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Low-Resolution Solution Structures of Munc18:Syntaxin Protein Complexes Indicate an Open Binding Mode Driven by the Syntaxin N-Peptide

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Low-Resolution Solution Structures of Munc18:Syntaxin Protein Complexes Indicate an Open Binding Mode Driven by the Syntaxin N-Peptide Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide Michelle P. Christiea,1, Andrew E. Whittena,1,2, Gordon J. Kinga, Shu-Hong Hua, Russell J. Jarrotta, Kai-En Chena, Anthony P. Duffb, Philip Callowc, Brett M. Collinsd, David E. Jamese, and Jennifer L. Martina,2 Divisions of aChemistry and Structural Biology and dMolecular Cell Biology, Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland 4072, Australia; bNational Deuteration Facility, Australian Nuclear Science and Technology Organisation, Lucas Heights, New South Wales 2234, Australia; cLarge Scale Structures Group, Institut Laue-Langevin, 3800 Grenoble, France; and eDiabetes and Obesity Research Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved May 4, 2012 (received for review October 14, 2011) When nerve cells communicate, vesicles from one neuron fuse with closed conformation inactivates Sx1a by preventing H3 interact- the presynaptic membrane releasing chemicals that signal to the ing with SNARE partners, SNAP25 on the plasma membrane and next. Similarly, when insulin binds its receptor on adipocytes or vesicle associated membrane protein 2 (VAMP2, also known as muscle, glucose transporter-4 vesicles fuse with the cell membrane, synaptobrevin) on the vesicle membrane. Conversely, when the allowing glucose to be imported. These essential processes require intramolecular Habc interaction is removed, Sx1a can adopt an the interaction of SNARE proteins on vesicle and cell membranes, as open conformation and H3 is then free to participate in SNARE well as the enigmatic protein Munc18 that binds the SNARE protein complex assembly by forming a SNARE binary complex with Syntaxin. Here, we show that in solution the neuronal protein SNAP25 and a SNARE ternary complex with VAMP2, leading to Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a membrane fusion (Fig. 1). Proteins that differentially promote N-peptide is present. Conversely, the adipocyte protein Syntaxin4 the closed or open Sx1a conformation could thus negatively or does not bind its partner Munc18c unless the N-peptide is present. positively regulate synaptic SNARE complex formation, mem- Solution-scattering data for the Munc18-1:Syntaxin1a complex in brane fusion, and neurotransmission. In the homologous fusion the absence of the N-peptide indicates that this complex adopts the system in adipocytes and muscle, glucose-transporter (GLUT)4- inhibitory closed binding mode, exemplified by a crystal structure containing vesicles are trafficked to the plasma membrane to fa- of the complex. However, when the N-peptide is present, the cilitate glucose uptake in response to insulin signaling. The solution-scattering data indicate both Syntaxin1a and Syntaxin4 equivalent Sx in this system is Sx4. adopt extended conformations in complexes with their respective Sec1/Munc18 (SM) proteins are also highly conserved and Munc18 partners. The low-resolution solution structure of the open probably regulate all eukaryotic vesicle transport processes, al- Munc18:Syntaxin binding mode was modeled using data from though a variety of interactions, binding modes, and functions cross-linking/mass spectrometry, small-angle X-ray scattering, and have been described. Thus, SM proteins are thought to play small-angle neutron scattering with contrast variation, indicating a major inhibitory role by keeping Sxs in the closed conforma- significant differences in Munc18:Syntaxin interactions compared tion, as exemplified by a crystal structure of the neuronal with the closed binding mode. Overall, our results indicate that the Munc18-1 in complex with Sx1a (7). A closed binding mode is neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate also observed in the crystal structure of a primordial Munc18:Sx and functionally distinct binding modes, closed and open, depend- complex suggesting that closed binding, and inhibition of Sx, is ing on the presence of the N-peptide, whereas Munc18c:Syntaxin4 important in the pathway leading to SNARE assembly (8). adopts only the open binding mode. SM proteins have also been shown to interact with SNARE binary and SNARE ternary complexes (9–11), and these inter- membrane fusion | protein interactions | small-angle neutron scattering | actions require an open Sx binding mode as well as the presence small-angle X-ray scattering of an N-peptide, the 10–30 residues at the very N terminus of Sx (11–14). The two binding modes (closed and N-peptide) are embrane trafficking is an essential and highly regulated thought to be associated with different aspects of SM protein Mprocess whereby cargo-carrying vesicles are directed to function. For example, in the synaptic fusion system, Munc18-1 dock and fuse with specific cell membranes. The process requires may act as a chaperone by binding closed Sx1a during trafficking soluble N-ethylmaleimide-sensitive factor attachment receptor to the plasma membrane (14–17). Munc18-1 can then bind and (SNARE) proteins on vesicle and target membranes. These in- stimulate SNARE complex assembly at the plasma membrane, teract to form high-affinity SNARE complexes required for to facilitate membrane fusion (17, 18). Similarly, in Caeno- docking and membrane fusion (1, 2). Structurally, the SNARE rhabditis elegans, the closed SM:Sx binding mode is required for complex is a parallel four-helix bundle comprising helices con- tributed from several partner SNAREs (3). This helical bundle promotes vesicle fusion by bringing the vesicle and plasma Author contributions: M.P.C., A.E.W., G.J.K., K.-E.C., D.E.J., and J.L.M. designed research; membranes into close proximity and by providing the energy M.P.C., A.E.W., G.J.K., S.-H.H., R.J.J., K.-E.C., A.P.D., P.C., B.M.C., and J.L.M. performed research; A.P.D. contributed new reagents/analytic tools; M.P.C., A.E.W., G.J.K., S.-H.H., required for membrane fusion (4, 5). K.-E.C., P.C., B.M.C., and J.L.M. analyzed data; and M.P.C., A.E.W., B.M.C., and J.L.M. wrote In the neuronal synapse, neurotransmitters are released from the paper. nerve cells by SNARE-mediated fusion of vesicles with the The authors declare no conflict of interest. plasma membrane in response to action potentials. In this pro- This article is a PNAS Direct Submission. cess, one of four SNARE helices is contributed by Syntaxin (Sx) 1M.P.C. and A.E.W. contributed equally to this work. 1a, a protein localized at the plasma membrane by a trans- 2 To whom correspondence may be addressed. E-mail: [email protected] or membrane helix. In addition to its membrane-anchored SNARE [email protected]. helix (H3), Sx1a has an Habc domain that can bind to the H3 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. helix (6) forming a closed Sx1a conformation (Fig. 1A). This 1073/pnas.1116975109/-/DCSupplemental. 9816–9821 | PNAS | June 19, 2012 | vol. 109 | no. 25 www.pnas.org/cgi/doi/10.1073/pnas.1116975109 Downloaded by guest on September 27, 2021 structural changes were speculated to show how SM proteins might transit from binding closed Sx to binding open Sx (30). Using protein pull-down experiments, chemical cross-linking, isothermal titration calorimetry, and small-angle scattering, we investigated the binding of two SM:Sx pairs: neuronal Munc18-1: Sx1a and adipocyte Munc18c:Sx4, sharing ∼40–50% identity. We found that in the absence of the N-peptide, Sx1a binds tightly to Munc18-1 in a closed conformation consistent with the published crystal structure. In the presence of the N-peptide, the interac- tion affinity is similar but a different, more extended Sx1a con- formation is bound to Munc18-1. On the other hand, Munc18c did not bind tightly to Sx4 lacking the N-peptide but did bind to Sx4 when the N-peptide was present; the binding mode in solution is similarly extended. We modeled the low-resolution structures of the complexes using scattering and cross-linking data that together suggest an open binding mode. Results Differential Role of the Sx N-Peptide. We used isothermal titration calorimetry (ITC) to evaluate the binding of Munc18 proteins to cognate Sxs with and without the N-peptide. The Munc18-1 results recapitulate those of others (14, 28, 32) showing that Sx1a and Sx1a lacking its N-peptide (Sx1aΔN) bind to Munc18-1 with similar high affinities (Kd, 1.4 and 10 nM, respectively; see Table S1 for full details). Fig. 1. Sx constructs and interactions. We used Sx1a and Sx4 with the We also investigated the interaction between the homologous transmembrane anchor replaced with a polyhistidine tag (Sx-His) and with proteins from adipocytes, Munc18c and Sx4, that regulate the the N-peptide removed (SxΔN-His). (A) Schematic showing different forms of trafficking of GLUT4 vesicles to the cell surface in response to membrane-anchored Sx: closed Sx, open Sx, binary complex with SNARE insulin signaling. We found that the Munc18c:Sx4 interaction (Kd partner SNAP (green), and ternary complex with SNARE partners [SNAP25 in 95 nM; Table S1) is weaker than Munc18-1:Sx1a. Strikingly, we green; VAMP2 (or synaptobrevin) in yellow]. The four-helix bundle of the were unable to detect any interaction between Munc18c and SNARE ternary complex is required for membrane fusion, and open Sx is Sx4ΔN by ITC, even with very high concentrations of Sx4ΔN(Fig. thought to be required to form SNARE complex. (B) Sx1a, Sx1aΔN, Sx4, and Δ Δ fi S1). Circular dichroism spectra of Sx4 N and Sx4 are equivalent, Sx4 N were immobilized on metal-af nity resin via the C-terminal poly- Δ histidine tags and incubated with detagged Munc18-1 or Munc18c.
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