Computational Analysis and Prediction of the Binding Motif and Protein Interacting Partners of the Abl SH3 Domain

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Abstract
Protein-protein interactions, particularly weak and transient ones, are often mediated by peptide recognition domains, such as Src Homology 2 and 3 (SH2 and SH3) domains, which bind to specific sequence and structural motifs. It is important but challenging to determine the binding specificity of these domains accurately and to predict their physiological interacting partners. In this study, the interactions between 35 peptide ligands (15 binders and 20 non-binders) and the Abl SH3 domain were analyzed using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. The calculated binding free energies correlated well with the rank order of the binding peptides and clearly distinguished binders from non-binders. Free energy component analysis revealed that the van der Waals interactions dictate the binding strength of peptides, whereas the binding specificity is determined by the electrostatic interaction and the polar contribution of desolvation. The binding motif of the Abl SH3 domain was then determined by a virtual mutagenesis method, which mutates the residue at each position of the template peptide relative to all other 19 amino acids and calculates the binding free energy difference between the template and the mutated peptides using the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. A single position mutation free energy profile was thus established and used as a scoring matrix to search peptides recognized by the Abl SH3 domain in the human genome. Our approach successfully picked ten out of 13 experimentally determined binding partners of the Abl SH3 domain among the top 600 candidates from the 218,540 decapeptides with the PXXP motif in the SWISS-PROT database. We expect that this physical-principle based method can be applied to other protein domains as well. One of the central questions of molecular biology is to understand how signals are transduced in the cell. Intracellular signal transduction is mainly achieved through cascades of protein-protein interactions, which are often mediated by peptide-binding modular domains, such as Src Homology 2 and 3 (SH2 and SH3). Each family of these domains binds to peptides with specific sequence and structural characteristics. To reconstruct the protein-protein interaction networks mediated by modular domains, one must identify the peptide motifs recognized by these domains and understand the mechanism of binding specificity. These questions are challenging because the domain-peptide interactions are usually weak and transient. Here, the authors took a physical-principles approach to address these difficult questions for the SH3 domain of human protein Abl, which binds to peptides containing the PXXP motif (where P is proline and X is any amino acid). They generated a position-specific scoring matrix to represent the binding motif of the Abl SH3 domain. Analysis on the binding free energy components suggested insights into how the binding specificity is achieved. Most known protein interacting partners of the Abl SH3 domain were correctly identified using the position-specific scoring matrix, and other potential interacting partners were also suggested.