Membrane fusion: grappling with SNARE and SM proteins

Abstract

The two universally required components of the intracellular membrane fusion machinery, SNARE and SM (Sec1/Munc18-like) proteins, play complementary roles in fusion. Vesicular and target membrane-localized SNARE proteins zipper up into an alpha-helical bundle that pulls the two membranes tightly together to exert the force required for fusion. SM proteins, shaped like clasps, bind to trans-SNARE complexes to direct their fusogenic action. Individual fusion reactions are executed by distinct combinations of SNARE and SM proteins to ensure specificity, and are controlled by regulators that embed the SM-SNARE fusion machinery into a physiological context. This regulation is spectacularly apparent in the exquisite speed and precision of synaptic exocytosis, where synaptotagmin (the calcium-ion sensor for fusion) cooperates with complexin (the clamp activator) to control the precisely timed release of neurotransmitters that initiates synaptic transmission and underlies brain function.

Figure 1

Structure of SNARE and SM proteins and some proteins that grapple with them. (A) SNARE complex (also called cis-SNARE complex) of VAMP/synaptobrevin-2 (blue helix), Syntaxin-1A (red helix), and SNAP-25 (green and yellow helices for the N- and C-terminal domains, respectively; adapted from (23)). The Habc domain of Syntaxin-1A (brown helices, adapted from 68) is positioned arbitrarily. (B) An SM protein, highlighting its arch-like structure (Munc18-1, adapted from (38)). (C) Complexin, bound to the SNARE complex (Complexin-1, shown in magenta, adapted from (66)), has a helical region that binds at the interface of v- and t-SNARE in an anti-parallel orientation. (D) Synaptotagmin, the calcium sensor for synchronous synaptic transmission (Synaptotagmin-1, adapted from (69, 70)), with its membrane-proximal (C₂A) and membrane-distal (C₂B) C₂ domains labeled and the position of critical bound calcium ions (orange) shown. All proteins are at the same scale and the bilayer thickness is approximately on the same scale.

Figure 2

(A) The zippering model for SNARE-catalyzed membrane fusion. Three helices anchored in one membrane (the t-SNARE) assemble with the fourth helix anchored in the other membrane (v-SNARE) to form trans-SNARE complexes, or SNAREpins. Assembly proceeds progressively from the membrane-distal N-termini towards the membrane-proximal C-termini of the SNAREs. This generates an inward force vector (F) that pulls the bilayers together, forcing them to fuse. Complete zippering is sterically prevented until fusion occurs, so that fusion and the completion of zippering are thermodynamically coupled. (B) Therefore, when fusion has occurred, the force vanishes and the SNAREs are in the low energy cis-SNARE complex.

Figure 3

SM proteins are designed to bind four helix bundles. (A) The “closed” conformation of Syntaxin-1A, in which the SM protein Munc18-1 binds the four helix bundle composed of syntaxin’s own Habc domain (three helices, in brown) and its own SNARE motif helix (fourth helix, in red; adapted from (38)). This closed state has so far only been found with syntaxins involved in exocytosis. Inset: SM proteins are universally attached to Habc domains by a specialized sequence at the N-terminus of Habc (labeled as N-peptide; adapted from 38, 43, 44). (B) The “open” conformation of a t-SNARE complex, consisting of a t-SNARE and its cognate SM protein bound to the N-peptide of its syntaxin’s Habc domain. This is believed to be the universal state in which t-SNAREs are open (i.e. reactive) with cognate v-SNAREs to form trans-SNARE complexes (C) resulting in fusion. Positioning of the protein domains in B and C are arbitrary. Panel C illustrates SNAREs and SM proteins, the universal fusion machinery.