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The structure of the MUN domain of the synaptic protein Munc13-1 by Li, et al., in this issue of Structure (Li et al., 2011) shows that seemingly disparate regulators of SNARE-mediated membrane fusion are highly conserved at the structural level.
The synapse is a complicated place with vast numbers of regulators involved in multiple interactions that are finely tuned to respond rapidly to signals and to facilitate optimal learning and memory. At the pre-synaptic membrane, formation of fuseogenic SNARE complexes and subsequent synaptic vesicle fusion are the downstream targets of much of this regulation. A major goal of neurobiologists, biochemists and structural biologists is to elucidate the molecular mechanisms of these regulators and how their activities are integrated to ensure specific and timely release of neurotransmitter signals.
The regulator Munc18, of the Sec1/Munc18 (SM) family, binds tightly to the plasma membrane SNARE syntaxin-1. It has several roles, including chaperoning syntaxin during its life-cycle to prevent premature or inappropriate SNARE complexes, and helping to stimulate SNARE-mediated membrane fusion. However, the role of the Munc18-syntaxin interaction has been paradoxical. Although Munc18 has an established positive role in syntaxin function, it binds to a “closed”, intramolecularly inhibited conformation of syntaxin. Resolution of this predicament appears to involve another puzzling regulator, Munc13-1.
The Munc13 protein family was discovered as critical for neuronal regulation in C. elegans (unc-13). Munc13 is thought to function in the docking and priming steps of synaptic vesicle fusion, whereby vesicles are docked to the plasma membrane and primed such that they are ready to rapidly fuse upon stimulation and Ca2+ influx (Fig. 1a; (Wojcik and Brose, 2007) and references therein). Such priming necessitates the release of Munc18 from syntaxin and opening of the inhibited syntaxin conformation to facilitate SNARE complex assembly. The large Munc13 protein (~200 kDa) is composed of a number of putative interaction domains for binding phorbol esters/diacylglycerol, phospholipids, Ca2+, calmodulin and other regulators including RIM and DOC2. Moreover, a region termed the MUN domain (a.a. 859-1531) interacts with syntaxin, membrane anchored syntaxin/SNAP-25 SNARE complexes and Munc18.
A major clue toward understanding Munc13’s function was suggested by the demonstration that loss of unc-13 could be partially bypassed by a mutant of C. elegans syntaxin that is locked in a constitutively uninhibited conformation (Richmond et al., 2001). Recent biochemical and NMR data from the Rizo laboratory (Ma et al., 2011) provided the mechanistic insight—interaction of the Munc13 MUN domain with Munc18 and syntaxin leads to release of the inhibited Munc18-syntaxin complex and promotion of SNARE complex assembly. Clearly lacking, however, was high resolution structural data on the Munc13 protein that has frustrated researchers in the field for number of years.
In this issue, the Tomchick and Rizo groups reveal their successful crystal structure of an independently folded region of the MUN domain (MUN-CD) of Munc13 (Li et al., 2011). As suggested by previous computational predictions (Pei et al., 2009), this region folds into two stacked helical bundles that are structurally homologous to the exocyst subunit Sec6 (Fig. 2; rmsd 2.8 Å; (Sivaram et al., 2006) and other members of the CATCHR (Complex Associated with Tethering Containing Helical Rods) family of intracellular tethering factors (reviewed in (Yu and Hughson, 2010). Moreover, the cargo binding domains from type V myosins, yeast Myo2 and Myo4, also share this structure (Pashkova et al., 2006). Most surprisingly, the structural homology between these diverse proteins arises from sequences that are ≤10% identical. The sequence conservation is primarily due to hydrophobic core packing residues, which presumably form and stabilize the helical bundle structures. Intriguingly, the topology of the helices and the connecting loops within each structure is the same, strongly suggesting that each subunit has diverged from an ancient ancestor (Sivaram et al., 2006).
The CATCHR family is comprised of subunits from the exocyst, COG (Conserved Oligomeric Golgi), Dsl1 and GARP (Golgi Associated Retrograde Protein) families of tethering complexes (Yu and Hughson, 2010). They share common functionalities; subunits from each of the complexes interact with proteins and lipids on the vesicle and target membranes, usually Rab and Rho families of small GTPases, and with the SNAREs. CATCHR complexes have been proposed to physically tether vesicles to their target membranes. However, little direct evidence for tethering has been observed, and it is likely that the key roles for these complexes are in controlling the spatial and temporal specificity of SNARE-mediated fusion.
Thus, this new MUN domain structure illuminates a previously undiscovered connection between these diverse SNARE regulators. What does this mean for similarities of function? These bundles appear to be powerful modules for protein-protein interactions, some intra-complex, such as between exocyst subunits, and others reaching out to binding partners, such as Munc-13 and the SNAREs, and myosins and their cargo. Does the structural homology implicate Munc13 as a vesicle tether? Perhaps in a general sense, as Munc13 can interact with a number of proteins on the vesicle and plasma membranes to bridge the membranes for specific fusion. Most intriguing is the functional role for Munc13 in releasing Munc18’s inhibition of syntaxin; both the exocyst and COG complexes have been shown to interact with their cognate SM proteins (Wiederkehr et al., 2004; Laufman et al., 2009). It will be interesting to see if the characterization of the MUN domain provides insights into the function of subunits of the CATCHR complexes, which may play similar SNARE regulatory roles with their SM partners.
As the field moves forward, additional biochemical and structural developments will be critical to elucidate the intricate mechanisms of these proteins. It is curious that Munc13 has so many diverse functional domains and that the tethering complexes are composed of many (up to eight) large subunits. If the sole activity of these proteins is to bind and regulate SNARE complex assembly, this task could be performed with one or two reasonably sized proteins. However, it is clear that these must be large scaffolds to integrate a wide variety of signals to ensure the fidelity of trafficking. Therefore, the next attractive, although quite elusive, targets for structural studies are the full-length proteins, and co-complexes between different binding partners; thus far, only the full-length Exo70 subunit from the exocyst and co-structures from the small Dsl1 complex have been determined. Of course, a clear view of the mechanisms behind coordinated vesicle tethering and SNARE assembly requires that the models inspired by the structural work be tested by clever mutagenesis and cell biological studies in vivo. The MUN structure has indeed shown us the money, and provides us with an opportunity to spend it wisely in pursuit of these molecular mechanisms.
Research in the Munson lab is supported by National Institutes of Health Grant GM068803.
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