RESUMO
Despite the success of PD-1 blockade in cancer therapy, how PD-1 initiates signaling remains unclear. Soluble PD-L1 is found in patient sera and can bind PD-1 but fails to suppress T cell function. Here, we show that PD-1 function is reduced when mechanical support on ligand is removed. Mechanistically, cells exert forces to PD-1 and prolong bond lifetime at forces <7 pN (catch bond) while accelerate dissociation at forces >8pN (slip bond). Molecular dynamics of PD-1-PD-L2 complex suggests force may cause relative rotation and translation between the two molecules yielding distinct atomic contacts not observed in the crystal structure. Compared to wild-type, PD-1 mutants targeting the force-induced distinct interactions maintain the same binding affinity but suppressed/eliminated catch bond, lowered rupture force, and reduced inhibitory function. Our results uncover a mechanism for cells to probe the mechanical support of PD-1-PD-Ligand bonds using endogenous forces to regulate PD-1 signaling.
Assuntos
Antígeno B7-H1 , Simulação de Dinâmica Molecular , Proteína 2 Ligante de Morte Celular Programada 1 , Receptor de Morte Celular Programada 1 , Ligação Proteica , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/genética , Humanos , Ligantes , Antígeno B7-H1/metabolismo , Antígeno B7-H1/química , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/química , Proteína 2 Ligante de Morte Celular Programada 1/genética , Transdução de Sinais , Mutação , Animais , Linfócitos T/metabolismo , Células HEK293RESUMO
Selectins and integrins are key players in the adhesion and signaling cascade that recruits leukocytes to inflamed tissues. Selectin binding induces ß2 integrin binding to slow leukocyte rolling. Here, a micropipette was used to characterize neutrophil adhesion to E-selectin and intercellular adhesion molecule-1 (ICAM-1) at room temperature. The time-dependent adhesion frequency displayed two-stage kinetics, with an E-selectin-mediated fast increase to a low plateau followed by a slow increase to a high plateau mediated by intermediate-affinity binding of integrin αLß2 to ICAM-1. The αLß2 activation required more than 5â s contact to E-selectin and spleen tyrosine kinase (Syk) activity. A multi-zone channel was used to analyze αLß2 activation by P-selectin in separate zones of receptors or antibodies, finding an inverse relationship between the rolling velocity on ICAM-1 and P-selectin dose, and a P-selectin dose-dependent change from bent to extended conformations with a closed headpiece that was faster at 37°C than at room temperature. Activation of αLß2 exhibited different levels of cooperativity and persistent times depending on the strength and duration of selectin stimulation. These results define the precise timing and kinetics of intermediate activation of αLß2 by E- and P-selectins.