Unraveling the Importance of Protein-Protein Interaction: Application of a Computational Alanine-Scanning Mutagenesis to the Study of the IgG1 Streptococcal Protein G (C2 Fragment) Complex Irina S. Moreira, Pedro A. Fernandes, and Maria J. Ramos* REQUIMTE/Departamento de Quı ´mica, Faculdade de Cie ˆ ncias da UniVersidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal ReceiVed: August 23, 2005; In Final Form: March 12, 2006 Alanine-scanning mutagenesis of protein-protein interfacial residues is a very important process for rational drug design. In this study, we have used the improved MM-PBSA approach that combining molecular mechanics and continuum solvent permits one to calculate the free energy differences through alanine mutation. To identify the binding determinants of the complex formed between the IgG1 (immunoglobulin-binding protein G) and protein G, we have extended the experimental alanine scanning mutagenesis study to both proteins of this complex and, therefore, to all interfacial residues of this binding complex. As a result, we present new residues that can be characterized as warm spots and, therefore, are important for complex formation. We have further increased the understanding of the functionality of this improved computational alanine-scanning mutagenesis approach testing its sensitivity to a protein-protein complex with an interface made up of residues mainly polar. In this study, we also have improved the method for the detection of an important amino acid residue that frequently constitutes a hot spots tryptophan. Introduction Protein-protein interactions form the basis for most biologi- cal processes including events such as intercellular communica- tion and programmed cell death. 1 A diverse study of protein- protein complexes have shown that shape complementarity is important for complex formation and that it is usually ac- companied by a high degree of chemical complementarity. 1,2 The factors affecting protein shape complementarity include the size of the buried interface, alignment of polar and nonpolar residues, and number of buried waters. 3 Crystallographic structures and alanine scanning mutagenesis of protein-protein interfacial residues have generated a large amount of information that allowed the discovery of energeti- cally important determinants of specificity at intermolecular protein interfaces. They are compact, centralized regions of residues crucial for protein association and have been named hot spots. 1,4 Therefore, a hot spot has been defined as an amino acid residue where alanine mutations cause an increase in the binding free energy larger than 4.0 kcal/mol, even though lower values are used for statistical analyses. 5,6 The warm spots are those with binding free energy differences between 2.0 and 4.0 kcal/ mol, and the null spots are the residues with binding free energy differences lower than 2.0 kcal/mol. 5 There is some tendency for these residues (the hot spots or the warm spots) to be close together and organized in clusters. 5 Hot spots are generally located near the center of protein- protein interfaces, away from the solvent, and the residues that most frequently form a hot spot are tryptophan (21%), arginine (13%), and tyrosine (12%). Since most protein-protein interac- tion surfaces are flat, it has been proposed that the exclusion of the hot spot from water requires that these residues are surrounded by a set of contacts that are energetically unimportant forming an O-ring structure. 7 One of the most interesting features of hot spots is that they are complementary to each other, with buried charged residues forming salt bridges and hydrophobic residues from one surface fitting into depressions of the binding partner. 1 Hot spots present high functional and structural adaptability. Different proteins partners tend to bind to the same hot spot, which adapts to present the same residues in different structural contexts. 1,8 Alanine-scanning mutagenesis of protein-protein interfacial residues continues to induce interest since further understanding of the nature of binding in complexes, in terms of the diverse biophysical features of the process, is essential to a phenom- enological interpretation of the results. The reliable prediction of key residues in the interface has immediate applications in protein engineering, and it is an attractive alternative therapy for many diseases (structure-based drug design). 9 Even though binding free energies can be measured experi- mentally, the quantification of the free energy components, essential to a phenomenological interpretation of the results, is hard to obtain. 10 Theoretical and computational methods rep- resent a technique capable of resolving this type of problem. 11 Therefore, great effort has been invested in attaining a compu- tational method to predict ΔΔG binding upon alanine mutation that is simultaneously detailed and fully atomistic and with a high rate of success. In a recent paper we have published a method capable of achieving an overall success rate of 80% and a 100% success rate in residues for which alanine mutation causes an increase in the binding free energy higher than 4.0 kcal/mol (hot spots). 11 In this paper, we stress the advantage of a computational approach to a comprehensive molecular thermodynamic view of affinity and, consequently, to detect drug resistant mutations. Our study defines the energetic contributions of all residues of * Corresponding author. E-mail: mjramos@fc.up.pt. 10962 J. Phys. Chem. B 2006, 110, 10962-10969 10.1021/jp054760d CCC: $33.50 © 2006 American Chemical Society Published on Web 05/12/2006