Activation pathway of Src kinase reveals intermediate states as targets for drug design

Abstract

Unregulated activation of Src kinases leads to aberrant signalling, uncontrolled growth and differentiation of cancerous cells. Reaching a complete mechanistic understanding of large-scale conformational transformations underlying the activation of kinases could greatly help in the development of therapeutic drugs for the treatment of these pathologies. In principle, the nature of conformational transition could be modelled in silico via atomistic molecular dynamics simulations, although this is very challenging because of the long activation timescales. Here we employ a computational paradigm that couples transition pathway techniques and Markov state model-based massively distributed simulations for mapping the conformational landscape of c-src tyrosine kinase. The computations provide the thermodynamics and kinetics of kinase activation for the first time, and help identify key structural intermediates. Furthermore, the presence of a novel allosteric site in an intermediate state of c-src that could be potentially used for drug design is predicted.

Figure 1. Conformational changes associated with the c-src activation

The (A) inactive and (B) active crystal structures show structural changes in the activation loop (A-loop; red) and C-helix (orange), switching of the electrostatic network formed between Lys295, Glu310, Arg409 and Tyr416, and alignment of residues L325, M314, F405 and H384 (shown in licorice and surface representation) to form a hydrophobic regulatory spine (R-spine) in active state (D) as compared to the inactive state conformation (C). R-spine forms a continuous hydrophobic region linking the two lobes of the catalytic domain and it is critical for the catalytic activity of the kinase.

Figure 2. Conformational landscape of c-src tyrosine kinase

The conformational landscape generated using RMSD of A-loop residues (404-424) and difference of the distance between Glu310-Arg409 and Lys295-Glu310 residue pairs as the order parameters reveals multiple intermediates along the activation pathway. The free energy values are reported in kcal mol⁻¹. The landscape is obtained by summing over all the micro-states of the 2000 state MSM from i=1 to N using the following equation $W(x,y)=k_B\mathit{\text{Tln}}[{\sum }_i^N{\pi }_i{h}_i(x,y)]$ where π_i is the probability of state i in the MSM, and h_i(x, y) is the normalized histogram of the variables x and y restricted to the MSM state i.

Figure 3. Kinetics of the c-src kinase activation

MSM of the kinase conformational change reveals novel intermediate states along the activation pathway and provide a measure of the activation/deactivation timescales. (A) Variation of key structural metrics as a function of time along the activation trajectory obtained using the MSM. MSM trajectories are calculated using a kinetic Monte Carlo algorithm to generate a trajectory of (τ=5 ns) microstate jumps, and selecting at random (uniformly) a simulation snapshot to report observables at each time step. RMSD of the activation loop is calculates using heavy atoms of residues 404-424. The following atoms were used for the calculations of distances between residues: Lys295(NZ atom in the ${\text{NH}}_3^{+}$ group), Glu310 (CD atom in the COO⁻ group) and Arg409 (CZ atom in the Guanidinium group). Different colors represent the different conformational states of the c-src kinase. Inactive(B), intermediate states I₁ (C), I₂(D), and active(E) states are shown in magenta, green, black and blue respectively with active state also marked with an asterisk. (F) These four conformational states could be further subdivided into states with different conformations of DFG-motif and R-spine.

Figure 4. Mechanism of ANS induced stabilization of intemediate I₂

(left) ANS binding to the allosteric site adjacent to C-helix in c-src kinase stabilizes the intermediate conformation by blocking the interactions between Lys295 and Glu310. The hydrogen bond formation between Lys295 and Glu310 is required for the locking of the C-helix in the active conformation. The sulfonate group in the ANS forms a hydrogen-bond with the Lys295 thereby locking it in its inactive conformation. (right) ANS binding also pushes the C-helix away from the ATP binding pocket. Superimposition of the crystal structures of the inactive (cyan) and active (green) states of ATP-bound c-src kinase with the ANS-bound src-kinase (orange) reveals the distinct conformation of the c-helix in presence of ANS.

Figure 5. Mutual Information between residues in c-src kinase

Hierarchical clustering of significant mutual information values identifies four regions (shown as squares) within the catalytic domain which have significant dynamical correlation between them. The most strongly coupled cluster involves the residues in the A-loop region which cooperatively unfold during the activation process. Colors indicate the log of the mutual information value.