The Conformational Transition Pathways and Hidden Intermediates in DFG-Flip Process of c-Met Kinase Revealed by Metadynamics Simulations

Abstract

Protein kinases intrinsically translate their conformations between active and inactive states, which is key to their enzymatic activities. The conformational flipping of the three-residue conservative motif, Asp-Phe-Gly (DFG), is crucial for many kinases’ biological functions. Obtaining a detailed demonstration of the DFG flipping process and its corresponding dynamical and thermodynamical features could broaden our understanding of kinases’ conformation-activity relationship. In this study, we employed metadynamics simulation, a widely used enhanced sampling technique, to analyze the conformational transition pathways of the DFG flipping for the c-Met kinase. The corresponding free energy landscape suggested two distinct transition pathways between the “DFG-in” and “DFG-out” states of the DFG-flip from c-Met. On the basis of the orientation direction of the F1223 residue, we correspondingly named the two pathways the “DFG-up” path, featuring forming a commonly discovered “DFG-up” transition state, and the “DFG-down” path, a unique transition pathway with F1223 rotating along the opposite direction away from the hydrophobic cavity. The free energies along the two pathways were then calculated using the Path Collective Variable (PCV) metadynamics simulation. The simulation results showed that, though having similar free energy barriers, the free energy cuve for the DFG-down path suggested a two-step conformational transition mechanism, while that for the DFG-up path showed the one-step transition feature. The c-Met DFG flipping mechanism and the new intermediate state discovered in this work could provide a deeper understanding of the conformation-activity relationship for c-Met and, possibly, reveal a new conformational state as the drug target for c-Met and other similar kinases.

Design, Synthesis, and Evaluation of New Mesenchymal–Epithelial Transition Factor (c-Met) Kinase Inhibitors with Dual Chiral Centers

“…As the backbone RMSD value of two complexes in Figure 9 , the RMSD value of the system for the ( R , S )-12a complex increased to around 2.0 Å at 4 ns and settled smoothly around 2.2 Å until the end of the simulation, while the RMSD plot of ( R , R )-12a demonstrated the initial complex system fluctuations till 5.5 ns, and then the plot stabilized around 1.7 Å, indicating that the system equilibrated at the end of the simulation. These results indicated that the conformational changes of the docked complexes were within the acceptable limit of 1–3 Å during the 10 ns simulation [ 44 ].…”

Molecular Insights on the Conformational Transitions and Activity Regulation of the c-Met Kinase Induced by Ligand Binding

“…Our previous work found two pathways between the most populated state, “DFG-in,” and the flipped state, “DFG-out,” in apo c-Met based on the well-tempered metadynamics simulations. The first pathway passed the well-known “DFG-up” intermediate, and the other pathway underwent a newly characterized intermediate state, “DFG-down.” The two different pathways between the “DFG-in” and “DFG-out” states of apo c-Met (Figure A) were further demonstrated in this work by the BE-MetaD simulations, an efficient sampling technology of the free energy landscapes.…”

“…Our previous work 36 found two pathways between the most populated state, "DFG-in," and the flipped state, "DFG-out," in apo c-Met based on the well-tempered metadynamics simulations. The first pathway passed the well-known "DFGup" intermediate, and the other pathway underwent a newly characterized intermediate state, "DFG-down."…”

“…Kalodimos et al employed nuclear magnetic resonance (NMR) spectroscopy to study the interconversions between the active and two inactivate intermediates and revealed the contributions of the A-loop and DFG-motif to the activity of the Abl1 kinase . In our previous work, we demonstrated that the DFG-motif converts between the “in” and “out” (inactive) conformation of c-Met. Two pathways were observed between the “DFG-in” and “DFG-out” states.…”

Design, synthesis and biological evaluation of sulfonylamidines as potent c-Met inhibitors by enhancing hydrophobic interaction

“…Among them, type II inhibitors, such as cabozantinib (4), not only bind the ATP binding site occupied by type I inhibitors but also extend over the gatekeeper Ile1145 into the adjacent allosteric hydrophobic back pocket in the inactive "DFG out" conformation. [20][21][22][23] Moreover, the available literature has demonstrated that the newly resulting hydrophobic interactions may have the potential to overcome the acquired resistance to type I inhibitors induced by the mutation of the active site of c-Met. 24,25 Taking into account the above insights, our group recently reported a series of sulfonylamidine-based derivatives as potent c-Met inhibitors, of which compound 7 was found to be an optimal compound in terms of potency and safety.…”