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. 2017 Nov 15;7(1):15604.
doi: 10.1038/s41598-017-10697-0.

Millisecond Dynamics of BTK Reveal Kinome-Wide Conformational Plasticity Within the Apo Kinase Domain

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Free PMC article

Millisecond Dynamics of BTK Reveal Kinome-Wide Conformational Plasticity Within the Apo Kinase Domain

Mohammad M Sultan et al. Sci Rep. .
Free PMC article

Abstract

Bruton tyrosine kinase (BTK) is a key enzyme in B-cell development whose improper regulation causes severe immunodeficiency diseases. Design of selective BTK therapeutics would benefit from improved, in-silico structural modeling of the kinase's solution ensemble. However, this remains challenging due to the immense computational cost of sampling events on biological timescales. In this work, we combine multi-millisecond molecular dynamics (MD) simulations with Markov state models (MSMs) to report on the thermodynamics, kinetics, and accessible states of BTK's kinase domain. Our conformational landscape links the active state to several inactive states, connected via a structurally diverse intermediate. Our calculations predict a kinome-wide conformational plasticity, and indicate the presence of several new potentially druggable BTK states. We further find that the population of these states and the kinetics of their inter-conversion are modulated by protonation of an aspartate residue, establishing the power of MD & MSMs in predicting effects of chemical perturbations.

Conflict of interest statement

M.M.S., R.A.D., R.U., and F.L. declare no competing financial interests. VSP is a consultant and SAB member of Schrodinger, LLC and Globavir, sits on the Board of Directors of Omada Health, and is a General Partner at Andreessen Horowitz.

Figures

Figure 1
Figure 1
BTK exists in several thermodynamically stable states. Within the MD ensemble, BTK catalytic domain (a) samples several states, including active (DFGin/C-helixin) (b), Src-like (A-loop folded/DFGin/C-helixout) (c) and DFGout (d). The transition from active (b) to Src like (c) is defined by the outward rotation of the C-helix (orange) and folding of the A- loop (red). The C-helix rotation breaks a critical salt bridge between Glu445-Lys430 and forms salt bridges between Glu439-Arg468 (yellow) and Glu445-Arg544(orange). In the DFGout state, Phe540(purple), part of the DFG motif (blue) rotates away from the core of the protein towards the ATP binding site. The R-spine (purple surface) forms continous hydorphobic contacts in the active state but is broken in the other states.
Figure 2
Figure 2
BTK’s apo domain contains kinome-wide conformational plasticity. Comparison of 9% of MD generated structures (a) against publically available kinase domain structures (b) projected along three key degrees of freedom as outlined in Möbitz et al.. We used the data and classification scheme provided in ref. to generate (b). The top y-coordinate tracks the C-helixin to C-helixout transition while the bottom y-coordinate tracks the DFGin to DFGout transition. The common x-axis subdivides the conformations into pharmacologically relevant states of the DFG motif. The white circles in (a) correspond to the starting configurations for the MD simulations. The points are colored according to their Möbitz classification and detailed in Supplementary Fig. 16. For BTK’s free energies along these coordinates, see Supplementary Fig. 9.
Figure 3
Figure 3
MSMs predict a multistate ensemble whose populations are modulated via DFG protonation. Thermodynamics of the BTK-ASP and BTK-ASH ensembles projected along the two dominant tICs (a) show a stable Src-like state. For standard errors along each coordinate, see Supplementary Fig. 12. Simple four state cartoon model (b) of the kinase dynamics. Kinetics of several molecular switches as a function of time along a MSM trajectory for BTK-ASP (c). The MSM trajectory was generated using a Monte Carlo algorithm to simulate a trajectory of 800 μs from the Markovian transition matrix. At each step, we randomly selected a simulation structure assigned to that state to report the instantaneous observables. The root mean squared deviation (RMSD) of the A-loop is calculated using the heavy atoms of residue Asp539-Phe559. For A-loop RMSD to the extended state, see Supplementary Fig. 17. We used the delta carbon of the Glu439 and zeta carbon of Arg468, and the delta carbon of Glu445 and zeta nitrogen of catalytic Lys430 to calculate the distances in the next two panels to quantify C-helix in to out transition. We used Thr410-Val415, and Phe540, Met449, His519, and Leu460 heavy atoms to quantify the P-loop, and R-spine RMSD. The R-spine is only completely formed when the C-helix (orange trace) is rotated inwards. The DFG RMSD is calculated using heavy atoms from Asp539-Gly541. For all RMSD calculations, we used a double helical inactive state as the reference state. The lighter color traces give the instantaneous value for the observable and the dark traces provide moving averages across 10 frames. The color corresponds to the color scheme used in Fig. 1 to highlight structural motifs in BTK.
Figure 4
Figure 4
BTK’s deactivation proceeds via an intermediate state. Starting from the active state (a), the C-helix swings out to form an intermediate (b) characterized by a disordered A-loop and a stable Arg468-Glu439 salt bridge. The activation loop then folds into a Src-like double helical inactive state (c). The double helical state is stabilized by a secondary salt bridge between the catalytic Glu445 and Arg544. The P-loop has been omitted in all three panels for the sake of clarity. The heat map (d) shows the projection of the centroids of these states unto our free energy landscape. Panel (d) has been reproduced from Fig. 3 for clarity.
Figure 5
Figure 5
BTK DFG flips via the C-lobe, and proceeds after the formation of a helical intermediate state (a). Snapshots (b) going from red to white to blue, from the DFGout to DFGin trajectory showing the transient outward rotation of Met449 and Phe517 for the DFG flip. The DFGout to DFGin cross-over (c, panel 1) is preceded by the folding of residues Ser543-Leu547 (c, panel 2) and transient outward rotation of both Met449 and Phe517 (c, panel 3). Projection of the 3 selected frames from (b) onto the top two tICs (d) gives us the approximate free energies of the DFGout, intermediate and DFGin states.

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