Exploring conformation changes of Janus kinase 2 pseudokinase mediated by mutations through Gaussian accelerated molecular dynamics and principal component analysis

The pseudokinase domain (JH2) of the protein tyrosine kinase (Janus kinase 2, JAK2) regulates the activity of a tyrosine kinase domain (JH1) in JAK2, which is further affected by mutations in the JH2. In this work, Gaussian accelerated molecular dynamics (GaMD) simulations followed by construction of free energy landscapes (FELs) and principal component analysis (PCA) were performed to study effect of two mutations V617F and V617F/E596A on the conformations of the ATP-bound JH2. The dynamic analyses reveal that mutations affect the structural flexibility and correlated motions of the JH2, meanwhile also change the dynamics behavior of the P-loop and αC-helix of the JH2. The information from FELs unveils that mutations induce less energy states than the free JH2 and the WT one. The analyses of interaction networks uncover that mutations affect the salt bridge interactions of ATP with K581, K677 and R715 and alter hydrogen bonding interactions (HBIs) of ATP with the JH2. The changes in conformations of the JH2 and ATP-JH2 interaction networks caused by mutations in turn generate effect on the activity regulations of the JH2 on the JH1. This work is expected to provide significant theoretical helps for deeply understanding the function of the JH2 and drug design toward JAK2. Communicated by Ramaswamy H. Sarma</p

Cross-correlation matrices of the fluctuations of the coordinates for C_α atoms around their mean positions during the equilibrium phase of the simulations.

<p>The extent of correlated motions and anticorrelated motions are color-coded for unbound A-FABP(A), 8CA(B), F8A(C) and I4A(D).</p

Hydrogen bonding energy calculated based on an empirical equation.

<p>Hydrogen bonding energy calculated based on an empirical equation.</p

Interactions of key residues in A-FABP with the inhibitor 8CA.

<p>Fig. A represents frequency distribution of the H atom…acceptor distance, Fig. B depicts the position of inhibitor 8CA relative to key residues, Fig. C shows the hydrophobic contacts as a function of the simulation time.</p

Molecular structures of the three inhibitors 8CA (A), F8A(B) and I4A(C).

<p>The structural difference is labeled by red circle.</p

Interactions of key residues in A-FABP with the inhibitor F8A.

<p>Fig. A represents frequency distribution of the H atom…acceptor distance, Fig. B depicts the position of inhibitor F8A relative to key residues, Fig. C shows the hydrophobic contacts as a function of the simulation time.</p

The polar interactions between inhibitors and the key residues (kcal/mol).

<p>The polar interactions between inhibitors and the key residues (kcal/mol).</p

Plot of the RMSF of C_α atoms in A-FABP through the equilibrium phase of MD simulation.

<p>Plot of the RMSF of C_α atoms in A-FABP through the equilibrium phase of MD simulation.</p

Binding free energies of wild-type and mutant A-FABP to inhibitors calculated by the SIE method^a.

a<p>All energies are in kcal·mol⁻¹,</p>b<p>ΔEnergy = Energy^complex–Energy^A-FABP–Energy^inhibitor,</p><p>ΔG^exp were derived from the experimental values in Ref (Barf et al. 2009) using the equation ΔG≈–RTlnIC50,</p><p>ΔΔG_bind = ΔG^mutant–ΔG^complex.</p

The root-mean-square deviation (RMSD) of the backbone atoms relative to their crystal structure as a function of time for 8CA (black), F8A(red), I4A(blue) and unbound A-FABP (dark cyan).

<p>The root-mean-square deviation (RMSD) of the backbone atoms relative to their crystal structure as a function of time for 8CA (black), F8A(red), I4A(blue) and unbound A-FABP (dark cyan).</p