Self-association of the H3 Region of Syntaxin 1A

Abstract

Intracellular membrane fusion requires SNARE proteins found on the vesicle and target membranes. SNAREs associate by formation of a parallel four-helix bundle, and it has been suggested that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A, designated H3, contributes one of the four helices to the SNARE complex. In the crystal structure of syntaxin 1A H3, four molecules associate as a homotetramer composed of two pairs of parallel helices that are anti-parallel to each other. The H3 oligomer observed in the crystals is also found in solution, as assessed by gel filtration and chemical cross-linking studies. The crystal structure reveals that the highly conserved Phe-216 packs against conserved Gln-226 residues present on the anti-parallel pair of helices. Modeling indicates that Phe-216 prevents parallel tetramer formation. Mutation of Phe-216 to Leu appears to allow formation of parallel tetramers, whereas mutation to Ala destabilizes the protein. These results indicate that Phe-216 has a role in preventing formation of stable parallel helical bundles, thus favoring the interaction of the H3 region of syntaxin 1a with other proteins involved in membrane fusion. Intracellular membrane fusion requires SNARE proteins found on the vesicle and target membranes. SNAREs associate by formation of a parallel four-helix bundle, and it has been suggested that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A, designated H3, contributes one of the four helices to the SNARE complex. In the crystal structure of syntaxin 1A H3, four molecules associate as a homotetramer composed of two pairs of parallel helices that are anti-parallel to each other. The H3 oligomer observed in the crystals is also found in solution, as assessed by gel filtration and chemical cross-linking studies. The crystal structure reveals that the highly conserved Phe-216 packs against conserved Gln-226 residues present on the anti-parallel pair of helices. Modeling indicates that Phe-216 prevents parallel tetramer formation. Mutation of Phe-216 to Leu appears to allow formation of parallel tetramers, whereas mutation to Ala destabilizes the protein. These results indicate that Phe-216 has a role in preventing formation of stable parallel helical bundles, thus favoring the interaction of the H3 region of syntaxin 1a with other proteins involved in membrane fusion. Eukaryotic cells transport cargo between different intracellular compartments, and release selective cargo into the extracellular space. This task requires that transport vesicles bud from the membranes of organelles and fuse specifically with target membranes. An extensively studied example of this process is synaptic vesicle exocytosis, in which neurotransmitter-filled vesicles fuse with the plasma membrane of a presynaptic neuron to release neurotransmitter into the synaptic cleft (1Jahn R. Sudhof T.C. Annu. Rev. Biochem. 1999; 68: 3250-3262Crossref Scopus (1034) Google Scholar). The process of directed docking and fusion of vesicles is mediated by several proteins; these include the soluble NSF 1The abbreviations used are:NSFN-ethylmaleimide-sensitive factorSNARESNAP receptorv-SNAREvesicle SNAREt-SNAREtarget SNAREβ-Meβ-mercaptoethanolMADmultiwavelength anomalous dispersionPAGEpolyacrylamide gel electrophoresisCDcircular dichroismGSTglutathione S-transferaser.m.s.d.root mean square deviationSNAPsoluble NSF attachment protein attachment proteins (SNAPs), the SNAP receptor proteins (SNAREs) NSF, Sec1 proteins, and the Rab small GTPases. In neurons, the SNARE proteins involved in this process are VAMP2, syntaxin 1a, and SNAP-25. N-ethylmaleimide-sensitive factor SNAP receptor vesicle SNARE target SNARE β-mercaptoethanol multiwavelength anomalous dispersion polyacrylamide gel electrophoresis circular dichroism glutathione S-transferase root mean square deviation soluble NSF attachment protein The SNARE proteins are believed to be directly involved in the intracellular membrane fusion event. SNAREs are associated with vesicle (v-SNARE) and target (t-SNARE) membranes and can interact with each other by forming stable four-helix bundles (SNARE complexes). The synaptic vesicle v-SNARE VAMP2 contains a small cytoplasmic domain followed by a C-terminal transmembrane anchor, and the cytoplasmic domain is largely unstructured in isolation (2Fasshauer D. Otto H. Eliason W.K. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 3Hazzard J. Sudhof T.C. Rizo J. J. Biomol. NMR. 1999; 14: 203-207Crossref PubMed Scopus (75) Google Scholar). The t-SNARE SNAP-25 is attached to the plasma membrane via palmitoylation of four cysteine residues and displays a significant amount of disorder in isolation in solution (2Fasshauer D. Otto H. Eliason W.K. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 4Fasshauer D. Bruns D. Shen B. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 4582-4590Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). However, both VAMP2 and SNAP-25 adopt helical conformations when part of the SNARE complex. The t-SNARE syntaxin 1a consists of an N-terminal domain termed Habc, a region of ∼9 kDa termed H3, and a C-terminal transmembrane anchor. The Habc domain is a three-helix bundle (5Fernandez I. Ubach J. Dulubova I. Zhang X. Sudhof T.C. Rizo J. Cell. 1998; 94: 841-849Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 6Lerman J.C. Robblee J. Fairman R. Hughson F.M. Biochemistry. 2000; 39: 8470-8479Crossref PubMed Scopus (94) Google Scholar), and the H3 region forms an amphipathic helix that associates with either partner SNAREs or the Habc domain (7Sutton R.B. Fasshauer D. Jahn R. Brünger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1953) Google Scholar, 8Misura K.M.S. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (625) Google Scholar). The three SNARE proteins have a modest affinity for each other in binary complexes (∼1 μm), but the ternary SNAP-25·VAMP2·syntaxin 1a SNARE complex is extremely stable (9Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (672) Google Scholar). The x-ray crystal structure of the “core” SNARE complex, which consists of the cytoplasmic portion of VAMP2, two fragments of SNAP-25, and the H3 region of syntaxin 1a, reveals that these three proteins form a 110-Å-long parallel four-helix bundle (7Sutton R.B. Fasshauer D. Jahn R. Brünger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1953) Google Scholar). Syntaxin 1a H3 and VAMP2 each contribute one helix to the complex, and SNAP-25 contributes two helices. When bound to the regulatory protein nSec1, syntaxin 1a exists as a four-helix bundle (8Misura K.M.S. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (625) Google Scholar). The N-terminal half of H3 contributes one helix to the bundle, and the C-terminal half consists of a mixture of short helices and random coils. Because only the H3 region participates in the ternary 7 S complex, it has been proposed that syntaxin 1a must undergo large conformational changes to allow Habc to dissociate from H3, and allow syntaxin 1a to bind VAMP2 and SNAP-25 (10Rice L.M. Brennwald P. Brunger A.T. FEBS Lett. 1997; 415: 49-55Crossref PubMed Scopus (60) Google Scholar, 11Dulubova I. Sugita S. Hill S. Hosaka M. Fernendez I. Sudhof T.C. Rizo J.A. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (561) Google Scholar). It has also been observed that syntaxin 1a can form stable binary complexes with SNAP-25 in vitro and that these complexes may be a precursor to SNARE complex assembly (4Fasshauer D. Bruns D. Shen B. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 4582-4590Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 12Nicholson K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nature Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (177) Google Scholar). Syntaxin 1a associates with many other proteins in the presynaptic cell (13Fujita Y. Shirataki H. Sakisaka T. Asakura T. Ohya T. Kotani H. Yokoyama S. Nishioka H. Matsuura Y. Mizoguchi A. Scheller R.H. Takai Y. Neuron. 1998; 20: 905-915Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 14Rettig J. Sheng Z.-H. Kim D.K. Hodson C.D. Snutch T.P. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7363-7368Crossref PubMed Scopus (273) Google Scholar, 15Woodman P.G. Biochim. Biophys. Acta. 1997; 1357: 155-172Crossref PubMed Scopus (54) Google Scholar), and it is possible that other conformations of the protein exist but have been the role of syntaxin 1a in membrane have the x-ray crystal structure of the H3 region of syntaxin The structure reveals that H3 forms a and that a conserved may the of the four helices with to each other. Mutation of this to or different on the H3 as assessed gel filtration chemical and circular dichroism The structure also for SNARE complex assembly and may a for SNARE precursor The H3 region of syntaxin 1a into the as Y. Scheller R.H. J. 1996; PubMed Google Scholar), and the into the of The cells in for in the of and with by and by in the of The with of for each of and for The with β-mercaptoethanol and the fusion protein with The to a or and the to with a of kDa of the and syntaxin 1a H3 region by by of protein in a and and solution and to an of The crystals to and have cell a The contains two of the H3 with a which half of the four-helix the crystals and to an of the protein crystals to The for both the and proteins the possible of or crystal but with a to the also This crystal form but the used for The of this crystal contains four of H3, to the four-helix from crystals from to and and on an from a four of the a pairs and and with 1997; PubMed Scopus Google Scholar). are in is the of and is the mean of of as for the in are for the for the for the is the of and is the mean of of as for the in a in are for the for the for the for structure protein but this protein to to by crystals in many different It is that the and of the crystals but the crystals be into different this cysteine into the H3 protein the to allow for attachment of to significant of protein when in or and of the proteins as for the protein. three of these proteins and these three proteins used for of the proteins in several different but to the proteins in solution in a of with and the proteins to a gel filtration to attachment of the by The only by with the protein. between the of protein and H3 a of this the and J. on the to The the helical but of to the multiwavelength anomalous dispersion on the and to with the used as a The and as in the P. 1996; Scholar), with an of The and an The contains two parallel which associate with a pair to form the four-helix are in is the a or the between two is the for the is the a or the between two is the for the in a with the M. A. PubMed Scopus Google Scholar). the A.T. P. J. M. L.M. T. Biol. 1998; PubMed Scopus Google Scholar). of the by the of the that used for The cysteine is present in a portion of the but the bound in a of a crystal a random of the and used as a for The to with a target A. 1996; Scopus Google against the of and the against the with a target In this the is that the that the tetramer is of the of the two crystal forms the to be into the cell by of and factor the to the molecules and molecules in that and that a chemical The contains residues for for for for and one are in and a of the that in the of the The contains the from the from a of the that in the of the The contains the from the from and the and and the and in in of molecules are for the of syntaxin 1a H3 in the by and D. in the adopt an helical for for for and for to to to The a of the that in the of the The contains the from the and the and are for the of syntaxin 1a H3 in the by and D. in the adopt an helical for for for and for D. in a H3 protein on a in and and into for cross-linking and as and to H3 protein from to for each and cross-linking for by an of and and for to the of the and and syntaxin 1a H3 proteins, protein by to a or in a and or The with protein of dichroism and on protein protein against a and or and The protein for an The for of the from in helical and in protein by the the from to as the of the of the and a a and The by the of the protein to in and for each The by the to in with a each The H3 region of syntaxin 1a, of residues in as a glutathione S-transferase fusion protein and of the in the from a a mixture of and crystals in a that two molecules in the which to The against a to from a but crystal form that contains four of the H3 in the and are in of the four of H3 in the of the crystal forms an amphipathic helix the of the parallel by two helices associates in an anti-parallel with parallel The of the helices form both the parallel and anti-parallel This a four-helix bundle with The of the bundle is other anti-parallel four-helix bundles are with helices anti-parallel to one with the two parallel helices the J. Biol. PubMed Scopus Google Scholar). The observed in the H3 tetramer by which helices can associate into a are conformational the four helices in the H3 and are as are and D. and are and with for residues and In contains an in the and are and The N-terminal of and are C-terminal and as a the of the four-helix bundle are as as the The that the H3 region forms an helical assembly it of to the H3 homotetramer structure with the SNARE complex structure (7Sutton R.B. Fasshauer D. Jahn R. Brünger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1953) Google Scholar). The between these two is that of the helices are parallel to each other in the SNARE complex, whereas the H3 tetramer consists of anti-parallel pairs of parallel helices. is that the H3 region in the complex structure is the The syntaxin 1a in the crystal structure of the SNARE complex is or in different in the whereas in the H3 tetramer the is either or crystal the solution of syntaxin proteins I. Sugita S. Hill S. Hosaka M. Fernendez I. Sudhof T.C. Rizo J.A. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (561) Google Scholar, L.M. Brunger A.T. Nature Struct. Biol. 1999; PubMed Scopus Google Scholar), in which the H3 region is from residues This may from the that in the interaction with Habc the half of the H3 the the of the H3 in the the of the or of the helical into the crystal may the present structure the present in from from crystals to a that the of each of H3 is from residues to in the structure that these residues are only from the H3 tetramer on from the SNARE complex with an of and from the H3 tetramer on H3 from the SNARE complex with an of on of two parallel helices in the H3 tetramer with two helices in the SNARE complex This is to in between the are between and for the H3 tetramer and between and for the complex The H3 parallel also have as of a as the in the SNARE complex structure and The of the H3 four-helix bundle is composed of and these residues the of the SNARE complex (7Sutton R.B. Fasshauer D. Jahn R. Brünger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1953) Google Scholar). However, each contributes one to the of the four-helix bundle, and in each of parallel helices these residues are in the and are to one The pair of residues from parallel helices packs against two residues by the anti-parallel helices In the SNARE complex structure Gln-226 forms part of a with of and and of Phe-216 forms part of a it is against small residues present in SNAP-25 and VAMP2 are present on the of the H3 The H3 is and displays a large of The H3 tetramer contains only two small of in the part of the to from and from B. both the H3 tetramer and the SNARE complex have and both form In both bind and NSF to form a S 15Woodman P.G. Biochim. Biophys. Acta. 1997; 1357: 155-172Crossref PubMed Scopus (54) Google and and NSF are that in transport and are thus of with many different SNARE and NSF may SNARE protein on and thus for the of these of the H3 tetramer also reveals a docking for a small is bound in a between of and of this is the of the helix in This structure be to the is from in the crystal the used as the in this The is bound to a part of the of a in the crystal but found in the The to be several other that be attached to solution but the of the observed H3 the of H3 in solution by gel filtration and chemical The for an H3 tetramer is the H3 an of kDa on a gel filtration The crystal structure reveals that the H3 tetramer is a and the from the may be in part to the of the three different cross-linking also the of H3 in solution for in of the of or is the of the as by is which is with a small of tetramer can be The with the only of to protein cross-linking results as by results with the are and are each protein the from to protein and are in protein. that the H3 kDa the The crystal structure of the tetramer is with the observed cross-linking The three residues in one of the H3 are the N-terminal and and the C-terminal The are a of cross-linking of pairs of from parallel helices. the of from and are in the crystal and the of and from parallel helices and are in the Modeling indicates that small changes in the pair cross-linking of the used in these In the with the cross-linking are to between and each of the H3 and from parallel helices are cross-linking of each but only one of the from the anti-parallel helices is in to form to either of the or The of the H3 oligomer assessed by circular dichroism to both and to the and as to the as the protein to with D. Eliason W.K. Brunger A.T. Jahn R. Biochemistry. 1998; PubMed Scopus Google Scholar, P. D. D. Scheller R.H. Biochemistry. 1997; PubMed Scopus Google Scholar), the indicate that the H3 is The for the H3 oligomer is and It has been that can or helical structure by the between J. Biol. 1994; PubMed Scopus Google Scholar). The may be to the formation of between residues to one on the of the helix and and and residues are in the of several parallel helix the SNARE complex Kim T. PubMed Scopus Google Scholar, Zhang T. Kim T. PubMed Scopus Google Scholar), and other proteins W.A. Biochemistry. PubMed Scopus Google Scholar), and the Nature. PubMed Scopus Google Scholar). the of the SNARE complex and the for in the of bundle the H3 an In the H3 two Gln-226 residues form with each other and are against two Phe-216 residues present on the anti-parallel pair Modeling that four residues form a of in an of four H3 helices. However, this that four Phe-216 residues against one in the and it appears that this significant into the In the SNARE complex, Phe-216 of syntaxin 1a packs against several and from SNAP-25, and from VAMP2 of the H3 region of several different proteins reveals that the and as as to each are highly conserved T. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. R.B. Brunger A.T. Jahn R. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). It has been suggested that the of the large Phe-216 with residues to the of helices in the complex D. R.B. Brunger A.T. Jahn R. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The anti-parallel H3 tetramer that Phe-216 may also formation of stable but parallel syntaxin tetramers, thus the H3 region to bind other proteins as and SNAP-25. In the of the large Phe-216 it is possible that four H3 helices have a parallel this H3 Phe-216 Ala and Phe-216 Leu to the proteins that the proteins to form the complexes as the are stable the or have an structure that prevents formation of the as the protein. In the of it is these the by the residues and allow a parallel tetramer to the of the proteins and to the protein. filtration reveals that the protein the as the protein the can be to tetramers, and are present the of In the protein a on a gel filtration When this is to either or cross-linking a is observed but the protein largely The of the and and and with the protein. the has a H3 the of is with of and this two of anti-parallel and parallel with and is a to the protein to and to This the helical bundles and allow formation of only the stable from only one of two This the that the mutation the H3 to form anti-parallel and parallel tetramers, and that the parallel is The protein as by the to have helical structure and the This is with that the protein a to a on the of the protein in to the or proteins; the this the gel filtration for the three proteins The of the and protein the tetramer but the protein from the to the This indicates that the protein forms and this in may the of the these results that Phe-216 of the prevents parallel tetramer formation and that the of Leu parallel tetramer formation. The cross-linking and the of the may a of and anti-parallel tetramers, with the stable the the of Ala prevents formation of structure the and of by as for the protein. The H3 region of syntaxin 1a can form binary complexes with partner t-SNARE SNAP-25, and this interaction may be an in the formation of the SNARE complex (4Fasshauer D. Bruns D. Shen B. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 4582-4590Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 12Nicholson K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nature Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (177) Google Scholar). In syntaxin 1a and SNAP-25 interact to form a binary complex that affinity for either (2Fasshauer D. Otto H. Eliason W.K. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, L.M. Brunger A.T. Nature Struct. Biol. 1999; PubMed Scopus Google Scholar, J. Scheller R.H. Neuron. 1994; 13: Full Text PDF PubMed Scopus Google Scholar), and this interaction is the in forming the SNARE K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nature Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (177) Google Scholar). The of the binary complex is but indicate that the complex is a parallel four-helix and syntaxin 1a and SNAP-25 indicate that these proteins form a complex, whereas from the syntaxin 1a and SNAP-25 proteins the of a or complex (2Fasshauer D. Otto H. Eliason W.K. Jahn R. Brunger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). from and the of SNAP-25 N-terminal in the binary complex (4Fasshauer D. Bruns D. Shen B. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 4582-4590Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 12Nicholson K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nature Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (177) Google Scholar), but it is the C-terminal are also that the 1a binary complex forms a four-helix bundle it is possible that the parallel syntaxin helices observed in the H3 tetramer structure two of syntaxin 1a in a complex with SNAP-25. The syntaxin may SNAP-25 in thus a for and SNARE complex formation. syntaxin 1a and SNAP-25 are present in a it is that both the and C-terminal of SNAP-25 bind to the syntaxin the of the two proteins is of it is that only the N-terminal region of SNAP-25 forms the binary complex with syntaxin 1a indicates to formation of either of parallel binary but and be to the and of the syntaxin complex. has that the syntaxin cytoplasmic domain forms and a small amount of tetramer in solution J.C. Robblee J. Fairman R. Hughson F.M. Biochemistry. 2000; 39: 8470-8479Crossref PubMed Scopus (94) Google Scholar). This to the H3 It that the assembly observed in the H3 crystal structure the of H3 as as the assembly of syntaxin The H3 used to but is for residues This is syntaxin to be stable of syntaxin J.C. Robblee J. Fairman R. Hughson F.M. Biochemistry. 2000; 39: 8470-8479Crossref PubMed Scopus (94) Google Scholar). The for syntaxin is and the is J.C. Robblee J. Fairman R. Hughson F.M. Biochemistry. 2000; 39: 8470-8479Crossref PubMed Scopus (94) Google Scholar). The and cross-linking by J.C. Robblee J. Fairman R. Hughson F.M. Biochemistry. 2000; 39: 8470-8479Crossref PubMed Scopus (94) Google μm), to used in the have to in the H3 gel filtration However, only one that the H3 region may in the of the Habc This may be to the interaction of H3 with the Habc region in the cytoplasmic domain of In a of fusion is observed A. Nature. 1997; PubMed Scopus Google Scholar), that complexes of syntaxin fusion J.C. Robblee J. Fairman R. Hughson F.M. Biochemistry. 2000; 39: 8470-8479Crossref PubMed Scopus (94) Google Scholar). The results that proteins are to form of fusion is mediated by proteins this of fusion is different from fusion parallel four-helix It is possible that anti-parallel syntaxin form between molecules in and an the membranes to allow fusion to However, indicate that be stable SNARE complex The highly conserved Phe-216 to formation of stable parallel H3 This may be to that as the binary SNAP-25 complex or the SNARE complex, can be from a It is that of for parallel four-helix bundle assembly to of by the large It is also possible that the Phe-216 Ala protein forms parallel four-helix bundles The of this protein is for a stable parallel four-helix bundle as the SNARE complex but this may be in part to of the by the small The of Phe-216 on the assembly of syntaxin H3 that has on the assembly of helical The of Phe-216 may be both in of SNARE helices D. R.B. Brunger A.T. Jahn R. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), and also to of syntaxin R. and T. for T. and the the for M. and H. the for and with A. for with and A. R. A. and J. for