Structure-Based Energetics of Human Bim Peptide Binding to Myeloid Cell Leukemia 1

Mcl-1 is an antiapoptotic protein in the Bcl-2 family that is often overexpressed in cancer, making it a key therapeutic target. The Mcl-1 protein strongly binds (nM affinity) to the pro-apoptotic protein Bim and reduces the concentration of free Bim protein, facilitating the survival of cancer cells. Therefore, inhibiting Mcl-1 with small molecules by competing with and displacing the Bim protein offers significant potential for cancer treatment. The X-ray structure of human Mcl-1 in complex with the wild-type Bim peptide (BH3 domain of Bim protein) reveals that the peptide, in an amphipathic α-helical conformation, fits into the hydrophobic cleft of the Mcl-1 protein. We performed alchemical free energy simulations to quantitatively assess how amino acid mutations at 11 positions on the 23-residue peptide influence the stability of Mcl-1:Bim complex. A total of 54 mutant peptides, including aliphatic and nonstandard amino acid substitutions without altering the charge, were compared to the template Bim peptide. Mcl-1 incurs a large penalty of 3 kcal/mol for alanine mutations at residues (L10A and I13A), which are positioned in the middle of the peptide within its hydrophobic cleft of the protein. This penalty arises due to the loss of hydrophobic interactions. However, Mcl-1 showed weak discrimination (less than 1.5 kcal/mol) for other alanine mutations in the peptide, with the strength of discrimination decreasing for the residues near the terminals. These results support previous experimental observations showing a significant decrease in Mcl-1 affinity due to L10A and I13A mutations in Bim, with a lesser impact on affinity from other alanine mutations. Interestingly, the F17V mutation stabilizes the protein-peptide complex by 3 kcal/mol, indicating that the F17V-Bim mutant is a promising Mcl-1 inhibitor. Mcl-1 strongly disfavors the D15 → E15 mutation in the Bim peptide with a penalty of about 6.5 kcal/mol due to loss of salt bridge interactions. Notably, adding one carbon to the aliphatic arm in the Bim (D15 → E15) can significantly disrupt the electrostatic interactions. This highlights the necessity of achieving an optimal hydrophobic-hydrophilic balance for effective Mcl-1:peptide interactions. The study connects thermodynamics and structures to clarify the mechanism of peptide recognition by Mcl-1, providing valuable insights for designing Bim variants with enhanced affinity.