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 DFGout conformations represents a mainstream strategy for developing highly selective type II inhibitors. RTKs can adopt various DFGout 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 DFGout 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 DFGout 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 DFGout subconformational space to shift populations from nonclassical to classical DFGout 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 DFGout conformations. Such a role of JM is particularly favorable for creating druggable DFGout conformations that can be exploited for designing high-selectivity type II inhibitors.