Fig 1.
Schematic representation of the protein constructs used in this study.
In vivo full-length Sx consists of an N-peptide preceding an N-terminal α-helical bundle (the Habc domain), a SNARE motif (the H3 helix) and a C-terminal transmembrane region. We used soluble Sx1 and Sx4 constructs lacking the transmembrane domain for experiments reported here. ΔN indicates Sx constructs lacking the N-peptide. Munc18 and SNAP25 and VAMP2 constructs used in these experiments are also shown. The positions of engineered fusion tags and protease cleavage sites are as indicated.
Fig 2.
Munc18 proteins bind non-cognate Sxs.
Sx11-261-His or Sx41-275-His were incubated with detagged Munc18a or Munc18c for 2 h, 24 h or 48 h before the Sx was immobilized onto TALON™ Co2+ affinity beads for 1h and then washed. Samples of the beads were then run on SDS-PAGE and stained with Coomassie Blue to determine if detagged Munc18 had been pulled down by cognate and non-cognate Sx partners. Detagged Munc18a and Munc18c were also incubated for the same time periods on beads without bound Sx to monitor non-specific binding (control lanes). Solid vertical lines on the gel image denote the removal of intervening lanes or placing two different gels adjacent to each other.
Table 1.
ITC derived thermodynamic parameters for Munc18:Sx-His and Munc18:ΔNSx-His interactions.
Values reported are average and standard deviation from at least three experiments.
Fig 3.
Isothermal titration calorimetry data.
The raw data (upper part of each panel) and integrated normalized data (lower part of each panel) are shown from ITC experiments between HMunc18c or Munc18a-His and cognate/non-cognate Sx partners.
Fig 4.
Munc18 proteins bind weakly to non-cognate ΔNSxs.
ΔNSx125-261-His or ΔNSx430-275-His were incubated with detagged Munc18a or Munc18c for 2h, 24 h or 48 h before ΔNSx was immobilized onto TALON™ Co2+ affinity beads for 1h and then washed. Samples of the beads were then run on SDS-PAGE and stained with Coomassie Blue to determine if Munc18 proteins bound to ΔNSxs. Munc18 proteins were also incubated for the same time periods on beads without bound Sx to monitor non-specific binding (control lanes). Solid vertical lines on the gel image denote the removal of intervening lanes or placing two different gels adjacent to each other.
Fig 5.
Non-cognate Munc18 interactions with SNARE complexes.
(A) Cognate and non-cognate Munc18 proteins were first incubated with Sx11-261-His to form the Munc18:Sx11-261-His binary complex in the presence of beads. The beads were washed, then incubated with the Sx1 SNARE partners, SNAP25 and VAMP2, overnight at 4°C before SNARE complex formation was assessed by the presence of SNAP25 and VAMP2 on the beads (analysed by SDS-PAGE with Coomassie Blue staining). (B) Pre-formed SNARE complex comprising Sx11-261-His:SNAP25:VAMP2 was captured by beads and then incubated with detagged Munc18a or detagged Munc18c for 2 h at 4°C. The SNARE complex was captured using affinity beads, and the presence of bound Munc18 was evaluated by SDS-PAGE analysis and Coomassie Blue staining. Each image shown is representative of multiple replicate experiments, and the solid lines through the gel indicate where images of different gels have been joined.
Fig 6.
Specificity of Munc18:Syntaxin interactions.
Munc18a (cyan) binds Sx1 (magenta) via two tight binding modes (left hand side). One binding mode occurs in the presence of the Sx1 N-peptide, the other in its absence. “Non-cognate” Munc18c (gray) also binds tightly to Sx1, though its interaction with Sx1 lacking the N-peptide is very weak (indicated by dotted line). Munc18c binds more tightly than Munc18a to Sx4 (orange) but neither Munc18 recognises Sx4 lacking its N-peptide. These findings indicate that Munc18a and Munc18c bind Sxs differently. Specifically Munc18a has two tight binding modes/sites for Sx1 one of which does not require the N-peptide binding interaction. Munc18c has one tight binding mode/site for Sx4 or Sx1 that requires the Sx N-peptide.