the two membranes2,3. These ‘SNARE complexes’ were shown to involve parallel interactions between membrane-proximal SNARE motif sequences that form long coiled coils4,5. As the neuronal SNARE complex was thought to be highly stable6, these findings led naturally to the proposal that the high energy of SNARE complex formation could be used to drive membrane fusion4,5. This proposal was supported by reconstitution experiments with purified recombinant components7 and cell-cell fusion assays with ‘flipped SNAREs’ expressed on cell surfaces8. Moreover, the crystal structure of the neuronal SNARE

complex revealed, at atomic resolution, a bundle of four parallel α-helices that should bring the two membranes within distances close to those required for fusion9. Hence, there is currently little doubt that SNARE complex formation is crucial for fusion. However, in attempts to understand how SNAREs function in fusion, the answer to a key question has remained elusive: what is the energetics of SNARE complex formation? On page 890 of this issue, Li et al.10 describe a first, crucial step in answering this question, revealing a very high stabilization energy (35 kBT) for a partially assembled neuronal SNARE complex.

The high stability of SNARE complexes has been widely assumed in the literature

on the basis of available data, but it has not been demonstrated from a strictly thermodynamic point of view, as the observed stability could have, at least in part, a kinetic origin. For instance, denaturation experiments have shown that disassembly of the neuronal SNARE complex requires very high temperatures or concentrations of chaotropic agents, suggesting a very high stability, but the complex can be reassembled only at much lower temperatures or concentrations of chaotropic agents11. This dramatic hysteresis indicates that equilibrium could not be reached in these disassembly-reassembly experiments (even after very long incubations), a limitation that prevented the determination of stabilization

Josep Rizo and Han Dai are in the Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390, USA, and Han Dai is currently in the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115, USA. e-mail: jose@arnie.swmed.edu

How much can SNAREs flex their muscles?Josep Rizo & Han Dai

The high stability of SNARE complexes is probably crucial for their role in membrane fusion, but it has been difficult to measure. A surface-forces apparatus has now been used to measure the stabilization energy of a partially assembled SNARE complex, and the result (35 kBT) is among the highest protein-folding free energies ever observed. Moreover, this approach offers a bright future for further structural and energetic studies of membrane fusion machineries.

Figure 1 Example of a force-versus-distance profile observed during an approach-separation cycle in one of the SFA experiments of Li et al.10. The force normalized to the mean radius of curvature of the two surfaces is plotted as a function of the separation distance between the two bilayers. The different phases observed during the cycle, as explained in the text, are numbered from 1 to 6. Figure courtesy of David Tareste.

6, Adhesive jump

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energies. Deuterium exchange experiments have also suggested a very high stability for the neuronal SNARE complex12, but this finding could again have a kinetic origin. Finally, atomic-force microscopy has yielded information about the forces required to disrupt SNARE complexes13,14, but not about their energetics.

To obtain energetic information on SNARE complex assembly, Li et al.10 used a surface-forces apparatus (SFA)15. This contains two closely apposed cylindrical lenses whose surfaces are covered by thin films. The instrument allows accurate measurement of the minimal separation distance between the surfaces and the force between them, which, after normalization to the mean radius of curvature of the two lenses, is converted to free energy per unit area between two planar surfaces. In the experiments of Li et al.10, the surfaces were covered with phospholipid bilayers containing the v-SNARE synaptobrevin on one lens and the heterodimeric t-SNARE complex formed by syntaxin and SNAP-25 on the other10. The transmembrane regions of synaptobrevin and syntaxin were deleted to avoid potential complications with fusion, and C-terminal cysteines were added to allow covalent linkage to maleimide-containing phospholipids in the membranes.

A basic SFA experiment involved simultaneous measurement of distances and forces at regular intervals during repeated cycles of approach and separation of the two membranes. During the approaching phase, a slowly increasing long-range repulsive force arising from steric clashes between unassembled v- and t-SNAREs was observed between 20 and 8 nm (Fig. 1, phases 1 and 2). This repulsive force showed a plateau around 8 nm that can be attributed to the start of SNARE complex assembly (phase 3). A steep repulsive force then occurred below 5 nm, owing to compression of the assembled complexes and remaining unassembled SNAREs (phase 4). In the separation phase, the pushing force coincided with that in the approaching phase at short distances but then diverged, continuing to decrease steeply without any plateau and becoming a pulling force at distances above 4–5 nm (phase 5). Finally, the bilayers jumped out of contact at distances of about 8–10 nm (this is referred to as the adhesive jump; phase 6), when the pulling force overcame the force binding the two bilayers; this pulling force can thus be related to the adhesive energy of the assembled SNARE complexes.

The data are of impressive quality, and the force-distance profiles for each approaching-separation cycle in a given experiment are superimposable, showing the reversibility of the events taking place in each cycle. Still, the adhesive energy was found to increase with the contact time between the two surfaces (defined as the time elapsed between the end of the approaching phase and the adhesive jump), reaching a plateau at contact times of about 60 min. This observation indicates that reaching a true equilibrium (where SNARE complexes are maximally assembled) takes a considerable amount of time, but equilibrium can be reached, which is crucial for derivation of thermodynamic data. Note that the design of the SFA experiments should accelerate both the assembly and the disassembly rates, and hence equilibrium should be reached sooner in these experiments than in previous solution denaturation experiments11. Thus, the SFA experiments start with preformed syntaxin–SNAP-25 heterodimers on a surface that is forced to be in close proximity to the v-SNARE surface during the approaching phase, which should increase the on rates of complex assembly. In contrast, in the solution experiments11, there was no preformed t-SNARE heterodimer, and assembly required the productive association of multiple polypeptide chains through random collisions in solution. Furthermore, the disassembly rates, which are extremely slow for the soluble SNARE complexes11, should be accelerated by the pulling forces in the SFA experiments.

Li et al.10 observed a large range of adhesive energies in separate experiments, but they were able to satisfactorily attribute such high variability to different SNARE densities in each experiment, as there was a correlation between the adhesive energy and the long-range repulsive force measured during the approaching phase. Moreover, modeling these repulsive forces with a simple polymer theory allowed the authors to determine the SNARE surface densities and hence to convert the measured adhesive energies (per unit area) to binding energies per SNARE complex (35 ± 7 kBT). The homogeneity of the binding energies derived from experiments with very different SNARE densities provides a convincing validation for the application of polymer theory in calculating the densities and for the conclusion that a true equilibrium can be reached in the experiments.

Given the dimensions of the SNARE complex9, the data show that the complexes formed during the SFA experiments were not fully assembled at their membrane-proximal C termini (about 12–25 membrane-proximal residues of each SNARE were estimated to be unstructured or not fully structured)10. Hence, the measured stabilization energy of 35 kBT corresponds not to the full neuronal SNARE complex but rather to an intermediate where about 20%–40% of the complex is not fully assembled (Fig. 2). Nevertheless, this energy is already comparable to the highest folding energies observed in proteins. It is unclear why SNARE complexes were not fully

∆G = –35 kBTN

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Figure 2 Diagram summarizing the main result obtained with the SFA experiments of Li et al.10, which yielded the free energy of formation of a partially assembled neuronal SNARE complex between two planar membranes (35 kBT). Synaptobrevin is in red, SNAP-25 in green and syntaxin in yellow (SNARE motif) and orange (the N-terminal Habc domain). N marks the N termini of synaptobrevin and syntaxin. For the syntaxin–SNAP-25 heterodimer (left), the extent of assembly is unknown; the Habc domain is shown interacting with the SNARE motifs because this domain has been found to inhibit SNARE-induced liposome fusion20, but the interaction mode is unknown. Li et al.10 estimate that the SNARE complex intermediate observed in their SFA experiments is assembled about 60%–80%.

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assembled in these experiments. However, the observation of an intermediate is in accord with the observation of slower deuterium exchange rates at the N terminus of the SNARE complex than at its C terminus12, and with increasing evidence that the SNARE complex assembles from the N to the C terminus, forming at least one intermediate, in vitro and in vivo16.

The existence of an intermediate in neuronal SNARE complex assembly has important mechanistic implications, because it suggests that the assembly is not a fully cooperative, all-or-none process and that at least two regions of the SNARE complex may perform different actions. Hence, knowing the breakdown of free energies released upon assembly of the distinct functional regions of the SNARE complex seems even more important, from a mechanistic point of view, than determining the free energy of full SNARE complex assembly. As Li et al.10 point out, the value of 35 kBT they measured is close to the free energies calculated to be necessary for formation of stalk or hemifusion intermediates10. It is thus tempting to speculate that formation of the SNARE complex intermediate could lead to hemifusion, and assembly of the C terminus of the SNARE complex could give the final push for fusion. This is a valid view, but one can also argue that, at 35 kBT per

SNARE complex intermediate, a few of these complexes could provide much more than the energy necessary for full membrane fusion.

A key problem, however, is whether this energy can actually be used for membrane fusion. The intermediate observed using SFA is expected to bring two membranes within 4–5 nm of each other, and this action by itself should not lead to hemifusion or fusion unless it is coordinated with additional actions of the C-terminal residues. In regard to this, the results of the SFA experiments that Li et al.10 performed with neutral membranes suggest that basic residues at the SNARE ‘juxtamembrane’ regions interact with negatively charged membranes, which could bend the membranes to initiate hemifusion or fusion with help from the SNARE transmembrane regions17. Alternatively, the 35 kBT released upon formation of the SNARE complex intermediate could be used directly for hemifusion or fusion if the intermediate could be kept away from the center of the (future) fusion pore by interactions with a bulky protein such as Munc18-1 (ref. 18). Another possibility is that formation of the intermediate serves only to hold the membranes together, serving as a support for the truly fusogenic action of the SNARE C terminus, perhaps together with

additional factors such as complexins or the Ca2+ sensor synaptotagmin-1 (ref. 19). The SFA approach described by Li et al.10 provides a powerful tool to investigate these and other interesting possibilities related to the mechanisms of neurotransmitter release and membrane fusion in general.

1. Jahn, R. & Scheller, R.H. Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).

2. Sollner, T. et al. Nature 362, 318–324 (1993).3. Sollner, T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H.

& Rothman, J.E. Cell 75, 409–418 (1993).4. Hanson, P.I., Roth, R., Morisaki, H., Jahn, R. &

Heuser, J.E. Cell 90, 523–535 (1997).5. Lin, R.C. & Scheller, R.H. Neuron 19, 1087–1094

(1997).6. Hayashi, T. et al. EMBO J. 13, 5051–5061

(1994).7. Weber, T. et al. Cell 92, 759–772 (1998).8. Hu, C. et al. Science 300, 1745–1749 (2003).9. Sutton, R.B., Fasshauer, D., Jahn, R. & Brunger, A.T.

Nature 395, 347–353 (1998).10. Li, F. et al. Nat. Struct. Mol. Biol. 14, 890–896

(2007).11. Fasshauer, D., Antonin, W., Subramaniam, V. &

Jahn, R. Nat. Struct. Biol. 9, 144–151 (2002).12. Chen, X. et al. Neuron 33, 397–409 (2002).13. Yersin, A. et al. Proc. Natl. Acad. Sci. USA 100,

8736–8741 (2003).14. Liu, W. et al. Biophys. J. 91, 744–758 (2006).15. Israelachvili, J.N. Intermolecular and Surface Forces

(Academic Press, London, 1992).16. Sorensen, J.B. et al. EMBO J. 25, 955–966

(2006).17. McNew, J.A. et al. J. Cell Biol. 150, 105–117

(2000).18. Rizo, J., Chen, X. & Arac, D. Trends Cell Biol. 16,

339–350 (2006).19. Dai, H., Shen, N., Arac, D. & Rizo, J. J. Mol. Biol.

367, 848–863 (2007).20. Parlati, F. et al. Proc. Natl. Acad. Sci. USA 96,

12565–12570 (1999).

Decades after their discovery, prions remain one of the most controversial and enigmatic pathogens. Defined as infectious proteins that are devoid of any transmissible

nucleic acids, these agents are believed to self-replicate by a mechanism involving a conformational switch, in which the abnormal (misfolded) conformer binds the normal form of the same protein and coerces it into the abnormal conformation1–5. The existence of such a ‘protein-only’ infectious entity was originally proposed to explain the pathogenic process in transmissible spongiform encephalopathies (TSEs), a group of dreadful brain-wasting

disorders such as mad cow disease, Creutzfeldt-Jakob disease in humans, and scrapie in sheep1,2. However, the prion concept has since been expanded to include certain non-mendelian traits in yeast and other fungi, the inheritance of which can be explained by self-propagating changes in protein conformation4,5. Although progress in unraveling molecular details of mammalian prion biology has been excruciatingly slow, rapid strides

Prion strains under the magnifying glassNathan J Cobb & Witold K Surewicz

Prion ‘strains’, multiple conformations of the same misfolded protein, have captured great interest because of their role in transmission of mad cow disease to humans. Prion strains have also been observed in yeast, where self-propagating protein folds are responsible for inheritable traits. Recent findings reveal an exciting new insight into the structural basis of this phenomenon.

Nathan J. Cobb and Witold K. Surewicz are in the Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA. e-mail: witold.surewicz@case.edu

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