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Neurotoxins in Snake Venom
Cobra Green Mamba
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General Characteristics of Protein
Fasciculins
Fasciculins : a family of closely related peptides isolated
from Mamba venom toxins (FAS-I, FAS-II, and FAS-III)
Function : Inhibits ACETYLCHOLINESTERASE
(AChE), which is an enzyme to degrade neurotransmitter
ACh. In skeletal muscle, fasciculations are observed
initially, followed by flaccid paralysis.
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FAS-II from Eastern Green MambaSequence:
tm(cyshtttsrailtnc)gens(cyrksrrhppkmvlgrgc)g(cppgddnlevk)cctspdkcny
4 S-S bonds: :3 22 2:17 39 3:41 52 4:53 - 59
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Hydrophobic,Hydrophilic,&Transmembrane
CharacteristicsNo Tranmembrane segments predicted by T-MAP
GREASE
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Which Toxins?
Fasciculin1from Eastern Green Mamba(Dendroaspis angusticeps)
Cobratoxinfrom Taiwan Cobra (Naja naja atra)
Fasciculin inhibits mammalianand fish
acetylcholinesterases at picomolar concentrations,but is a relatively weak inhibitorof avian,
reptile, and insectacetylcholinesterases.
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About Fasciculin
Small protein(61 amino acids)
3-finger shaped Cross-linked by 4 disulfide bridges
(S atoms are in Cystine amino acid)
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Other Representations of 1FAS
Secondary sheet structures are rendered in orange.
Backbone BallStick Ribbons
Space Sticks Strand
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Mode of Action
These snake neurotoxins act on the
neuromuscular junction (next slide)
and block neuromuscular transmission. Fasciculin interferes with this process by
binding to Acetylcholinesterase (AChE).
Cobratoxin binds to the Acetylcholinereceptors on the muscle cell.
Result: Death by respiratory paralysis.
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Neuromuscular Junction
Vesicles containing
Acetylcholine (ACh)
ACh receptors
Acetylcholinesterase
(AChE)
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ACh Receptor Channel Opens
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Structure/Function of Fasciculin
Fasciculin (yellow)
docked with AChE.
Acetylcholinesterase with
Acetylcholine bound.
Red= AChE active site
Yellow= ACh molecule
One loop covers theAChE active site.
Two other loops fitinto a crevice and
surround a protrusion.
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AChE-Fas Interface
fasciculin (light gray)AChE (dark gray)
Mutations to areas with
highly complementary
shapes reduce the toxicity
of Fasciculin.
Redspheres -> are points on the interface that
suggest docking of a protrusion into a crevice.
Yellowand greenspheres indicate the docking of
flat surfaces.
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Other Toxins Act on Same Process
Inactivate Acetylcholinesterase(like Fasciculin) Sarin nerve gas
several insecticides such as Malathion
Block the Acetylcholine receptor(like Cobratoxin)
Taiwan banded krait snake venom(a-bungarotoxin)
Poison Arrow" neurotoxinfrom the skin of a Columbian frog (histrionicatoxin)
Curare, a paralytic agent used medically (d-tubocurarine)
Neuromuscular junction:
Black Widow Spider venom - triggers Acetylcholine release
Botulism toxin - blocks Acetylcholine release
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Even More Detail Fas-AChE
Loop II of fasciculin contains a cluster of hydrophobicresidues that interact with the peripheral anionic site of theenzyme and occlude substrate access to the catalytic site.
Loop I fits in a crevice near the lip of the gorge to
maximize the surface area of contact of loop II at the gorgeentry. The fasciculin core surrounds a protruding loop onthe enzyme surface and stabilizes the whole assembly.
The aromatic residues, Trp286, Tyr72, and Tyr124, havethe most marked influence on fasciculin binding. Theseresidues are unique to the susceptibleacetylcholinesterases.
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Expression and Activity of Mutants of
Fasciculin-IIThe availability of a crystal structure of a FasII-acetylcholinesterase
complex affords an opportunity to examine in detail the interaction ofthe toxin with its target site.
Sixteen mutations:
t m c y s h t t ts ra I l t n c g e n s c y r ks r r hp pk m vlg
r g c
g c p p g dd n l e v kc c t s p d k c n y
L2: r27-p30-p31subset dominates the inhibitory activity &
interacts with the peripheral anionic siteof the enzyme AchE.
2ndL
3rdL
1stL
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L1: t8-t9-r11subset is fully exposed at the tip and external edge of
L1,which fits in a crevice near the lip of the mAChEcatalytic gorge
and maximizes the surface area of contact of loopII at the gorge
entry.
L3: lackof interaction of residues d45and k51with mAChE
Expression and Activity of Mutants of
Fasciculin-II
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Conserved subsequences
------ S-S bonds & r r h p p k m v l
PSIBLAST (Position Specific Iterative BLAST) E-value
ACETYLCHOLINESTERASE TOXIN C. 2e-20
TOXIN C13S1C1 PRECURSOR in Eastern green mamba 2e-06
TOXIN F-VIII PRECURSOR 1e-05
SHORT NEUROTOXIN 1 (NEUROTOXIN ALPHA). 3e-05
TOXIN S5C4 8e-05
SHORT NEUROTOXIN 1 2e-04
SHORT NEUROTOXIN 1 (NEUROTOXIN 4.11.3). 4e-04
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Fasciculin vs. CobratoxinQuery: TXF7_DENAN Length = 61
Reference: Query= (61 letters); NXL1_NAJKA Length = 71
Score = 23.5 bits (49), Expect = 3e-04 Identities = 10/26 (38%), Positives =
14/26 (53%), Gaps = 1/26 (3%)
Query: 35 LGRGCGCPPGDDYLEVKCCTSPDKCN 60
LG CP CC S D CN
Sbjct: 39 LGCAATCPTVKTGVDIQCC-STDNCN 63
Score = 14.2 bits (25), Expect = 0.18 Identities = 3/8 (37%), Positives = 6/8
(74%)
Query: 50 VKCCTSPD 57
C PD
Sbjct: 1 IRCFITPD 8
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Cobratoxin(71amino acids)
S-S bonds:
1:3 - 20
2:14 - 41
3:26 - 30
4:45 - 56
5:57 - 62
http://www.imb-jena.de/cgi-bin/htmlit.pl?color=ffffff&id=GIF&src=2ctx.gif&name=Image%20Library%20Thumb%20Nail%202CTX -
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CLUSTALW OUTPUTSDSCNR_526500 TICYSHTTTSRAILKDC-GENSCYRKSRRHPPKMVLGRGCGCPPGDDYLEVKCCTSPDKC
TXF7_DENAN TMCYSHTTTSRAILTNC-GENSCYRKSRRHPPKMVLGRGCGCPPGDDYLEVKCCTSPDKCSDSCNR_493149 TMCYSHTTTSRAILTNCPGETNCYKKSRRHPPKMVLGRGCGCPTVAPGIKLNCCTT-DKC
SDSCNR_488281 RICYNHQSTTRATTKSC-EENSCYKKYWRDHRGTIIERGCGCPKVKPGVGIHCCQS-DKC
SDSCNR_495320 RICYNHQSTTPATTKSC-GENSCYKKTWSDHRGTIIERGCGCPKVKQGIHLHCCQS-DKC
:**.* :*: * ..* *..**:* . :: ****** : ::** : ***
SDSCNR_526500 NY
TXF7_DENAN NY
SDSCNR_493149 NY
SDSCNR_488281 NY
SDSCNR_495320 NN
*
Key:SDSCNR:526500 ACETYLCHOLINESTERASE TOXIN C [D. polylepis (Black mamba)], 61 AA
TXF7_DENAN
SDSCNR:493149 TOXIN (SYNTHETIC CHIMERA)_r-chii, 61 AA
SDSCNR:488281 short neurotoxin 1 - black mamba, 60 AA
SDSCNR:495320 short neurotoxin 1 - eastern Jameson's mamba, 60 AA
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Tree 15928
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Tree 21882
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Tree 22694
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ReferenceThe binding sites of inhibitory monoclonal antibodies on
acetylcholinesterase. Identification of a novel regulatory site at theputative "back door". J Biol Chem. 1999 Sep 24;274(39):27740-6.
Protein-protein association: investigation of factors influencing
association rates by brownian dynamics simulations.J Mol Biol. 2001 Mar 9;306(5):1139-55.
"Biochemistry and Molecular Biology of Snake Neurotoxin" J.
Chin. Chem. Soc., Vol. 46, No. 3, 1999 Chen-chung Yang andLong-sen Chang Department of Life Science, National Tsing
Hua University, Hsinchu, Taiwan 30043 and Department of
Biochemistry, Kaohsiung Medical College, Kaohsiung, Taiwan
807, R.O.C.