1. Calculation of interaction energy between voltage-gated potassium channel Kv1.2 and blocker agitoxin Valery N. Novoseletsky Maria A. Bolshakova Konstantin V. Shaitan Moscow 2013 Molecular modeling group Bioengineering department Biology Faculty M.V.Lomonosov Moscow State University

2. 2 Pore-forming membrane proteins that open or close in response to changes in the transmembrane voltage. Potassium channels are the most widely distributed type of ion channel and are found in virtually all living organisms. Voltage-Dependent Potassium Channels Action potential is a short- lasting event in which the electrical membrane potential of a cell rapidly rises and falls, following a consistent trajectory. During action potentials, voltage-dependent potassium channels play a crucial role in returning the depolarized cell to a resting state.

3. 3 (Long, Campbell, MacKinnon, 2005) Crystal Structure of a Mammalian Voltage-Dependent K + Channel

4. 4 Transmembrane domain scheme: helices S1-S4 form the voltage-sensing domain, helices S5 and S6 take place in the ions conduction. Crystal Structure of a Mammalian Voltage-Dependent K + Channel

5. 5 Side view: voltage-sensing domain, pore domain and selective filter. Crystal Structure of a Mammalian Voltage-Dependent K + Channel

6. Scorpion venom toxin – agitoxin 6 Agitoxin structure was determined by NMR (Kresel, Kasibhatha, 1995). Structure has highly conserved motif consisting of - 3 antiparallel β-sheets - α-helix - 3 disulfide bridges Agitoxin specifically blocks Kv1.2 channel with high affinity (Kd < 1 nmol/L)

7. Extracellular and membrane view. Highly conserved residues involved in binding are marked (pdb-codes 1AGT for agitoxin, 2A79 for channel). 7 Kv1.2 channel complex with agitoxin

8. Molecular system preparation 8

9. Potential of mean force with umbrella sampling 1. Pulling the toxin away from the channel using steered molecular dynamics 9 Toxin-channel distance increases with time from 2.5 nm at 0 ps to 7 nm at 500 ps.

10. 2. Frame selection 3. Configuration space sampling 10 Potential of mean force with umbrella sampling

11. Dependence of PMF on toxin-channel distance in 0.1 M NaCl solution. 11 Potential of mean force with umbrella sampling 4. PMF extraction and ΔG calculation

12. Dependence of PMF on toxin-channel distance in 0.1 M NaCl solution. Binding energy is equal to the max PMF value and consists 22 ± 2 kcal/mol. 12 Potential of mean force with umbrella sampling 22 ± 2 kcal/mol 5. Error estimation

13. Effect of ionic strength on the free energy of binding 13 Binding energy drops with the increase of ionic strength. 0, 025M 0, 05M 0,1M 0,2M 0,4M z (nm) 25 20 15 10 5 0

14. Comparison of the results * Zachariae et al, 2008 ** Our data *** Khabiri et al, 2011 14 ComplexExperimentCalculation Kv 1.2 - agitoxin ∆G = 10,3 ± 0,2 kcal/mol* (160 мМ NaCl, 4.5 мМ KCl, 2 мМ CaCl₂, 1 мМ MgCl₂) ∆G = 22 ± 2 kcal/mol** (100 мМ NaCl) Kv 1.3 - charibdotoxin ∆G = 10,6 ± 0,2 kcal/mol*** (164.5 мМ KCl, 2 мМ CaCl₂, 1 мМ MgCl₂) ∆G = 27 ± 1 kcal/mol*** (160 мМ KCl) Kv 1.3 - charibdotoxin ∆G = 11,4 ± 0,2 kcal/mol*** (160 мМ NaCl, 4.5 мМ KCl, 2 мМ CaCl₂, 1мМ MgCl₂) ∆G = 26 ± 1 kcal/mol*** (4.5 мМ KCl)

15. Conclusion 1. Structural model of agitoxin-channel complex was constructed 2. Free energy binding was calculated using potential of mean force and umbrella sampling 3. Obtained results differ from experimental data. Probable reason is in the mistake of system preparation 15

16. Thank you for attention! 16