The interaction of <scp>PD</scp> ‐1/ <scp>PD</scp> ‐ <scp>L1</scp> and its inhibition by anti‐ <scp>PD</scp> ‐ <scp>L1</scp> antibody at the single‐molecule level

Programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) checkpoint signaling and its blockade by checkpoint inhibitors are dependent on molecular interactions at the binding interface. In this work, the two complete complex structures in the protein native state of PD-1 with PD-L1, and the anti-PD-L1 antibody atezolizumab were investigated by atomic force microscopy (AFM) single-molecule force spectroscopy and predicted by AlphaFold modeling. AFM revealed that the PD-1/PD-L1 binding interface displayed greater stability than the atezolizumab/PD-L1 complex due to hydrogen bonding, while the hydrophobic effect enhanced binding flexibility at the atezolizumab/PD-L1 interface. The two complexes exhibited different bond lifetimes reflecting binding interface stability and transition distance related to the interface flexibility. This work provides relevant methodology to evaluate single-molecule macromolecular interactions. Impact statement Our research developed a novel and close-to-native physiological platform to evaluate protein interactions from structural, mechanical, and kinetic perspectives at the single-molecule level. This could be applied in the design of more effective checkpoint inhibitory molecules and provides relevant methodologies for evaluating single-molecule macromolecular interactions.