Application of metadynamics for investigation of human interferon gamma mutants

1. Application of metadynamics for investigation of human interferon gamma mutants Peicho Petkov, Elena Lilkova, Petko Petkov, Nevena Ilieva, Leandar Litov 2nd Regional Conference “Supercomputing Applications in Science and Industry” Sunny Beach, 21.09.2011

2. Introduction • Newton’s equations: • Potential energy (forcefield): • Free energy: Torsion Bond strength Sum over all bonds Bond angle Sum over all angles Coulomb interaction Van der Waals interactions

3. Metadynamics Metadynamics influences the evolution of the system by addition of a time-dependent external potential, constructed as a sum of gaussians, centered along the trajectory of a set of collective variables.

4. Metadynamics external potential Basic assumption:

5. Laio A, and Gervasio FL, Metadynamics: a method to simulate rare events and reconstruct the free energy in biophysics, chemistry and material science. 2008, Rep. Prog. Phys. 71, 126601 (22pp).

6. Collective variables • The CVs are explicit functions of the coordinates of the particles (or a group of particles) of the investigated systems. • The set of CVs should be able to clearly distinguish between the initial and the final state and preferably the intermediates. • Ideally, the CVs should describe all the slow events that are relevant to the investigated process, but in the same time their number should be small.

7. Human Interferon Gamma (hIFN) PDB ID: 1fg9 Amino acids forming H-bonds with receptor residues

8. Task Aberrant IFNγ expression is associated with many autoinflammatory and autoimmune diseases. The task is to find a possible way to inhibit its activity by: • Blocking the binding sites of hIFNγ • Find a ligand binding hIFNγ and blocking its activity • Blocking the binding receptors (hIFNγRα) on the cell surface • With mutated hIFNγ proteins, lacking biological activity • With some other ligand

9. Mutation site - 87Lys-88Lys-89Lys IFNγ accomplishes its multiple biological effects by activating STAT transcription factors, which are translocated to the nucleus through a specific nuclear localization sequence (NLS) in the IFNγ molecule. Two putative NLS have been pointed out in the hIFNg, one of which is located in helix E(residues 83-89). 100 random mutations These residues do not take part in the interaction between hIFNγ and its cell-surface receptor, but participate in inducing biological effect in the cell.

10. Metadynamics model • Colective variables – the backbone torsional angles φandψ of the amino acid which is on position 87; • The reconstructed free energy profiles in the space, defiend by φandψwere assessed by: • Similarity with respect to the FES profile of the native hIFNγ: • Height of the free energy barrier G, separating the α-helical and extended conformation regions

11. Free energy surface ofhIFNγ

12. Mutant 120 – Gln Ala Gly

13. Mutant 61 – His Pro Leu

14. Selected stable mutants (S<0.155 kCal/moland ΔG > 13.0 kCal/mol)

15. Conclusions • Biomolecular processes having long characteristic times and involving large-scale special rearrangement of many atoms are still challenging to simulate. • Advanced sampling techniques as metadynamics now allow such phenomena to be studied more efficiently. Metadynamics is a very powerful and effective method for accelerating rare events and reconstructing free energy surfaces of complex systems. • We used metadynamics to investigate the effect of 100 random mutations of three aminoacids in the molecule of human interferon gamma on the stability of the local secondary structure. • As a result 12 mutants were selected for further experimental investigations, which are assessed to maintain the local secondary structure of helix E and show similar or better stability than the native protein.

16. Thank You for Your Attention!

17. Back-up slides

18. Backbone torsional angles

19. hIFNγ Ramachandran plot

20. Ramachandran plot

21. Accuracy of the reconstructed FES • The error in the reconstructed free energy is independent on the underlying FES; • The accuracy and the efficiency of the reconstructed FES depend on the width of the Gaussians δs and on the ratio ω/τG; • The accuracy also depends on parameters, characterizing the investigated physical system and the chosen CVs; • The most important parameter that controls the efficiency and the error of FES reconstruction is the filling speed δs/S.

22. Advanced Metadynamics Techniques • Multiple walkers metadynamcis The same metadynamics simulation is run simultaneously on multiple replicas of the system, called walkers. The walkers interact with each other by sharing and contributing to the construction of the same metadynamics bias. • Parallel tempering metadynamcis Multiple replicas of the system are simulated at different temperatures. At time intervals τx an exchange of the coordinates of two replicas at adjacent temperatures is attempted and accepted according to a Metropolis criterion • Bias exchange metadynamcis Several replicas are simulated in parallel at the same temperature, but each replica is biased with a history-dependent potential, acting on just one or two CVs. At fixed time intervals, swaps of the history dependent potentials between pairs of replicas are attempted and accepted according to a Metropolis criterion.