Neurotoxins in Snake Venom

1. Neurotoxins in Snake Venom Cobra Green Mamba by Tim Reed, Katie Eaton, Cathy Peng, BettyLou Doern

2. 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.

3. 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.

4. 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.

5. FAS-I from Eastern Green Mamba Sequence: tm(cyshtttsrailtnc)gens(cyrksrrhppkmvlgrgc)g(cppgddylevk)cctspdkcny 4 S-S bonds: 1:3 – 22 2: 7 – 393:41 – 52 4:53 - 59

6. FAS-II from Eastern Green Mamba Sequence: tm(cyshtttsrailtnc)gens(cyrksrrhppkmvlgrgc)g(cppgddnlevk)cctspdkcny 4 S-S bonds: 1:3 – 22 2:17 – 393:41 – 52 4:53 - 59

7. Hydrophobic,Hydrophilic,&Transmembrane Characteristics • No Tranmembrane segments predicted by T-MAP • GREASE

8. 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.

9. About Fasciculin • Small protein (61 amino acids) • 3-finger shaped • Cross-linked by 4 disulfide bridges (S atoms are in Cystine amino acid)

10. Other Representations of 1FAS Secondary sheet structures are rendered in orange.

11. 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”.

12. Neuromuscular Junction Vesicles containing Acetylcholine (ACh) ACh receptors Acetylcholinesterase (AChE)

13. ACh Receptor Channel Opens

14. Acetycholinesterase Cleans Up AChE hydrolizes ACh, so that the process can start again.

15. Structure/Function of Fasciculin

16. AChE-Fas Interface

17. 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

18. 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.

19. 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: • t m c y s h t t t s r a I l t n c g e n s c y r k s r r hp pk m vl g r g c • g c p p g d d n l e v k c c t s p d k c n y • L2: r27-p30-p31 subset dominates the inhibitory activity & interacts with the peripheral anionic siteof the enzyme AchE. 1st L 2nd L 3rd L

20. 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 mAChEcatalytic gorge and maximizes the surface area of contact of loopII at the gorge entry. L3: lackof interaction of residues d45 and k51 with mAChE

21. 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

22. 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

23. Cobratoxin(71amino acids) S-S bonds: 1: 3 - 20 2: 14 - 41 3: 26 - 30 4: 45 - 56 5: 57 - 62

24. CLUSTALW OUTPUT SDSCNR_526500 TICYSHTTTSRAILKDC-GENSCYRKSRRHPPKMVLGRGCGCPPGDDYLEVKCCTSPDKC TXF7_DENAN TMCYSHTTTSRAILTNC-GENSCYRKSRRHPPKMVLGRGCGCPPGDDYLEVKCCTSPDKC SDSCNR_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

25. Tree 15928

26. Tree 21882

27. Tree 22694

29. 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.