Protein Flexibility and Dissociation Pathway Differentiation Can Explain Onset of Resistance Mutations in Kinases

Abstract

Understanding how mutations render a drug ineffective is a problem of immense relevance. Often the mechanism through which mutations cause drug resistance can be explained purely through thermodynamics. However, the more perplexing situation is when two proteins have the same drug binding affinities but different residence times. In this work, we demonstrate how all-atom molecular dynamics simulations using recent developments grounded in statistical mechanics can provide a detailed mechanistic rationale for such variances. We discover dissociation mechanisms for the anti-cancer drug Imatinib (Gleevec) against wild-type and the N368S mutant of Abl kinase. We show how this point mutation triggers far-reaching changes in the protein's flexibility and leads to a different, much faster, drug dissociation pathway. We believe that this work marks an efficient and scalable approach to obtain mechanistic insight into resistance mutations in biomolecular receptors that are hard to explain using a structural perspective.

Keywords: Kinase; Kinetics; Molecular Dynamics; Protein Flexibility; Resistance Mutations.

Figure 1:

Crystallographic binding mode: Imatinib (gray) bound to the catalytic domain of Abl kinase (PDB id 1OPJ). Kinase domain is divided into N-terminal lobe (N-lobe) and C-terminal lobe (C-lobe), with the inhibitor (Imatinib) binding site located between the lobes. Abl-kinase has conserved structural features like A-loop (pink ribbon), P-loop (blue ribbon), αC helix (yellow ribbon), hinge (purple ribbon), and DFG motif (yellow sticks). D381 in the DFG motif, and the salt bridge involving E286 in the αC helix, and K271 cover and block the binding tunnel in front. The site of mutation (N368) lies behind the DFG motif and is shown in a gray stick representation.

Figure 2:

A) and B) show order parameters OPs obtained through AMINO for WT and mutant respectively. C) and D) show the respective RC constructed from these OPs through SGOOP. E) shows the residence times (red bar) obtained by biasing the respective RC for WT and mutant system compared against experimental measurements (blue bars). The red bars represent the fitted residence time while the standard deviation is shown as a black error bar. See main text for further details of the OPs.

Figure 3:

Mutational effect on the substrate release pathway (red arrows in the top panel): A) WT (top left) and mutant (top right) panels. Ligand dissociation trajectory is depicted by ligands in licorice representation sampled every 100 ps. The overall direction of the substrate release is depicted by transparent pink arrows. Bar plot and the error bar representing the mean and standard deviation of the closest distance between the center of mass of Imatinib and B) hinge and C) αC helix for WT (blue bar) and mutant (orange bar) trajectories.

Figure 4:

A) Left: Starting state of WT Abl (Crystal structure). H-bond between Y253 (P-loop) and N322 (hinge) is shown as a blue dashed line. Right: Abl conformation transiting to the pre-release state involves disrupted H-bond between Y253 and N322 forming an open ligand release pathway depicted by a black arrow. B) Left: H-bond between D381 from the DFG motif and Imatinib (gray sticks) in N368S Abl is shown as a thick red line. Right: Conformational changes in N368S Abl as it transits to the pre-released state involve the disruption of the interaction network of E286, K271, Imatinib, and DFG motif. C) and D) respectively show the closest distance (with error bars) between C) Cα atoms of Y253 and N322 for WT Abl (blue) and N368S Abl (orange); and D) Cα atoms of K271 and D381 for WT Abl (blue) and N368S Abl (orange).

Figure 5:

Flexibility of DFG motif is modulated by H-bond interaction of N368 and the DFG loop. A) Crystal structure pose of Imatinib (gray licorice representation) in WT (left panel) and N368S (right panel). DFG motif is represented by yellow licorice. H-bond between N368 (gray licorice) and A380 is shown as blue dashed line. Bar plot and the error bar representing the mean and standard deviation of B) N368 and DFG motif for WT Abl (blue bar) and N368S Abl (orange bar) C) Root mean square deviation (RMSD) of DFG motif from the crystal structure position for WT Abl (blue bar) and N368S Abl (orange bar).