Insertion of the Membrane-proximal Region of the Neuronal SNARE Coiled Coil into the Membrane

In the neuron, solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins assemble into an α-helical coiled coil that bridges the synaptic vesicle to the plasma membrane and drives membrane fusion, a required process for neurotransmitter release at the nerve terminal. How does coiled coil formation drive membrane fusion? To investigate the structural and energetic coupling between the coiled coil and membrane, the recombinant SNARE complex in the phospholipid bilayer was studied using fluorescence quenching and site-directed spin labeling EPR. Fluorescence analysis revealed that two native Trp residues at the membrane-proximal region of the coiled coil are inserted into the membrane, tightly coupling the coiled coil to the membrane. The EPR results indicate that the coiled coil penetrates into the membrane with an oblique angle, providing a favorable geometry for the basic residues to interact with negatively charged lipids. The result supports the proposition that core complex formation directly leads to the apposition of two membranes, which could facilitate lipid mixing. Trp residues and basic residues are abundant at the membrane-proximal region of transmembrane SNARE proteins, suggesting the generality of the proposed mechanism for the SNARE complex-membrane coupling. In the neuron, solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins assemble into an α-helical coiled coil that bridges the synaptic vesicle to the plasma membrane and drives membrane fusion, a required process for neurotransmitter release at the nerve terminal. How does coiled coil formation drive membrane fusion? To investigate the structural and energetic coupling between the coiled coil and membrane, the recombinant SNARE complex in the phospholipid bilayer was studied using fluorescence quenching and site-directed spin labeling EPR. Fluorescence analysis revealed that two native Trp residues at the membrane-proximal region of the coiled coil are inserted into the membrane, tightly coupling the coiled coil to the membrane. The EPR results indicate that the coiled coil penetrates into the membrane with an oblique angle, providing a favorable geometry for the basic residues to interact with negatively charged lipids. The result supports the proposition that core complex formation directly leads to the apposition of two membranes, which could facilitate lipid mixing. Trp residues and basic residues are abundant at the membrane-proximal region of transmembrane SNARE proteins, suggesting the generality of the proposed mechanism for the SNARE complex-membrane coupling. solubleN-ethylmaleimide-sensitive factor attachment protein receptor 7-Br2-PC, 1-palmitoyl-2-stearoyl-(6,7)-dibromo-sn-glycero-3-phosphocholine 12-Br2-PC, 1-palmitoyl-2-stearoyl-(11,12)-dibromo-sn-glycero-3-phosphocholine 1,2-dioleoyl phosphatidylserine electron paramagnetic resonance nickel (II)-ethylenediamine-N,N′-diacetic acid n-octylglucoside phosphate-buffered saline pH 7.4, 0.057 (v/v) Tween 20, 10 mM l-methionine 1-palmitoyl-2-oleoyl phosphatidylcholine 25-kDa, solubleN-ethylmaleimide-sensitive factor attachment protein transmembrane domain(s) vesicle-associated membrane protein 2 (1-oxyl-2,2,5,5-tetramethylpyrrolinyl-3-methyl) methanethiosulfonate spin label Neurotransmitter release at synapses requires the fusion of neurotransmitter-containing vesicles to the presynaptic plasma membrane. Membrane fusion is, however, an exceedingly difficult process to go through without the assistant of specific proteins, because of the protective nature of the biological membranes. In the neuron, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)1 proteins play an essential role in promoting membrane fusion (1Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Google Scholar). It is proposed that assembly of the SNARE complex initially bridges two membranes, induces lipid mixing, and leads to the hemifusion state and the fusion pore, of which the detailed mechanism is largely unknown (2Rothman J.E. Nature. 1994; 372: 55-63Google Scholar, 3Jahn R. Südhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Google Scholar, 4Lin R.C. Scheller R.H. Annu. Rev. Cell Dev. Biol. 2000; 16: 19-49Google Scholar, 5Brunger A.T. Curr. Opin. Struct. Biol. 2001; 11: 163-173Google Scholar). Progress has been made in understanding the biophysical principles of SNARE assembly. SNARE assembly starts with the interaction of vesicle-associated membrane protein 2 (VAMP2 or synaptobrevin) with target plasma membrane SNAREs Syntaxin 1A and SNAP-25. Interactions between SNARE proteins are mediated by 舠SNARE motifs舡 that are essentially coiled coil sequences and are present in all SNARE proteins (3Jahn R. Südhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Google Scholar). For the SNARE complex, one SNARE motif each from Syntaxin 1A and VAMP2 and two from SNAP-25 assemble into a 110-Å-long four-stranded coiled coil (6Poirier M.A. Xiao W. Macosko J.C. Chan C. Shin Y.-K. Bennett M.K. Nat. Struct. Biol. 1998; 5: 765-769Google Scholar, 7Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Google Scholar). It is worthwhile to note that target plasma membrane SNAREs Syntaxin 1A and SNAP-25 also spontaneously assemble into a similar but less stable four-stranded coiled coil (8Xiao W. Poirier M.A. Bennett M.K. Shin Y.-K. Nat. Struct. Biol. 2001; 8: 308-311Google Scholar, 9Margittai M. Fasshauer D. Pabst S. Jahn R. Langen R. J. Biol. Chem. 2001; 276: 13169-13177Google Scholar, 10Zhang F. Chen Y. Kweon D.-H. Kim C.S. Shin Y.-K. J. Biol. Chem. 2002; 277: 24294-24298Google Scholar). How does coiled coil formation lead to membrane fusion? There are two features of the SNARE coiled coil that might be important. First, the helices are all aligned parallel, suggesting the co-location of two membrane attachment points, which sets up a favorable geometry for membrane fusion (6Poirier M.A. Xiao W. Macosko J.C. Chan C. Shin Y.-K. Bennett M.K. Nat. Struct. Biol. 1998; 5: 765-769Google Scholar, 7Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Google Scholar, 11Hanson P.I. Roth R. Morisaki H. Jahn R. Heuser J.E. Cell. 1997; 90: 523-535Google Scholar, 12Lin R.C. Scheller R.H. Neuron. 1997; 19: 1087-1094Google Scholar, 13Katz L. Hanson P.I. Heuser J.E. Brennwald P. EMBO J. 1998; 17: 6200-6209Google Scholar). Second, the coiled coil is highly stable (14Fasshauer D. Bruns D. Shen B. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 4582-4590Google Scholar, 15Fasshauer D. Otto H. Eliason W.K. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 28036-28041Google Scholar). Therefore, coiled coil formation might have the capacity to overcome the repulsive force between two apposing membranes. Although this mechanistic model appears to be structurally and energetically attractive, there are caveats that require careful consideration. For example, if the SNARE core were tethered with flexible linkers to membrane domains, coiled coil formation might not be able to bring about membrane apposition no matter how strong the pulling force it generates because the energy would be dissipated. To validate this model, a direct coupling between the coiled coil and membranes appears to be necessary. Previously, Brunger and co-workers proposed a hypothetical model for the coiled coil-to-membrane coupling (7Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Google Scholar). In this model, the coiled coil is linked to transmembrane domains (TMD) as continuous helices. This model arbitrarily assumes some bending flexibility of helices in short amino acid stretches at the membrane-proximal region. Furthermore, helix-disrupting mutations or amino acid insertions in the linker region have little or only moderate effect on the SNARE fusion activity, inconsistent with this model (16McNew J.A. Weber T. Engelman D.M. Sollner T.H. Rothman J.E. Mol. Cell. 1999; 4: 415-421Google Scholar,17Wang Y. Dulubova I. Rizo J. Südhof T.C. J. Biol. Chem. 2001; 276: 28598-28605Google Scholar). How then is the coiled coil energetically coupled to membranes? The answer to this fundamental question hinges on structural information of the connection of the coiled coils to the membranes. Recent EPR investigations of intact SNAREs using site-directed spin labeling EPR have yielded new results that not only confirm the existence of coupling between the coiled coil and the membrane but also suggest a tentative mechanism of the SNARE core-membrane coupling. EPR analysis indicated that the linker region of Syntaxin 1A, enriched with basic amino acid residues, is unstructured but laterally inserted into the membrane, tightly coupling the coiled coil to the membrane (18Kweon D.-H. Kim C.S. Shin Y.-K. Biochemistry. 2002; 41: 9264-9268Google Scholar,19Kim C.S. Kweon D.-H. Shin Y.-K. Biochemistry. 2002; 41: 10928-10933Google Scholar). Importantly, clusters of basic amino acid residues are found in the linker regions of all transmembrane SNAREs (20Weimbs T. Mostov K. Low S.H. Hofmann K. Trends Cell Biol. 1998; 8: 260-262Google Scholar), raising the possibility that SNARE linker regions generally insert into the membrane. Further, this tentative model offers a plausible explanation as to why this region is tolerant to helix-disrupting mutations. However, structure and membrane topology of the VAMP2 linker region is not experimentally confirmed yet. In this work, we report the EPR and fluorescence investigations of the membrane topology of the recombinant SNARE complex. Fluorescence quenching analysis revealed that the native Trp residues at positions 89 and 90 in VAMP2 are inserted into the acyl chain region of the bilayer. Further, the EPR results reveal that the core domain maintains the coiled coil structure up to residue 92, suggesting that the SNARE coiled coil is partially inserted into the head group region of the bilayer. The EPR data also suggest that the coiled coil penetrates into the membrane with an oblique angle. Taken together, the new results further establish the concept of the tight SNARE core-membrane coupling, providing structural basis for the force transmission from the core region to the membrane during SNARE assembly. 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1,2-dioleoyl phosphatidylserine (DOPS), 1-palmitoyl-2-stearoyl-(6,7)-dibromo-sn-glycero-3-phosphocholine (6,7-Br2-PC), and 1-palmitoyl-2-stearoyl-(11,12)-dibromo-sn-glycero-3-phosphocholine (11,12-Br2-PC) were purchased from Avanti Polar Lipids (Birmingham, AL). (1-Oxyl-2,2,5,5-tetramethylpyrrolinyl-3-methyl) methanethiosulfonate spin label (MTSSL) was obtained from Toronto Research Chemicals (North York, Canada). The paramagnetic reagent, nickel (II)-ethylenediamine-N,N′-diacetic acid (NiEDDA) was synthesized following the procedure described elsewhere (21Altenbach C. Greenhalgh D.A. Khorana H.G. Hubbell W.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1667-1671Google Scholar). Pfu Turbo DNA polymerase and Escherichia coli BL21-CodonPlus RIL were purchased from Stratagene (La Jolla, CA). n-Octylglucoside (OG) was from Roche Molecular Biochemicals. Bio-beads SM2 was obtained from Bio-Rad. Oligonucleotides for site-directed mutagenesis were obtained from Qiagen Operon Technologies (Alameda, CA). Ultrafree Centrifugal Devices Biomax-5K for proteins was obtained from Millipore (Bedford, MA). Nickel-nitrilotriacetic acid-agarose was purchased from Qiagen. Glutathione-agarose, human thrombin, 4-(2-aminoethyl)benzenesulfonyl fluoride, leupeptin, l-methionine, n-lauroyl sarcosine, Triton X-100, Tween 20, glycerol, l-methionine, isopropyl-ॆ-d-thiogalactopyranoside, dithiothreitol, ampicillin sodium salt, chloramphenicol, kanamycin, and the for were all purchased from VAMP2 the soluble SNARE motif of Syntaxin 1A Syntaxin and the SNARE motif of SNAP-25 are inserted in the as fusion proteins J.E. Biochem. Scholar). the the SNARE motif of SNAP-25 is in the as a To a for the specific native of VAMP2 was to of the were by site-directed mutagenesis and confirmed by DNA DNA fusion proteins were in coli BL21-CodonPlus RIL and using the were at in with ampicillin and the of the were further for at for at for VAMP2 but at for the Syntaxin 1A To the the was in pH 7.4, with Tween are 10 l-methionine, 2 fluoride, leupeptin, and 2 The were by on the For Triton (v/v) and n-lauroyl were to the for the was at for at The was then with in and the was at for The protein were with an of for and Syntaxin or with with Triton for The were the protein was to the the with without dithiothreitol, a of was The was initially with at for and to at was by with and the protein was from the with in the pH Triton The protein was coli and using acid-agarose the were at in with ampicillin and the of the were further for at For protein the was in pH 2 fluoride, on the was at for at The was with acid-agarose in A. The was at for the were with an of A. The protein was by protein using as a The spin labeling was by the with the at labeling was for all VAMP2 SNARE of SNAP-25 the were to the formation of complex D.-H. Chen Y. F. Poirier M. Kim C.S. Shin Y.-K. Biochemistry. 2002; 41: Scholar). was to acid-agarose of Syntaxin 1A and VAMP2 of were to the The Triton The was to at for with the recombinant SNARE complex was with with and The complex was using a the process was to pH with The protein was in the of vesicles with a were in without using an of of were with two of the SNARE complex. was to the to a of with an of the were the Bio-beads SM2 at for the the was The were at for to the protein and the of EPR were obtained using a with a and a The was at no of the The were at in The to the protein was with the EPR for the For the were obtained from the of the of the EPR as a of in the were obtained with with and with in the of the the is to of was The is the in the and of a paramagnetic The is to the the of the to the as and is to be to the W. The is on the from a of lipid at acyl chain For fluorescence membrane were from the recombinant SNARE complex with the lipid was the of the SNARE complex was For of the were to the membrane to the in the of and protein The fluorescence were with fluorescence The were at and the were in the of The fluorescence was obtained by the in this The of quenching was to the following and are the fluorescence in the and of is the for and is the of the The a for a of Scholar). fluorescence the membrane were with Triton to the quenching in a To the membrane of Trp residues, membrane two of lipid were or was in of of the at The of SNARE complex was by the procedure described The of quenching was as a of the of The of two Trp residues was to the analysis Biochemistry. Scholar, A. Biochemistry. Scholar). The of the Trp residue from the bilayer is by the 2 the from the bilayer to the for is the of the by the of the lipid are the fluorescence of the and and is the in the of the two or The of the region was to be Biochemistry. Scholar). For the of the data for the were For fluorescence the recombinant complex was from soluble Syntaxin 1A, and two SNARE from SNAP-25 The of Syntaxin 1A was not this to the of two in one membrane and to the complex in which two are to two apposing membranes. In the recombinant SNARE complex, there are two native Trp of at the membrane-proximal region of VAMP2 and and to the coiled coil in the core structure This is for the of the coiled coupling using First, to residues are to the or we Trp fluorescence in the of an that is and into the For the SNARE complex, the fluorescence as the suggesting that Trp residues are In for the complex, little in the of This result that Trp residues in the SNARE complex are from the suggesting the possibility of into the membrane. the of Trp residues into the membrane is in which are to the acyl chain of the In the of a Trp fluorescence is only Trp is in with the acyl chain region of the membrane. we would a the of the lipid a in was suggesting that Trp residues are inserted into the membrane. The of Trp residues was on quenching by the lipid lipid with at the and and the lipid Trp residues are the of lipids. there are two Trp residues, the be an of two In and to the coiled coil are inserted in the membrane in the SNARE complex. To investigate the membrane topology of the SNARE complex further using site-directed spin labeling residues of VAMP2 the were with to which a spin label was to the region with EPR For EPR the recombinant complex was from soluble Syntaxin 1A, and two SNARE from SNAP-25. recombinant were of the complex, which is one of the core complex, as confirmed with not of the SNARE complex into vesicles of the EPR were for at The EPR is to the of the EPR in are all There are structural that might have to the of the W.L. A. Langen R. M.A. Curr. Opin. Struct. Biol. 1998; 8: Scholar, M.A. K. Hubbell W.L. Biochemistry. Scholar, L. Hubbell W.L. Trends Biochem. Sci. 2002; The of the because of the α-helical could have the of the with of the protein would the of the the basis of the structure (7Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Google Scholar), we that the four-stranded coiled coil structure up to residue 92, which to between helices. the fluorescence it is that and are inserted into the acyl chain region of the bilayer. Therefore, we that a of the region is in the membrane, which into the membrane and the head group region. It is that the of all to the EPR to the possibility of between SNARE we EPR in which the by the interaction is Shin Y.-K. Proc. Natl. Acad. Sci. U. S. A. 92: Scholar). of the EPR with the confirmed that SNARE are from each the not the possibility of of the SNARE complex. It is to note that EPR for positions are less In EPR for positions for the to the linker region of Syntaxin 1A (18Kweon D.-H. Kim C.S. Shin Y.-K. Biochemistry. 2002; 41: 9264-9268Google Scholar, C.S. Kweon D.-H. Shin Y.-K. Biochemistry. 2002; 41: 10928-10933Google Scholar). The EPR is a for the of the in a of the the M.A. K. Hubbell W.L. Biochemistry. Scholar). However, the is not to information the and the Furthermore, the of the protein into the bilayer the for the SNARE complex. we the EPR to the structure and the membrane topology of the linker region. For the the EPR the to a paramagnetic as to the of the or the to a paramagnetic as to for example, the into the membrane J.C. Kim Shin Y.-K. J. Mol. Biol. 1997; Scholar, Y.-K. C. F. Hubbell W.L. Scholar, C. T. Khorana H.G. Hubbell W.L. Scholar). In and for the SNARE complex are the residue an of we an of however, there are and for the which might a structure as Further, the that to be in to of the is less an of and has been found for α-helical at which the fusion of and a J.C. Kim Shin Y.-K. J. Mol. Biol. 1997; Scholar, Nat. Struct. Biol. 2001; 8: Scholar, Shin Y.-K. Biochemistry. 1999; Scholar). the of to has been to be a to the For a we a of this the with a of In the which is as the of the of to is as a of residue a of the the In it appears that there is a of the in the region of residues To this we the data with a of the residue which the α-helical In this we also into the of the the as a to the The EPR data are with the in the nature of the the EPR results into the of the α-helical with the structure from the structure in which the residue of VAMP2 is The also that the is with to the membrane the of the could not be from the For the of to has been to a to the membrane However, EPR for positions and highly with might be to some of the in the complex. This the of the membrane analysis in the region of residues To it is that and are inserted into the membrane from the lipid EPR for positions of It is also highly that residues are from the with the α-helical For we the with obtained from the in the membrane of the lipid the similar of the SNARE complex. The analysis revealed that positions are all inserted into the membrane to the of the the EPR data and the fluorescence we that the of the coiled coil is inserted into the membrane at an oblique angle. Further, we that residues might be unstructured inserted into the membrane. The of VAMP2 residues and in the SNARE complex the membrane is not into the that Trp residues are found in membrane proteins the S.H. Nat. Struct. Biol. Scholar). It is further by the that the membrane-proximal region of VAMP2 has phospholipid S. C. R. K. M. Proc. Natl. Acad. Sci. U. S. A. 2000; Scholar). The of Trp to the membrane the of the structure and topology of membrane proteins Shin Y.-K. Biochemistry. Scholar). Trp residues are also found in in the linker region of transmembrane SNARE proteins (20Weimbs T. Mostov K. Low S.H. Hofmann K. Trends Cell Biol. 1998; 8: 260-262Google Scholar), of Trp residues in specific of SNAREs and which is to membrane The EPR results suggest that a of the SNARE coiled coil penetrates into the membrane with an oblique angle. a topology a favorable geometry for basic residues and to be able to interact with the membrane to the each basic residue to the energy of the J. M. H. S. J. Scholar). Therefore, we that the would be to the energy from Trp residues and basic residues, we that the SNARE complex is highly stable with the energy of as as that the of VAMP2 is to the membrane Engelman D.M. 2001; Scholar). However, the EPR results suggest that the coiled coil domain of the complex is with to the membrane, bending or at the linker region. The EPR results suggest that positions are strong for the connection between the and the The EPR analysis that positions are in the acyl chain region. In parallel, EPR the of the linker region and of Syntaxin 1A the membrane (18Kweon D.-H. Kim C.S. Shin Y.-K. Biochemistry. 2002; 41: 9264-9268Google Scholar, C.S. Kweon D.-H. Shin Y.-K. Biochemistry. 2002; 41: 10928-10933Google Scholar). It was found that this region is laterally inserted into the membrane, pulling the SNARE core the membrane. However, one be careful in the EPR EPR the of the the not report the of the native amino the native residues are charged amino For EPR the of for However, the of the chain be The on to the on to is for the model for the complex that up results is in In this model, the tight coupling between the SNARE core and two apposing membranes is by the between the basic residues and the negatively charged in the membrane and the of Trp residues into the membrane. that the energy of the from two to The of the energy for membrane fusion is as as P.I. J. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar), a of which from the of a the lipid and the hemifusion that coiled coil formation generates force to overcome this energy The question is, how is the force from the coiled coil to the membrane To the coiled coil be structurally coupled to the membrane the force by the coiled coil formation would be dissipated. The EPR analysis that there is structural coupling between the coiled coil and the membrane. Furthermore, we that this coupling to However, it appears that the coupling force is the membrane force during fusion Therefore, one SNARE complex might not have the capacity to the membrane force during This energetic be overcome by the of one SNARE complex D.-H. Chen Y. F. Poirier M. Kim C.S. Shin Y.-K. Biochemistry. 2002; 41: Scholar, M. Otto H. Jahn R. 1999; Scholar, R. J. B. D. J. Biol. Chem. 2000; Scholar, H. K. T. H. H. T. Cell. 2001; Scholar). In it has been that SNARE for the fusion Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar). of the SNARE coiled coil to the membrane appears to have the complex. In the the the of which is as as (7Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Google Scholar), is between two membranes. the coiled coil were not inserted into the membrane, the of two membranes would be by the existence of the coiled coil in the In it has been that the SNARE complex might membrane apposition A. Trends Biochem. Sci. 2001; Scholar). this two membranes would on the and would not to fusion Annu. Rev. Scholar). The oblique of the of the coiled coil would a to this The of Trp and the basic residues is by the using the VAMP2 it has been that the of human Further, similar of human has been for the S. C. L. K. K. C. M. EMBO J. 2002; Scholar). SNARE proteins are to the membrane fusion in the However, membrane fusion is by the There is that the vesicle protein is a Nat. Mol. Cell. Biol. 2002; Scholar, T.C. J. Biol. Chem. 2000; 277: Scholar). Although have that not only to the membrane in a but also with SNARE and of into the region of the similar to the linker regions of SNARE complex J. J. Biol. Chem. 2000; Scholar). that the interaction between SNARE and might in the membrane further

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.