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 t-SNARE_Syntaxin_1A |
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Stiffness of the Juxtamembrane Region of the t-SNARE Syntaxin 1A
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Volker Knecht and
Helmut Grubmüller
For exocytosis and for the selective transport of macromolecules between the various organelles of eukaryotic cells the merging of a transport vesicle membrane with a target membrane is an essential step.
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According to the SNARE hypothesis, this process is mediated through the assembly of vesicle (v) and target (t) SNARE proteins [1] (Fig. a). The cytosolic core of the SNARE complex involved in synaptic transmission has been resolved by X-ray crystallography and consists of the v-SNARE synaptobrevin-II (magenta) and the t-SNAREs syntaxin 1A (blue) and SNAP-25 (brown) which form a four-stranded parallel coiled coil [2] (Fig. b,c).
A 5-residue basic linker of unknown structure (red) connects the syntaxin 1A H3 helix (blue) with its c-terminal membrane anchor (green) which can be assumed to have alpha-helical conformation. For the initial step of membrane fusion, two scenarios are currently considered and discussed controversally [3,4]: (b) The linker is very flexible and represents a hinge region between the two adjacent syntaxin domains. (c) The linker is stiff enough to provide a mechanical coupling of the two syntaxin domains; as the SNAREs assemble, the target membrane is bent towards the vesicle membrane.
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The present simulation study aims to contribute to this discussion by computing the stiffness of the linker.
The stiffness of the linker is studied by molecular dynamics (MD) simulations including
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An 11-residue c-terminal part of the syntaxin 1A core helix (X-ray helix, turquoise). To stabilize the known secondary structure, internal restraints were imposed on the X-ray helix. Two linker residues adjacent to the X-ray helix were found to spontanously fold from loop into alpha-helical conformation and therefore for the presented simulations were also kept in alpha-helical conformation by internal restraints. The three remaining residues were modeled in random loop conformation.
- its 5-residue basic linker (grey loop) and its 23-residue alpha-helical transmembrane domain (yellow).
- The embedding lipid bilayer comprising 116 lipid molecules (green, the oxigen atoms of the polar region are shown as red spheres).
Three different lipid species were incorporated:
- Palmitate-Oleate(PO)-Phosphatidylcholine (PC): zwitterionic
- PO-Phosphatidylethanolamine (PE): zwitterionic
- PO-Phosphatidylserine (PS): acidic
These lipid species were combined to obtain two different lipid compositions:
- 70 POPC and 46 POPE (yielding a neutral bilayer)
- 70 POPC, 34 POPE, and 12 POPS (yielding a negatively charged bilayer).
The lipid bilayers were obtained by modifications of an equilibrated bilayer of POPC molecules.
- An explicit solvent environment (water molecules shown in blue). The solvent environment comprised 4720 water molecules and (dependent on the lipid composition) 6 chloride or 6 sodium ions such as to counterbalance the netto charge of the system.
All molecular dynamics simulations are carried out using the GROMACS simulation package [5]. The systems are simulated at 300K and a pressure of 1 bar with periodic boundary conditions.
The free energy for peptide bending is estimated from the equilibrium fluctuations. Preliminary results show the linker to be surprisingly stiff: To bend the linker by 20 degrees requires approx. 2 kcal/mol. Therefore our simulations so far support the mechanical coupling scenario.
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References
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T. Söllner, S. W. Whiteheart, M. Brunner, H. Erdjument-Bromage, S. Geromanos, P. Tempst, J. E. Rothman (1993) Nature 362, 318-324
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R. B. Sutton, D. Fasshauer, R. Jahn and A. T. Brunger (1998) Nature 395, 347-353
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J. A. McNew and T. Weber and D. M. Engelman and T. H. Söllner and J. E. Rothman (1999), Mol. Cell. 4 (3), 415-421
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J. A. McNew, T. Weber, F. Parlati, R. J. Johnston, T. J. Melia, T. H. Söllner and J. E. Rothman (2000), J. Cell Biol. 150 (1), 105-117
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H. J. C. Berendsen, J. P. M. Postma, A. DiNola and J. R. Haak Molecular dynamics with coupling to an external bath J. Chem. Phys. 81 (1984) pp. 3684-3690
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