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, Bulgaria2 Institute for Nuclear Research and Nuclear Energy, Bulgarian Academy of

Sciences, Sofia, Bulgaria

Overview

hIFN-γ and suppressing its abnormal biological activity

Protein docking with Rosetta Algorithm Scoring functions

hIFN-γ study with Rosetta Conclusions

hIFN-γ Powerful immunomodulator;

Autoimmune diseases due to abnormal expression;

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!

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;

High resolution calculation concept

Scoring function (1) Low resolution scoring function:

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 functions (2) High resolution scoring function:

S – scoring function terms; w – weights.

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;

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;

Blind prediction results

Refinement run results

Predicted structure

RMSD 0,74 Å

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;

Thank You

for

Your Attention!

Back-up slides

Metropolis acceptance criterion

Probability to go from state j to state i:

Table of weights