Hotspot Mutations in KIT Receptor Differentially Modulate Its Allosterically Coupled Conformational Dynamics: Impact on Activation and Drug Sensitivity

Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D) localized in crucial regulatory segments, the juxtamembrane region (JMR) and the activation (A-) loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts.Author Summary: Receptor tyrosine kinase KIT plays a crucial role in the regulation of cell signaling. This allosterically controlled activity may be affected by gain-of-function mutations that promote the development of several cancers. Identification of the molecular basis of KIT constitutive activation and allosteric regulation has inspired computational study of KIT hotspot mutations. In the present contribution, we investigated the mutation-induced effects on KIT conformational dynamics and intra-protein communication conditionally on the mutation location and the nature of the substituting amino acid. Our data elucidate that all studied mutations stabilize an inactive non-autoinhibited state of KIT over the inactive auto-inhibited state prevalent for the native protein. This shift in the protein conformational landscape promotes KIT constitutive activation. Our in silico analysis established correlations between the structural and dynamical effects induced by oncogenic mutations and the mutants auto-activation rates and drug sensitivities measured in vitro and in vivo. Particularly, the A-loop mutations stabilize the drug-resistant forms, while the JMR mutations may facilitate inhibitors binding to the active site. Cross-correlations established between local and long-range structural and dynamical effects demonstrate the allosteric character of the gain-of-function mutations mode of action.