cobra venom

8/11/2019 Cobra Venom

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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.

cobra venom

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