In-silico mutagenesis and binding free energy prediction
Following the experimental mutational analysis from previous studies [14, 37], we designed in-silico mutants of thehs CENP-H and hs CENP-M in an attempt to validate the predicted interactions between each subunit of the hypotheticalhs CENP-HIKM complex (Supplementary Figures S6 and S7). In order to validate predicted interface interactions between the C-terminal of the hs CENP-H and other subunits (C-terminal of thehs CENP-K and the N-terminal of the hs CENP-I), Hu et al . [14] constructed several mutants of the protein (L219A, V225A, L233A, K234A and L238A) based on residue conservation. The mutated residues correspond to ILE-205, ILE-211, LEU-219, ARG-220 and LEU-224 respectively in the th CENP-H. A dramatic reduction in binding affinity was recorded upon the mutation of each residue to alanine, indicating that the residues are essential for the protein-protein interaction of the complex. In a similar study by Basilico et al . [37], mutants of the hs CENP-M (L94A and L163E) were also designed based on residue conservation analysis and the mutation of both residues to alanine and glutamate respectively affected the interaction of the protein with the C-terminal of the hs CENP-I.
Having successfully designed the in-silico mutants of these proteins in line with reports from existing literature, we predicted the binding free energy changes using several predictive tools as reported in the materials and methods section. The reduction in binding free energy as a result of these mutations shows the consistency of our computational model with experimental reports (Table 1-4).
Table 1. The BeAtMuSiC-predicted change in binding affinity as a result of mutation. With the main input being the protein–protein complex structures, the output reports the mutated chains, the specific mutations, change in binding free energy (Kcal/mol) and solvent accessibility, both in partner and in complex. Solvent accessibility depicts the solvent-accessible surface ratio in the structure, based on DSSP computation.