Investigation of Binding Affinity of hIFN-gamma Mutated Forms with the Rosetta Suite of Programs

1. Investigation of Binding Affinity of hIFN-gamma Mutated Forms with the Rosetta Suite of Programs Damyan Grancharov1, Peicho Petkov1, Elena Lilkova1, Nevena Ilieva2, Leandar Litov1 1 Faculty of Physics, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria 2Institute for Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences, Sofia, Bulgaria

2. Overview • hIFN-γ and suppressing its abnormal biological activity • Protein docking with Rosetta • Algorithm • Scoring functions • hIFN-γ study with Rosetta • Conclusions

3. hIFN-γ • Powerful immunomodulator; • Autoimmune diseases due to abnormal expression;

4. Suppression of biological activity • Competitive binding of the hIFN-γR; • Mutated analogs of the native hIFN-γ that: • Interrupt the signal transduction pathway; • Preserve binding ability; • 12 mutated analogs out of 100 were selected; • Preservation of 3D structure = preservation of binding ability; • Protein docking is needed to test such a hypothesis!

5. Rosetta docking • Imitates the binding process in real life; • Low resolution search: • Centroid representation; • 500 random perturbations of the current conformation; • Low res scoring function; • High resolution calculation: • All atom representation; • High res scoring function; • 50 perturbations of random displacement and side-chain optimization; • Minimization; • Acceptance via Metropolis criterion;

6. High resolution calculation concept

7. Chemical terms: Spair – accounts for pairwise coupling of amino acids; Senv – accounts for the environment of amino acids; Structural terms: Scontact – “reward” term for VdW attractive interaction; Sbump – “penalty” term for VdW repulsive interaction; Salign – term to account for additional biological information; Scoring function (1) • Low resolution scoring function:

8. S – scoring function terms; w – weights. Scoring functions (2) • High resolution scoring function:

9. Scoring functions (3) • Satr – attractive Van der Waals interaction; • Srep – repulsive Van der Waals interaction; • Ssol – accounts for the presence of an implicit solvent; • Ssasa – accounts for the solvent accessible surface area; • Shb – accounts for the presence of hydrogen bonds; • Sdun – accounts for rotamer probabilities; • Spair – accounts for pairwise coupling of amino acids; • Selec – electrostatic interaction, divided into short and long ranged, attractive and repulsive;

10. hIFN-γ study with Rosetta • For the sake of testing the algorithm: • Native hIFN-γ was used; • “Blind prediction” protocol was used: • Partners were randomized; • 50000 models were generated; • Results plotted: Score vs RMSD; • Best model passed on to refinement; • Refinement run: • 1000 model were generated; • In the Score vs RMSD plot a “funnel” appeared;

11. Blind prediction results

12. Refinement run results

13. Predicted structure RMSD 0,74 Å

14. Conclusions • Close-to-native conformations can be obtained, paying the cost of extensive search: • Native hIFN-γ with receptor complex reproduced within 0,74 Å; • All 12 mutated forms will enter only a refinement procedure: • To be adjusted within the funnel in free energy space, encountered for the native hIFN-γ;

15. Thank You for Your Attention!

16. Back-up slides

17. Metropolis acceptance criterion • Probability to go from state j to state i:

18. Table of weights