Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide

Abstract

When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode.

Fig. 1.

Sx constructs and interactions. We used Sx1a and Sx4 with the transmembrane anchor replaced with a polyhistidine tag (Sx-His) and with the N-peptide removed (SxΔN-His). (A) Schematic showing different forms of membrane-anchored Sx: closed Sx, open Sx, binary complex with SNARE partner SNAP (green), and ternary complex with SNARE partners [SNAP25 in green; VAMP2 (or synaptobrevin) in yellow]. The four-helix bundle of the SNARE ternary complex is required for membrane fusion, and open Sx is thought to be required to form SNARE complex. (B) Sx1a, Sx1aΔN, Sx4, and Sx4ΔN were immobilized on metal-affinity resin via the C-terminal polyhistidine tags and incubated with detagged Munc18-1 or Munc18c. The gels at right and center show the proteins that were pulled down (bound) on equivalent amounts of resin after extensive washing. Resin-bound Sx1a and Sx1aΔN are both able to pull down Munc18-1, whereas Sx4 but not Sx4ΔN pulls down Munc18c. (Left) Proteins used in the experiment. Control shows the negative control interaction of Munc18 with resin. These data are representative of three replicates.

Fig. 2.

Munc18-1:Sx1a and Munc18-1:Sx1aΔN complexes differ. (A) SAXS data for Munc18-1:Sx1a (green) and Munc18-1:Sx1aΔN (gray). Inset shows the Guinier regions are linear. The calculated scattering profile from the closed Munc18-1:Sx1a crystal structure (solid line) is overlaid on the Munc18-1:Sx1aΔN SAXS data, showing an excellent correspondence (χ² = 0.6). By comparison, the Munc18-1:Sx1a scattering data fit less well to the crystal structure profile (χ² = 3.5). Data are shown on an absolute scale, where the Munc18-1:Sx1a scattering data have been offset by a factor of 10⁻¹ for clarity. Error bars represent propagated counting statistics. (B) Comparison of the low-angle portion of the scattering data for Munc18-1:Sx1aΔN and Munc18-1:Sx1a indicates a significant deviation, indicating differences between their structures. Data were normalized by protein concentration for this comparison. (C) Pair-distance distribution function, p(r), for Munc18-1:Sx1aΔN and Munc18-1:Sx1a derived from the scattering data using GNOM (46) indicates that D_max, the maximum dimension of the complex, is significantly larger for Munc18-1:Sx1a than for Munc18-1:Sx1aΔN.

Fig. 3.

Sx1a is extended when bound to Munc18-1. (A) SAXS data (gray) and neutron contrast variation data at 40% D₂O (red) and 100% D₂O (blue) for Munc18-1:Sx1a. Data are of high quality with linear Guinier regions (Inset) and yielding estimated molecular masses consistent with a 1:1 complex (Table S3). The calculated scattering profiles for the optimized model of the complex (solid lines) are overlaid on the data, with excellent visual correspondence. The χ² values are: 40%, 1.6; 100%, 4.5; X-ray, 0.4. The high value for the 100% D₂O is primarily because of small deviations of the fit to the data at low-q and are probably attributable to the misrepresentation of unstructured regions of Munc18, which dominates the signal in the 100% data. Data are shown on an absolute scale, where the 100% D₂O and X-ray scattering data have been off-set by factors of 10⁻¹ and 10⁻² for clarity. Error bars represent propagated counting statistics. (B) Pair-distance distribution function, p(r), derived from the scattering data using GNOM (46). This indicates that the increased maximum dimension of the Munc18-1:Sx1a complex is attributable to Sx1a (red curve), indicating that in the Munc18-1:Sx1a complex, Sx1a adopts an extended conformation.

Fig. 4.

An open binding mode of Munc18:Sx complexes. (A) Crystal structure of closed Sx1a (red) bound to Munc18-1. (B) Model of Munc18-1 (blue) in complex with Sx1a (red) complex, refined against solution scattering data and distance restraints, indicating that Sx1a adopts an open conformation when bound to Munc18-1. (C) Model of Munc18c (blue) in complex with Sx4 (red), refined against solution scattering data and distance restraints, indicating that Sx4 adopts an open conformation when bound to Munc18c. For both B and C, the position of the H3 helix is not definitive. (D) Schematic showing potential Munc18-1:Sx1a interactions. Munc18-1 interacts with closed Sx1a when the N-peptide is not engaged. Open Sx1a binding to Munc18-1 requires the Sx1a N-peptide. Munc18-1 binding to open Sx1a may precede SNARE complex formation with partner SNAREs SNAP25 (green) and VAMP2 (yellow). (E) It is not clear whether Sx4 exists in a closed conformation. Munc18c binds to open Sx4, and this requires an N-peptide interaction. Munc18c binding to open Sx4 may precede SNARE complex formation with SNAP23 (green) and VAMP2 (yellow).