Relative binding free energy calculations of inhibitors to two mutants (Glu46— Ala/Gln) of ribonuclease T1 using molecular dynamics/free energy perturbation approaches

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Abstract
We present free energy perturbation calculations on the complexes of Glu46— Ala46 (E46A) and Glu46— Gln46 (E46Q) mutants of ribonuclease T1 (RNaseT1) with inhibitors 2‘-guanosine monophosphate (GMP) and 2’adenosine monophosphate (AMP) by a thermodynamic perturbation method implemented with molecular dynamics (MD). Using the available crystal structure of the RNaseT1–GMP complex, the structures of E46A-GMP and E46Q-GMP were model built and equilibrated with MD simulations. The structures of E46A-AMP and E46Q-AMP were obtained as a final structure of the GMP—AMP perturbation calculation respectively. The calculated difference in the free energy of binding (ΔΔGbind) was 0.31 kcal/mol for the E46A system and —1.04 kcal/mol for the E46Q system. The resultant free energies are much smaller than the experimental and calculated value of ∼3 kcal/mol for the native RNase T1, which suggests that both mutants have greater relative adenine affinities than native RNaseT1. Especially E46Q is calculated to have a larger affinity for adenine than guanine, as we suggested previously from the calculation on the native RNaseT1. Thus, the molecular dynamics/free energy perturbation method may be helpful in protein engineering, directed toward increasing or changing the substrate specificity of enzymes.

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