Computational Alanine Scanning Mutagenesis: MM-PBSA vs TI

Understanding protein–protein association and being able to determine the crucial residues responsible for their association (hot-spots) is a key issue with huge practical applications such as rational drug design and protein engineering. A variety of computational methods exist to detect hot-spots residues, but the development of a fast and accurate quantitative alanine scanning mutagenesis (ASM) continues to be crucial. Using four protein–protein complexes, we have compared a variation of the standard computational ASM protocol developed at our group, based on the Molecular Mechanics/Poisson–Boltzmann Surface Area (MM-PBSA) approach, against Thermodynamic Integration (TI), a well-known and accurate but computationally expensive method. To compare the efficiency and the accuracy of the two methods, we have calculated the protein–protein binding free energy differences upon alanine mutation of interfacial residues (ΔΔG_bind). In relation to the experimental ΔΔG_bind values, the average error obtained with TI was 1.53 kcal/mol, while the ASM protocol resulted in an average error of 1.18 kcal/mol. The results demonstrate that the much faster ASM protocol gives results at the same level of accuracy as the TI method but at a fraction of the computational time required to run TI. This ASM protocol is therefore a strong and efficient alternative to the systematic evaluation of protein–protein interfaces, involving hundreds of amino acid residues in search of hot-spots.