Probing Domain Swapping for the Neuronal SNARE Complex with Electron Paramagnetic Resonance † Dae-Hyuk Kweon, ‡ Yong Chen, ‡ Fan Zhang, Michelle Poirier, § Chang Sup Kim, and Yeon-Kyun Shin* Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State UniVersity, Ames, Iowa 50011 ReceiVed February 7, 2002; ReVised Manuscript ReceiVed March 11, 2002 ABSTRACT: Highly conserved soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins control membrane fusion at synapses. The target plasma membrane-associated SNARE proteins and the vesicle-associated SNARE protein assemble into a parallel four-helix bundle. Using a novel EPR approach, it is found that the SNARE four-helix bundles are interconnected via domain swapping that is achieved by substituting one of the two SNAP-25 helices with the identical helix from the second four-helical bundle. Domain swapping is likely to play a role in the multimerization of the SNARE complex that is required for successful membrane fusion. The new EPR application employed here should be useful to study other polymerizing proteins. Neurotransmitter release at synapses requires fusion of synaptic vesicles with the presynaptic plasma membrane. In this process, the target plasma membrane proteins syntaxin 1A and SNAP-25 1 (synaptosome-associated protein of 25000 Da), and the VAMP2 (vesicle-associated membrane protein 2) interact with each other to form the ternary SNARE complex (1, 2). The C-terminal domain of syntaxin, two separate helix domains from SNAP-25, and a soluble domain of VAMP2 are assembled into a core structure that is a 110 Å long parallel four-stranded coiled coil (3, 4). In full-length SNAP-25 two helix domains are, however, flanked by a 56 amino acid region. This putative loop appears to be suf- ficiently long to transverse the coiled coil, connecting the two SNAP-25 helices from tail to head. Interestingly, when the flanking loop region of SNAP-25 is present in the complex, several higher molecular mass bands are observed in the SDS-PAGE than that of the core complex (5, 6) (Figure 1a). The flanking loop region might play an essential role in causing the oligomerization of the SNARE complex. Multimerization has been observed for the native SNARE complex as well (7). Multimerization of the SNARE complex is required for membrane fusion as it is for viral membrane fusion systems (8, 9). One possible mechanism for the SNARE complex oligo- merization is domain swapping (10, 11): a domain in one molecule is replaced with the identical domain from the second molecule (Figure 1b). Such swapping scheme may propagate to make a polymeric protein chain (Figure 1b). The polymerization via domain swapping has been proposed as the main mechanism for promoting the disease-causing fibril formation for proteins including human cystatin C (12) and prion proteins (13). Thus, what is learned from the SNARE oligomerzation might help to understand better the mechanisms of protein polymerization. EXPERIMENTAL PROCEDURES Plasmid Construction and Mutagenesis. Recombinant glutathione S-transferase (GST) fusion proteins were ex- † Support for this work is provided by National Institutes of Health Grant GM51290. * Corresponding author. E-mail: colishin@iastate.edu. Phone: (515) 294-2530. Fax: (515) 294-0453. ‡ Contributed equally to this paper. § Current address: Department of Psychiatry, Johns Hopkins Uni- versity School of Medicine, Baltimore, MD 21205. 1 Abbreviations: EPR, electron paramagnetic resonance; GST, glu- tathione S-transferase; IPTG, isopropyl -D-galactopyranoside; LB, Luria-Bertini broth; MTSSL, (1-oxy-2,2,5,5-tetramethylpyrroline-3- methyl)methanethiosulfonate; OD 600, optical density at 600 nm; PBST- Met, phosphate-buffered saline, pH 7.4, 0.05% (v/v) Tween-20, and 10 mM L-methionine; SNAP-25, synaptosome-associated protein of 25000 Da; SNARE, soluble N-ethylmaleimide sensitive factor attach- ment protein receptor; VAMP2 (or v-SNARE), vesicle-associated membrane protein 2. FIGURE 1: (a) Sodium dodesyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of the SNARE complexes. Ternary SNARE complexes are resistant to SDS. Lane S is the size marker. The lane 1 shows the appearance of the high molecular weight bands for the SNARE complex made of VAMP2 (amino acids 1-94), syntaxin 1A (amino acids 191-266), and SNAP-25 (amino acids 1-206), consistent with the western blot analysis in a previous report (3). Lanes 2-4 show SDS-PAGE analysis of the trypsin- digested SNARE complex for 3, 10, and 30 min, respectively. The trypsin-treated core SNARE complex migrates near 35 kDa, similar to the previous report (3). (b) Hypothetical models of the SNARE complex. Bottom: nondomain swapped SNARE complex. Middle: domain swapped dimer. Top: polymerized SNARE complex. The components of the four-helix bundle are color coded {green, syntaxin 1A; blue, VAMP2; red, N-terminal helix of SNAP-25 [SNAP-25(N)]; yellow, C-terminal helix of SNAP-25 [SNAP-25(C)]}. 5449 Biochemistry 2002, 41, 5449-5452 10.1021/bi0256476 CCC: $22.00 © 2002 American Chemical Society Published on Web 04/06/2002