Transcript Neurotoxins in Snake Venom - California State University

Neurotoxins in Snake Venom
Cobra
Green Mamba
by Tim Reed, Katie Eaton, Cathy Peng, BettyLou Doern
Why Toxins?
Why is all this information available?
• While DNA cannot be altered in an individual, the
effects of the proteins can be regulated. This
includes blocking receptor sites and inhibiting
enzymes via an inhibitor. Toxins are studied to
better understand how inhibitors work.
• Specifically, they have helped us gain a greater
understanding of muscle and nerve function.
Why Fasciculin?
•
•
•
•
Most snakes have multiple types of venom.
Cobras have both a short and long neurotoxin as
well as a cardiotoxin.
Kraits have three different types of neurotoxins.
Sea snakes have a neurotoxin as well as two blood
toxins (one causes lysis blood cells, the other is an
anti-coagulant).
Mambas have only Fasciculin.
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.
FAS-I from Eastern Green Mamba
Sequence:
tm(cyshtttsrailtnc)gens(cyrksrrhppkmvlgrgc)g(cppgddylevk)cct
spdkcny
4 S-S bonds: 1:3 – 22 2: 7 – 39
3:41 – 52 4:53 - 59
FAS-II from Eastern Green Mamba
Sequence:
tm(cyshtttsrailtnc)gens(cyrksrrhppkmvlgrgc)g(cppgddnlevk)cct
spdkcny
4 S-S bonds: 1:3 – 22 2:17 – 39
3:41 – 52 4:53 - 59
Hydrophobic,Hydrophilic,&Transmembrane
Characteristics
•No Tranmembrane segments predicted by T-MAP
•GREASE
Which Toxins?
• Fasciculin1 from Eastern Green Mamba
(Dendroaspis angusticeps)
• Cobratoxin from Taiwan Cobra (Naja naja atra)
Fasciculin inhibits mammalian and fish
acetylcholinesterases at picomolar concentrations,
but is a relatively weak inhibitor of avian,
reptile, and insect acetylcholinesterases.
About Fasciculin
• Small protein
(61 amino acids)
• 3-finger shaped
• Cross-linked by 4 disulfide bridges
(S atoms are in Cystine amino acid)
Other Representations of 1FAS
• Backbone
• BallStick
• Ribbons
• Space
• Sticks
• Strand
Secondary sheet structures are rendered in orange.
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 Acetylcholine
receptors on the muscle cell.
• Result: “Death by respiratory paralysis”.
Neuromuscular Junction
Vesicles containing
Acetylcholine (ACh)
ACh receptors
Acetylcholinesterase
(AChE)
ACh Receptor Channel Opens
Acetycholinesterase Cleans Up
AChE hydrolizes ACh, so that the process
can start again.
Structure/Function of Fasciculin
Fasciculin (yellow)
docked with AChE.
Acetylcholinesterase with
Acetylcholine bound.
Red = AChE active site
Yellow = ACh molecule
• One loop covers the
AChE active site.
• Two other loops fit
into a crevice and
surround a protrusion.
AChE-Fas Interface
fasciculin (light gray)
AChE (dark gray)
Mutations to areas with
highly complementary
shapes reduce the toxicity
of Fasciculin.
Red spheres -> are points on the interface that
suggest docking of a protrusion into a crevice.
Yellow and green spheres indicate the docking of
flat surfaces.
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" neurotoxin
from 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
Even More Detail Fas-AChE
• Loop II of fasciculin contains a cluster of hydrophobic
residues that interact with the peripheral anionic site of the
enzyme 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 gorge
entry. The fasciculin core surrounds a protruding loop on
the enzyme surface and stabilizes the whole assembly.
• The aromatic residues, Trp286, Tyr72, and Tyr124, have
the most marked influence on fasciculin binding. These
residues are unique to the susceptible
acetylcholinesterases.
Expression and Activity of Mutants of
Fasciculin-II
•The availability of a crystal structure of a FasII-acetylcholinesterase
complex affords an opportunity to examine in detail the interaction of
the toxin with its target site.
•Sixteen mutations:
tmcyshtttsraIltncgenscyrksrrhppkmvlg
rgc
1st L
2nd L
gcppgddnlevkcctspdkcny
3rd L
L2: r27-p30-p31 subset dominates the inhibitory activity &
interacts with the peripheral anionic site of the enzyme AchE.
Expression and Activity of Mutants of
Fasciculin-II
L1: t8-t9-r11 subset is fully exposed at the tip and external edge of
L1,which fits in a crevice near the lip of the mAChE catalytic gorge
and maximizes the surface area of contact of loop II at the gorge
entry.
L3: lack of interaction of residues d45 and k51 with mAChE
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
Fasciculin vs. Cobratoxin
Query: 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
Cobratoxin(71amino acids)
S-S bonds:
1: 3 - 20
2: 14 - 41
3: 26 - 30
4: 45 - 56
5: 57 - 62
CLUSTALW OUTPUT
SDSCNR_526500
TXF7_DENAN
SDSCNR_493149
SDSCNR_488281
SDSCNR_495320
TICYSHTTTSRAILKDC-GENSCYRKSRRHPPKMVLGRGCGCPPGDDYLEVKCCTSPDKC
TMCYSHTTTSRAILTNC-GENSCYRKSRRHPPKMVLGRGCGCPPGDDYLEVKCCTSPDKC
TMCYSHTTTSRAILTNCPGETNCYKKSRRHPPKMVLGRGCGCPTVAPGIKLNCCTT-DKC
RICYNHQSTTRATTKSC-EENSCYKKYWRDHRGTIIERGCGCPKVKPGVGIHCCQS-DKC
RICYNHQSTTPATTKSC-GENSCYKKTWSDHRGTIIERGCGCPKVKQGIHLHCCQS-DKC
:**.* :*: * ..* *..**:*
.
:: ******
: ::** : ***
SDSCNR_526500
TXF7_DENAN
SDSCNR_493149
SDSCNR_488281
SDSCNR_495320
NY
NY
NY
NY
NN
*
Key:
SDSCNR:526500
TXF7_DENAN
SDSCNR:493149
SDSCNR:488281
SDSCNR:495320
ACETYLCHOLINESTERASE TOXIN C [D. polylepis (Black mamba)], 61 AA
TOXIN (SYNTHETIC CHIMERA)_r-chii, 61 AA
short neurotoxin 1 - black mamba, 60 AA
short neurotoxin 1 - eastern Jameson's mamba, 60 AA
Tree 15928
Tree 21882
Tree 22694
Reference
The binding sites of inhibitory monoclonal antibodies on
acetylcholinesterase. Identification of a novel regulatory site at the
putative "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 and
Long-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.