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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!