Abstract
SNARE proteins are required for intracellular membrane fusion. In the neuron, the plasma membrane SNAREs syntaxin 1a and SNAP25 bind to VAMP2 found on neurotransmitter-containing vesicles. These three proteins contain "SNARE regions" that mediate their association into stable tetrameric coiled-coil structures. Syntaxin 1a contributes one such region, designated H3, and SNAP25 contributes two SNARE regions to the fusogenic complex with VAMP2. Syntaxin 1a H3 (syn1aH3) and SNAP25 can form a stable assembly, which can then be bound by VAMP2 to form the full SNARE complex. Here we show that syn1aH3 can also form a stable but kinetically trapped complex with the N-terminal SNARE region of SNAP25 (S25N). The crystal structure of this complex reveals an extended parallel four-helix bundle similar to that of the core SNARE and the syn1aH3-SNAP25 complexes. The inherent ability of syn1aH3 and S25N to associate stably in vitro implies that the intracellular fusion machinery must prevent formation of, or remove, any non-productive complexes. Comparison with the syn1aH3-SNAP25 complex suggests that the linkage of the N- and C-terminal SNAP25 SNARE regions is kinetically advantageous in preventing formation of the non-productive syn1aH3-S25N complex. We also demonstrate that the syn1aH3-S25N complex can be disassembled by alpha-SNAP and N-ethylmaleimide-sensitive factor.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The SNAREs syntaxin 1a, SNAP25, and VAMP2 participate in fusing neurotransmitter-filled vesicles with the plasma membrane of the cell, allowing neurotransmitter molecules to be released into the synaptic cleft
The Syntaxin 1a H3 (syn1aH3) construct consists of residues 191–266, so it includes all of the residues that interact with the partner SNAREs in the core complex [13] and the residues that link the SNARE region to the transmembrane anchor
Summary
Protein Expression and Purification—The following glutathione Stransferase (GST) or His fusion proteins were produced in Escherichia coli: rat GST-syn1aH3 [31] (residues 191–266), rat GST-syntaxin 1a (residues 1–266) [31], human His6-S25N [32], rat GST-S25C (residues 125–206), human GST-SNAP25 [32] (containing Cys to Ala mutations at positions 85, 88, 90, and 92 to facilitate purification), rat GSTVAMP2 (residues 1–93) [8], mouse GST-Vti1b (residues 142–199), human GST-syntaxin 7 H3 (residues 159 –239), human GSTSNAP23(N) (residues 1–78), rat GST-syntaxin 4 H3 [8], (residues 200 –273), His6-NSF [33], and His6-␣-SNAP [34]. Cells were harvested by centrifugation and resuspended in lysis buffer (250 mM NaCl, 20 mM Tris, pH 7.9, 2 mM 2-mercaptoethanol (-Me)), except for cells expressing His6-NSF. Cell lysates containing His6-tagged-proteins (except for His6-NSF) were loaded onto Ni-NTA-agarose columns (Qiagen), washed with 10 column volumes of lysis buffer, and eluted with increasing concentrations of imidazole. For purification of His6-NSF, cells were resuspended in NSF lysis buffer (100 mM Hepes/KOH, pH 7.1, 500 mM KCl, 10 mM ATP, 10% glycerol, 2 mM -Me, 1 mM phenylmethylsulfonyl fluoride, and one Complete-EDTA free (Roche Diagnostics) protease inhibitor mixture tablet per 50 ml of cell lysate). The eluted protein was loaded on a gel filtration column (S200 HR 26/60, Amersham Pharmacia Biotech) equilibrated in 25 mM Tris, pH 7.5, 300 mM KCl, 10% glycerol, 5 mM -Me, 1 mM ATP, and 1 mM EDTA.
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