Figure 1.
The domain organization of full length JAK2.
Figure 2.
The schematic of hierarchical protein-protein docking procedure.
Figure 3.
Starting models were generated by connecting sites identified by MutInf or by using dimer geometries from crystal structures.
(A) Two predefined packing models were constructed by manually joining the αC helix in the JAK2 kinase domain with two highly coupled sites in the JH2 pseudokinase domain identified by MutInf. (B) Four additional packing models were built by alignment to crystal structures of kinase dimeric forms (FGFR1, FGFR2, BRAF and PKB).
Figure 4.
Correlated motions couple active to putative allosteric sites in the JAK2 JH1 and JH2 domains.
(A) The pairwise matrix of the highly coupled residues in the inactive conformation of the JAK2 JH1 domain. (B) Strong correlations between the activation loop (green box) and the αC helix (red box) are observed in the JH1 domain (yellow in cartoon). (C) The pairwise matrix of the highly coupled residues in the active conformation of the JH2 domain. (D) The strong correlations of the loop between β7–β8 sheets near the hinge region (green box) with activation loop (red box) shown in the JH2 domain (blue in cartoon).
Table 1.
The structural and energetic properties of six predetermined models through 10 ns MD simulation.
Figure 5.
The V617F mutant in the JH2 domain packs closer to F595 than in the wild-type JH2 in 30 ns MD simulations.
(A) Centroid distances between F595 and V/F617 in JAK2-WT and JAK2-V617F show a closer packing in the V617F mutant than in the wild type. (B, C) Cartoon representations are shown for the last snapshot of JAK2-V617F and wild-type JH2, respectively, after 30 ns MD simulations, showing a contact in the V617F mutant that is not present after 30 ns for the wild-type pseudokinase. The F595, V/F617 and F594 are colored in orange, red and grey.
Figure 6.
Motions of the activation loop and αC-helix in JAK2-WT and JAK2-V617F in 30 ns MD simulation.
(A) The JAK2-V617F mutant increases the flexibility of the activation loop in the kinase domain by restricting the movement of αC helix region in the JH2. The activation loop in the kinase domain of JAK2-WT (black) and JAK2-V617F (red) is shown alongside the crystal structure (green) in cartoon representation using PyMOL. Other residues are shown in surface representation. (B) RMSDs of Cα atoms during 30 ns of MD simulation show displacement of the kinase domain activation loop residues 994–1028 in the V617F mutant (black) but not the wildtpype (red). (C) Conformations of the αC helix in kinase domain after 30 ns MD for V617F mutant (black) and wild type (red). (D) RMSDs for the kinase αC helix (residues 586–606) in JAK2-WT and JAK2-V617F during 30 ns MD simulation show that the position of this helix is relatively stable in both cases. RMSDs are computed over Cα atoms of JAK2 with respect to the initial model after superposition of the kinase domain.
Figure 7.
JAK2 JH1-JH2 complex model involves two sets of significant interfacial residues.
The JH2 (residues 523 to 816) is shown in blue cartoon while the kinase domain (residues 840 to 1132) is shown in yellow cartoon. The linker loop between two domains is colored pink. Interface 1 shows a site of electrostatic complementarity near the αC helix region of the JH2 and the αEF/αF loop region of kinase domain. The electrostatic complementarity is provided by R588, E592, E1028 and K1030. Interface 2 is composed of mainly hydrophobic residues, especially V706, L707, I901, R971, I973 and V1033.
Figure 8.
Mutation of interfacial residues changed JAK2 kinase activity.
(A) JAK2 mutants showed increased basal kinase activity. Activity of HA-tagged JAK2 mutants was measured by phospho-JAK2 antibodies. Total JAK2 level was measured by anti-HA antibodies. P-JAK2: phosphorylated JAK2. V: vector alone. WT: wild-type JAK2. (B) Hyperactive JAK2 mutants showed increased STAT5 activation. Activation of STAT5 was assessed using flow cytometry with Alexa647-conjugated antibodies to phospho-STAT5. Results were normalized to wild-type JAK2. (C) Hyperactive JAK2 mutants transformed BaF3/EpoR cells into factor-independent growth. Cell growth at each indicated day was measured by MTT assay. JAK2-V617F expressing cells became saturated on Day 5. WT: wild-type JAK2. (D) Mutations in JH1 reduced basal JAK2 kinase activity. Activation of STAT5 was determined as in (B). Results were normalized to wild-type JAK2.