Converging free energy estimates: MM‐PB(GB)SA studies on the protein–protein complex Ras–Raf

Abstract Estimating protein–protein interaction energies is a very challenging task for current simulation protocols. Here, absolute binding free energies are reported for the complex H‐Ras/C‐Raf1 using the MM‐PB(GB)SA approach, testing the internal consistency and model dependence of the results. Averaging gas‐phase energies (MM), solvation free energies as determined by Generalized Born models (GB/SA), and entropic contributions calculated by normal mode analysis for snapshots obtained from 10 ns explicit‐solvent molecular dynamics in general results in an overestimation of the binding affinity when a solvent‐accessible surface area‐dependent model is used to estimate the nonpolar solvation contribution. Applying the sum of a cavity solvation free energy and explicitly modeled solute–solvent van der Waals interaction energies instead provides less negative estimates for the nonpolar solvation contribution. When the polar contribution to the solvation free energy is determined by solving the Poisson–Boltzmann equation (PB) instead, the calculated binding affinity strongly depends on the atomic radii set chosen. For three GB models investigated, different absolute deviations from PB energies were found for the unbound proteins and the complex. As an alternative to normal‐mode calculations, quasiharmonic analyses have been performed to estimate entropic contributions due to changes of solute flexibility upon binding. However, such entropy estimates do not converge after 10 ns of simulation time, indicating that sampling issues may limit the applicability of this approach. Finally, binding free energies estimated from snapshots of the unbound proteins extracted from the complex trajectory result in an underestimate of binding affinity. This points to the need to exercise caution in applying the computationally cheaper “one‐trajectory‐alternative” to systems where there may be significant changes in flexibility and structure due to binding. The best estimate for the binding free energy of Ras–Raf obtained in this study of −8.3 kcal mol −1 is in good agreement with the experimental result of −9.6 kcal mol −1 , however, further probing the transferability of the applied protocol that led to this result is necessary. © 2003 Wiley Periodicals, Inc. J Comput Chem 2: 238–250, 2003