The Disordered JM Motif in RTKs Promotes Classical DFG_out Conformation Formation via the Dynamic Effect

Receptor tyrosine kinases (RTKs) are validated anticancer targets, and targeting their DFG_out conformations represents a mainstream strategy for developing highly selective type II inhibitors. RTKs can adopt various DFG_out conformations, but only the classical conformations with a fully formed back pocket are structurally validated to accommodate type II inhibitors. However, experimentally determined structures of kinases in a classical DFG_out conformation are scarce, which presents a significant obstacle for the structure-based design of selective RTK inhibitors. Recently, a conserved disordered motif N-terminal to the kinase domain of RTKs, called the juxtamembrane (JM) motif, has been reported to regulate the binding of inhibitors to the DFG_out conformation of VEGFR2, an RTK involved in angiogenesis. In this study, we performed extensive MD simulations to explore the impact of the disordered JM motif on the conformational space of the DFG motif in RTKs and to investigate how that influence may affect inhibitor binding to VEGFR2. We revealed that in VEGFR2, the disordered JM is highly dynamic and forms transient contacts with the kinase domain and consequently fine-tunes the DFG_out subconformational space to shift populations from nonclassical to classical DFG_out conformations. This dynamic model provides alternative structural explanations underpinning the reported regulatory effects of the JM motif on binding of the inhibitor to VEGFR2. Additionally, we demonstrated that in other RTKs beyond VEGFR2, the disordered JM similarly promotes classical DFG_out conformations. Such a role of JM is particularly favorable for creating druggable DFG_out conformations that can be exploited for designing high-selectivity type II inhibitors.