Figure 1.
Structural organization of KIT.
Stem Cell Factor (SCF) binding on the extracellular domain of KIT induces dimerization. Upon activation, KIT is autophosphorylated at tyrosine residues (magenta balls) that act as binding sites for downstream signaling kinases/mediators. The location of KIT gain-of-function mutations is indicated by residue numbers. Residues V560 and D816 (red balls), positioned in the JMR and in the kinase domain of the cytoplasmic region, are associated with highly malignant cancers. JMR, juxta-membrane region; KD, kinase domain; KID, kinase insert domain.
Figure 2.
The inactive-to-active state switch of KIT tyrosine kinase domain.
(a) The inactive auto-inhibited state with the A-loop and the JMR adjacent to the active site (1T45 [34] (c) and the active state with a solvent-exposed JMR and an extended A-loop conformation (1PKG [108]) with schematic representation of the secondary structures observed in the inactive and the active states. (b) The intermediate state, inactive not auto-inhibited, observed in KIT complex with imatinib (1T46 [34]). Protein surface of KIT (gray) with the A-loop (red) and the JMR (yellow) shown as cartoons. Imatinib is shown as sticks. The principal H-bonds stabilizing the β-sheets are shows by green doted lines. Positions of residues V560 and D816, if resolved, are indicated as balls. The catalytic residues, DFG, indicated as sticks are zoomed in the inserts.
Figure 3.
MD simulations of the KIT cytoplasmic domain in the inactive state.
The RMSDs (in Å) per residue were calculated from trajectories 1 (red) and 2 (green) of MD simulations of KITD816Y, KITD816N, KITD816H, KITV560G and KITV560G. Horizontal doted lines indicate the RMSDs mean values.
Figure 4.
Displacements and fluctuations of residues in KIT mutants.
(A) The RMSFs computed on the carbon and nitrogen backbone atoms over the total production simulation time of KIT mutants were compared to those in KITWT. (Top) RMSFs (Å) of KITWT [40] (graph in black) and the RMSFs difference (Δ RMSF, Å) between KITWT and mutants: (Middle) KITV560G (orange), KITV560D (purple), (bottom) KITD816V (red), KITD816H (green), KITD816Y (magenta) and KITD816N (blue). (B) The regions with significant average Δ RMSF values are displayed with different colors: JMR (yellow), A-loop (red), N- and C-lobe (cyan and blue) and KID (gray). The radii of the KIT cartoon representation is scaled relative to the RMSF values in KITWT.
Figure 5.
MD conformations of KIT cytoplasmic region in the native protein and its mutants.
(A) Superposed conformations were selected by RMSDs clustering. Ribbon diagrams display the proteins regions or fragments with different colors: JMR (yellow), A-loop (red), N- and C-lobe (cyan and blue), C-helix in the N-lobe (green), G-helix in C-lobe (purple) and KID (gray). The point mutations in positions 816 and 560 are shown as balls. The earlier reported KITWT and mutant KITD816V are encircled. (B) Superimposed structures of the most representative MD conformations in the native KIT (I) (in wheat), its mutant D816H (II) (in pale cyan) and the crystallographic structure 1T45 (III) (in gray). Ribbon diagrams display the proteins regions or fragments with different colors: (I) JMR in yellow, Cα-helix in green, A-loop in magenta; (II) JMR in orange, Cα-helix in cyan, A-loop in red; (III) JMR in sand, Cα-helix in pale green, A-loop in pink.
Figure 6.
A-loop conformations in KITWT and KITD816H/V/Y/N mutants.
(a) Cluster analysis of the MD conformations picked every 10 ps over the 96 ns of productive MD simulations were performed on the RMSDs computed for only the A-loop backbone atoms. Five different sets were produced through 5 independent runs of clustering (I–V). The composition of clusters is represented as colored barplots in a logarithmic scale: KITWT (gray), KITD816V (red) KITD816H (green), KITD816Y (blue), KITD816N (pink). The rank in the concatenated trajectory of the corresponding reference structure is reported in the bottom of each barplot and colored accoring to the population of the group (see below). (b–d). Superposition of the reference structures in each cluster analysis run. The reference structures are drawn as cartoons illustrating the A-loop conformation (b), the relative orientation of the residues Y823 (A-loop) and D792 (C-loop) (c) and the conformation of DFG motif (A-loop) (d). The A-loop is shown as cartoon diagrams, residues Y823 and D792 as sticks, F811 as thick lines; D/H/V/Y/N816 as sphere. The H-bonds are shown by dotted lines. The A-loop conformations observed in all simulated proteins including KITWT are shown in grey; conformations detected in all protein but one mutant are distinguished in black or blue; conformations detected only in the mutants are in yellow; the proper conformations of KITD816V mutant are in red; characteristic of the pair of mutants, KITD816V/Y or KITD816V/N are in purple or in pink.
Figure 7.
Variable of secondary structures in the cytoplasmic region of KITWT and mutants, KITD816H/Y/N and KITV560G/D.
Secondary structure assignments for the JMR (left) and the A-loop (right) were averaged over the two replica of each MD simulations (2×48 or 2×65 ns) of KITWT and mutants. For each residue, the proportion of each secondary structure type is given as a percentage of the total simulation time and shown with lines of different colors: 310-helices (red), antiparallel β-sheet (blue), turns (green), total structure (dashed gray).
Figure 8.
H-bond patterns in the A-loop and the JMR.
Top: H-bonds stabilizing the small 310-helix in the A-loop of KITWT (in two different projections) and coiled structure of the A-loop observed in mutant KITD816H. Bottom: H-bonds of the JMR and the N-lobe residues stabilizing the coiled structure of the JM-Switch in KITWT and KITV560D and nearly antiparallel β-sheet in KITV560G. All residues presented as sticks, each residue is labeled in KITWT and specified by color retained for the same residue in the mutants, only the side chains participating in the H-bonds are shown. The H-bonds are shown as dotted lines.
Figure 9.
Principal Component Analysis of KIT cytoplasmic region in the inactive state.
Calculation was performed on the backbone atoms of KITWT and mutants, KITD816H/Y/N and KITV560G/D, combining the MD trajectories 1 and 2 (total of 96-ns production simulation time for KITD816H/Y/N mutants, and 130-ns for KITV560G/D mutants) and taking the average MD conformations as references for the RMS fits. (a) A diagram gives the eigenvalues spectra of KITWT (black) and mutants, KITD816V (red), KITD816H (green), KITD816Y (purple), KITD816N (blue), KITV560G (orange) and KITV560D (mauve), in descending order. Atomic components in modes 1–3 for KITWT (b) and modes 1–2 for KIT mutants (c) are drawn as dark grey arrows on the protein cartoon representation. Some regions or fragments of the proteins are displayed with different colors: JMR (yellow), A-loop (red), N- and C-lobe (cyan and blue), the C-helix in the N-terminal (green), the C-terminal helix (purple) and KID (gray).
Figure 10.
Independent dynamic segments identified in KIT cytoplasmic region.
Structural mapping of the Independent Dynamic Segments (IDSs) identified in KITWT and KITmutants. The average conformations are presented as tubes. The size of the tube is proportional to the by-residue atomic fluctuations computed on the backbone atoms. IDSs are referred to as , where i = 1, 2…10, labeled in KITWT and specified by color retained for the IDSs in mutants. The largely modified or newly found IDSs in the mutants are referred to as
.
Figure 11.
Communication pathways of cytoplasmic region in KITWT.
(a) 2D graph of a global topology the inter-residues communications. Residues are represented by points, communication pathways (CPs) are depicted by bold lines and two connected residues by a thin line. Structural fragments involved in Independent Dynamic Segments (IDSs), are labelled as Si where i = 1, 2, …10. (b) 3D structural mapping of the communication efficiently of KIT residues. The average MD conformation is presented as cartoon. Residues in 2D (a) and 3D (b) representations are coloured from blue through green and yellow to red according to their communication efficiency, estimated as the number of residues to which they are connected by at least one CP. Residues V560, V559, D792, D816 and Y813 are encircled by magenta. Clusters corresponding to protein segments of interest are contoured by dotted lines. 2D and 3D graphs and drawn with Gephi and PyMOL modules incorporated in MONETA.
Figure 12.
Communication pathways of cytoplasmic region in KITWT and KITV560G/D mutants.
2D and 3D graphs were drawn with Gephi and PyMOL modules incorporated in MONETA. Left panel: 2D graphs of a global topology illustrating the inter-residues communications in KITWT and KIT mutants. Residues are represented by points, communication pathways (CPs) are depicted by bold lines and two connected residues (by at least on eCP) by a thin line. Residues are coloured from blue through green and yellow to red according to their communication efficiency. The colours are normalized according to the global range of these values for all studied proteins. The C-loop residues are contoured by dotted lines. Middle panel: Large-scale view of the CPs in KIT zoomed on CPs in the JMR and the A-loop. Residues are coloured from blue through green and yellow to red according to their communication efficiency. The colours are normalized according to the range of values for each protein, reflecing a relative communication efficiency of residues in each protein. The C-loop residues are contoured by dotted lines. Each residue is labelled by its number in KIT sequence. Right panel: Average MD conformation presented as cartoon. The residues forming IDSs in the A-loop and the JM-Switch are colored in red and yellow respectively. V559, D792 and Y823 are show in sticks, positions of V560G/D are indicated by black points. CPs initiated by either V559 or D792 are represented in bold lines, magenta in KITWT (CPwt only) and black in the mutants.
Figure 13.
Communication pathways of cytoplasmic region in KITWT and KITD816N/Y/H/V mutants.
2D and 3D graphs were drawn with Gephi and PyMOL modules incorporated in MONETA. Left panel: 2D graphs of a global topology illustrating the inter-residues communications in KITWT and KIT mutants. Residues are represented by points, communication pathways (CPs) are depicted by bold lines and two connected residues (by at least on eCP) by a thin line. Residues are coloured from blue through green and yellow to red according to their communication efficiency. The colours are normalized according to the global range of these values for all studied proteins. The C-loop residues are contoured by dotted lines. Middle panel: Large-scale view of the CPs in KIT zoomed on CPs in the JMR and the A-loop. Residues are coloured from blue through green and yellow to red according to their communication efficiency. The colours are normalized according to the range of values for each protein, reflecing a relative communication efficiency of residues in each protein. The C-loop residues are contoured by dotted lines. Each residue is labelled by its number in KIT sequence. Right panel: Average MD conformation presented as cartoon. The residues forming IDSs in the A-loop and the JM-Switch are colored in red and yellow respectively. V559, D792 and Y823 are show in sticks, positions of D816N/Y/H/V are indicated by black points. CPs initiated by either V559 or D792 are represented in bold lines, magenta in KITWT (CPwt only) and black in the mutants.
Figure 14.
Proposed mechanisms of the constitutive activation of KIT mutants and consequences on drug sensitivity.
The multi-states equilibrium of KIT cytoplasmic region in KITWT (upper panel), KITD816H/V/Y/N (middle panel) and KITV560G/D (lower panel). Each KIT conformation is represented as a molecular surface, except the JMR and the A-loop and imatinib drawn as cartoons and sticks respectively. In KIT mutants, the mutation position is shown by a ball. Equilibrium between two states is denoted by arrows of different thicknesses. Upper panel: In the absence of SCF, KITWT is mainly in the inactive autoinhibited state maintained by the JMR non-covalently bounded to the kinase domain. This state of KIT is the imatinib target. Middle panel: The A-loop mutations (D816V/H/Y/N) induce the inactive non-autoinhibited state of KIT evidenced by the JMR departure from the kinase domain. This effect conducts to deployment of the A-loop eventually leading to the constitutively active KIT state. The inactive non-autoinhibited state is not a suitable target for imatinib that inhibit the inactive autoinhibited state. Lower panel: The JMR mutations (V560G/D) greatly impact the JMR binding to the kinase domain and facilitate its departure, favoring the non-autoinhibited state, whereas the inactive conformation of the A-loop is still conserved. The inactive non-autoinhibited state of KIT is more consented in KITV560G/D than in KITWT and especially in KITD816V/H/Y/N, leaded to the increased sensitivity of KITV560G/D to inhibitor compared to KITWT. In each panel, the most preferred state of KIT in the presence of imatinib is encircled.