The design of mutants in protein functional regions, such as the ligand binding sites, is a powerful approach to recognize the determinants of specific protein activities in cellular pathways. For an exhaustive analysis of selected positions of protein structure large scale mutagenesis techniques are often employed, with laborious and time consuming experimental set-up. 'In silico' mutagenesis and screening simulation represents a valid alternative to laboratory methods to drive the 'in vivo' testing toward more focused objectives. We present here a high performance computational procedure for large-scale mutant modelling and subsequent evaluation of the effect on ligand binding affinity. The mutagenesis was performed with a 'saturation' approach, where all 20 natural amino acids were tested in positions involved in ligand binding sites. Each modelled mutant was subjected to molecular docking simulation and stability evaluation. The simulated protein-ligand complexes were screened for their impairment of binding ability based on change of calculated Ki compared to the wild-type. An example of application to the Endothelial Protein C Receptor residues involved in lipid binding is reported. The computational pipeline presented in this work is a useful tool for the design of structurally stable mutants with altered affinity for ligand binding, considerably reducing the number of mutants to be experimentally tested. The saturation mutagenesis procedure does not require previous knowledge of functional role of the residues involved and allows extensive exploration of all possible substitutions and their pairwise combinations. Mutants are screened by docking simulation and stability evaluation followed by a rationally driven selection of those presenting the required characteristics. The method can be employed in molecular recognition studies and as a preliminary approach to select models for experimental testing.
In silico saturation mutagenesis and docking screening for the analysis of protein-ligand interaction: the Endothelial Protein C Receptor case study
Chiappori F;D'Ursi P;Merelli I;Milanesi L;Rovida E
2009
Abstract
The design of mutants in protein functional regions, such as the ligand binding sites, is a powerful approach to recognize the determinants of specific protein activities in cellular pathways. For an exhaustive analysis of selected positions of protein structure large scale mutagenesis techniques are often employed, with laborious and time consuming experimental set-up. 'In silico' mutagenesis and screening simulation represents a valid alternative to laboratory methods to drive the 'in vivo' testing toward more focused objectives. We present here a high performance computational procedure for large-scale mutant modelling and subsequent evaluation of the effect on ligand binding affinity. The mutagenesis was performed with a 'saturation' approach, where all 20 natural amino acids were tested in positions involved in ligand binding sites. Each modelled mutant was subjected to molecular docking simulation and stability evaluation. The simulated protein-ligand complexes were screened for their impairment of binding ability based on change of calculated Ki compared to the wild-type. An example of application to the Endothelial Protein C Receptor residues involved in lipid binding is reported. The computational pipeline presented in this work is a useful tool for the design of structurally stable mutants with altered affinity for ligand binding, considerably reducing the number of mutants to be experimentally tested. The saturation mutagenesis procedure does not require previous knowledge of functional role of the residues involved and allows extensive exploration of all possible substitutions and their pairwise combinations. Mutants are screened by docking simulation and stability evaluation followed by a rationally driven selection of those presenting the required characteristics. The method can be employed in molecular recognition studies and as a preliminary approach to select models for experimental testing.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.