The First 3D Model of the Full-Length KIT Cytoplasmic Domain Reveals a New Look for an Old Receptor

Abstract

Receptor tyrosine kinases (RTKs) are key regulators of normal cellular processes and have a critical role in the development and progression of many diseases. RTK ligand-induced stimulation leads to activation of the cytoplasmic kinase domain that controls the intracellular signalling. Although the kinase domain of RTKs has been extensively studied using X-ray analysis, the kinase insert domain (KID) and the C-terminal are partially or fully missing in all reported structures. We communicate the first structural model of the full-length RTK KIT cytoplasmic domain, a crucial target for cancer therapy. This model was achieved by integration of ab initio KID and C-terminal probe models into an X-ray structure, and by their further exploration through molecular dynamics (MD) simulation. An extended (2-µs) MD simulation of the proper model provided insight into the structure and conformational dynamics of the full-length cytoplasmic domain of KIT, which can be exploited in the description of the KIT transduction processes. Receptor Tyrosine Kinases (RTKs) are cell-surface transmembrane proteins that control cell-signalling pathways 1 .

Folding and Intrinsic Disorder of the Receptor Tyrosine Kinase KIT Insert Domain Seen by Conventional Molecular Dynamics Simulations

“…The two descriptors, RMSD and RMSF, indicate the highly heterogeneous conformational composition of each data set obtained by cMD simulation of each KID entity. This data reveals the intrinsically disordered nature of KID from KIT, previously suggested in [ 11 ] based on a single trajectory of MD simulation. It is well known that a high content of polar and charged residues increases the propensity of a protein to be disordered [ 21 , 22 , 23 ].…”

“…The conserved α-helical architecture of the H1 helix, reported for KID embedded to KIT [ 11 ], is also observed in each studied KID entity, independently from its context—KID fused to KIT or cleaved polypeptide. Such structural consistency, together with conservation of its spatial position, suggests that this helix, immediately adjacent to the kinase domain, is the inner ordered motif of KID, which is critical for KIT function.…”

“…The kinase insert domain of KIT, composed of the 80 amino acids (aas) (F689–D768), was examined by conventional MD simulations (all-atom, with explicit water) as the cleaved polypeptide (KID C ) and as the subdomain of the kinase domain (KID D from KIT). The cleaved KID was simulated as a fully unconstrained entity (KID C ), and as a polypeptide with restrained distance between two Cα-atoms of terminal residues F689 and D768 (cleaved restrained KID, KID CR ), which is conserved in crystallographic structures of KIT [ 11 ]. The MD simulation run (of 1.8 µs for KID C and 2 µs for KIT and KID CR ) was repeated four (KID C ) and two (KIT and KID CR ) times with different randomised initial atomic velocities to extend conformational sampling of each studied entity and to examine the consistency and completeness of the produced KID conformations.…”

“…The intrinsic disorder of KID makes it difficult to study by experimental techniques [ 40 ], and the 3D numerical model is the unique description of KID from KIT [ 11 ]. By taking these 3D data as the initial structure for the study of its evolution over time, we obtained an accurate and detailed atomistic physical model which presents the intrinsically disordered kinase insertion domain of KIT as a set of heterogeneous conformations.…”

“…A first structural model of the full-length cytoplasmic domain of KIT has been published ( Figure 1 C) [ 11 ]. The primary analysis of the structural and dynamical properties of this model showed that the conformational variability of KIT is provided mainly by JMR, KID and C-terminal, which were interpreted rather as intrinsically disordered (ID) regions demonstrating significant structural and conformational plasticity.…”