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Biomolecular Structure Presentation Cassidy Dobson University of Massachusetts November 28, 2006 SNAREs Soluble N-ethylmaleimide-sensitive factor attachment protein receptor – Receptor protein – Attached to NSF – NSF = ATPase – First discovered by Rothman: protein inactivated ethylmaleimide cytoplasmic by N- SNAREs Coiled-coil proteins involved in membrane fusion – Cell Growth – Trafficking – Synaptic Transmission High sequence diversity – Conserved mechanism Rothman, J.E. and Warren, G. 1994. Implications of the SNARE hypothesis for intracellular membranetopology and dynamics. Curr. Biol. 4:220-233. SNARE complex formation Complex formation causes juxtaposition of two vesicles Receptor and Acceptor SNAREs poise vesicles for fusion SNARE proteins mediate fusion via a proposed “zipping up” mechanism – Overcomes energy barrier due to highly stable interaction Dissociation via NSF (ATPase) SNARE structure Hydrophillic ionic layer at center of 4 helix bundle Flanked by hydrophobic leucine zipper layers Ionic Layer of a Q SNARE Syntaxin 1A pre-complexed Syntaxin 1A w/ SNAP 25 Types of SNAREs Diverse in function, similar in structure – Generally categorized by presence of SNARE domain 60-70 amino acid stretch Heptad repeats Propensity to form coiled-coils – Heptad repeat: A type of tandem repeat sequence in which a group of seven amino acids occurs many times in a protein sequence SNARE homology Endosomal SNARE core complex (1GL2) Vti1a N-terminal domain (1VCS) Tlg1 (2C5J) Syntaxin 6 (1LVF) SNAP-23N (1NHL) “Old” SNARE nomenclature Originally referred to as v and t-SNARES – T-SNARE’s = Target membrane SNARES – V-SNARE’s = Vesicle membrane SNARES Homotypic fusion, multiple SNAREs in multiple transport systems – Now Q and R SNAREs – Depends on which residue contributes at zero ionic layer Syntaxin 1A and SNAP25 SNAREs involved in synaptic vesicle junction Transport neurotransmitters between synaptic cleft Neurons Action potential travels down axon of one neuron Signal given to following neuron by release of neurotransmitters into synaptic cleft Pre to post synaptic Pre-synaptic membrane Post-synaptic membrane The Neuronal SNARE complex 3 SNAREs involved – Syntaxin 1A – SNAP25 – Synaptobrevin or VAMP2 (vesicle-associated membrane protein) Each contribute one coil to the complex – SNAP25 contributes 2 coils Syntaxin 1A Q-SNARE Found at the pre-synaptic membrane – Big role in neurotransmitter release Binds SNAP-25 to form a trimeric complex at the pre-synaptic membrane N-terminal domain of syntaxin 1A Syntaxin 1A structure 35 KDa protein 3 domains Single C-terminal domain – Transmembrane spanning domain SNARE binding domain (H3) – Stretch of the 60-70 coiled coil amino acids Regulatory domain (Habc) – N-terminal – 3 Helix Bundle Structurally solved parts of syntaxin 1A ? C-terminal transmembrane domain SNARE domain (H3) N-terminal domain of syntaxin 1A (Habc) Syntaxin 1A PBD ID = 1HVV – Syntaxin 1A from Rat expressed in E. coli Solved by x-ray crystallography Resolution = 2.4 Å R-value = .262 R free = .238 Average B-factor = 44.8 Å2 SNAP25 Structure 25 KDa protein Contains 3 domains 2 SNARE binding domains – S25N and S25C Linker domain – 45 amino acids in length Targeted to PM by palmitolyation of 4 cysteines – Targets/Anchors SNAP25 to membrane Syntaxin 1A complexed with SNAP25 PBD ID = 1JTH – Complex of Syntaxin 1A associated with SNAP25 from rat expressed in E. coli Solved by x-ray crystallography Resolution = 2.0 Å R value = .265 R free = .284 Average B-factor = 37.4 Å2 MAD diffraction data Anisotropic temperature factor correction SAO map Syntaxin 1A “morph” Zipping up mechanism hard to model General principle – Greater stabilization of core as create the ionic layer – Electrostatics help with stabilization to overcome energy barrier of membrane fusion Divalent cations assist Snap’shots’ of change Syntaxin 1A pre bundled Where do these key residues go? SNARE domain rearrangement The whole SNARE mechanism Thank You!