The impact of PD-1 mutations on pembrolizumab binding: insights from molecular dynamics and MM-GBSA analysis.

[UNLABELLED] Programmed cell death protein 1 (PD-1) is a critical immune checkpoint receptor that regulates T-cell activity by interacting with its ligand, PD-L1. This interaction enables tumor cells to evade immune surveillance, making PD-1 a key target in cancer immunotherapy. Pembrolizumab, a monoclonal antibody targeting PD-1, disrupts this interaction, restoring immune responses against tumors. Given the importance of the PD-1/pembrolizumab interaction in treatment efficacy, this study investigates the impact of PD-1 interface mutations on pembrolizumab binding using molecular dynamics (MD) simulations and MM-GBSA free energy calculations. Structural analysis of the PD-1/Fab-pembrolizumab wild-type complex identified key residues involved in hydrogen bonding, hydrophobic interactions, and salt bridges that stabilize the complex. MD simulations over 50 ns provided insights into the dynamic behavior of these interactions, highlighting 16 PD-1 residues with significant energetic contributions to the binding. A mutation search using UniProt identified 28 likely benign PD-1 variants within these 16 key residues. MM-GBSA analysis following 50 ns of MD simulations for the 28 mutations revealed that 23 mutations reduced pembrolizumab binding affinity, while 5 mutations increased it. Notably, the Pro89Arg, Asp85Gly, and Asp85Asn mutations exhibited the most pronounced reductions in binding free energy, with decreases of 26.69, 20.33, and 18.38 kcal/mol, respectively. The Pro89Arg mutation disrupted key hydrophobic interactions with surrounding residues, while Asp85Gly and Asp85Asn abolished a critical salt bridge with Arg99 in the wild-type complex. Our findings provide a detailed characterization of PD-1/pembrolizumab binding and suggest that specific mutations may alter therapeutic efficacy. Further experimental studies are needed to validate these findings and assess their clinical significance.

[SUPPLEMENTARY INFORMATION] The online version contains supplementary material available at 10.1038/s41598-026-45077-0.