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. 2017 Apr;13(4):402-408.
doi: 10.1038/nchembio.2296. Epub 2017 Feb 6.

A water-mediated allosteric network governs activation of Aurora kinase A

Affiliations

A water-mediated allosteric network governs activation of Aurora kinase A

Soreen Cyphers et al. Nat Chem Biol. 2017 Apr.

Abstract

The catalytic activity of many protein kinases is controlled by conformational changes of a conserved Asp-Phe-Gly (DFG) motif. We used an infrared probe to track the DFG motif of the mitotic kinase Aurora A (AurA) and found that allosteric activation by the spindle-associated protein Tpx2 involves an equilibrium shift toward the active DFG-in state. Förster resonance energy transfer experiments show that the activation loop undergoes a nanometer-scale movement that is tightly coupled to the DFG equilibrium. Tpx2 further activates AurA by stabilizing a water-mediated allosteric network that links the C-helix to the active site through an unusual polar residue in the regulatory spine. The polar spine residue and water network of AurA are essential for phosphorylation-driven activation, but an alternative form of the water network found in related kinases can support Tpx2-driven activation, suggesting that variations in the water-mediated hydrogen bond network mediate regulatory diversification in protein kinases.

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Figures

Figure 1
Figure 1. Tpx2 induces a population shift towards the DFG-in state
(a) Crystal structures of protein kinases, aligned on the DFG motif, showing conserved water molecules (see Online Methods for pdb codes). The Q185 residue of AurA is shown. (b) Labeling scheme used to incorporate an infrared (IR) probe into AurA. (c) IR spectra of Q185CN in the apo form (black line and gray shading) in the presence of ADP (dashed pink line), Tpx2 (dashed blue line), and both ligands (solid blue line). Single representative spectra, normalized to the peak maxima. (d) Each panel shows the results of fitting IR spectra measured for an individual biochemical state at 25 °C. Thick lines are the experimental data colored as in panel c, thin dashed lines are numerical fits, and shaded peaks are individual fit components. Populations are derived from integrating the central gray peak (DFG-Out) and the outlying blue peaks (DFG-In). Single representative spectra and fitting results are shown. (e) The outlying peaks from the AurA spectra are compared with IR spectra of ethyl thiocyanate dissolved in aprotic solvents of varying polarity (DMSO, dimethyl sulfoxide, DMF, dimethylformamide, DCM, dichloromethane). (f) Models for the probe-water hydrogen bonds giving rise to the red-shifted (left) and blue-shifted (right) peaks in the Q185CN spectra. (g) Second derivatives of IR spectra of Q185CN bound to Tpx2 measured at temperatures from 5 to 40 °C. Arrows indicate features in the 2nd derivatives corresponding to peaks in the absorbance spectra. Their temperature-dependent amplitudes are shown in the inset.
Figure 2
Figure 2. Tpx2 promotes a nanometer-scale shift of the activation loop
(a) Left: schematics of AurA labeled with donor and acceptor dyes, showing the activation loop and the DFG aspartate for DFG-Out (gray) and DFG-In (blue) states. Right: emission spectra of donor-only (D-only) and donor + acceptor (D+A) samples in the presence of different concentrations of Tpx2. (b) Left panels: normalized donor peak intensities (donor/acceptor) are shown for titrations of Tpx2 and ADP performed with and without the other ligand. Right panels: binding constants for ADP and Tpx2, determined from fitting the data in the left panels. Mean values ± s.d.; n=3. (c) Ensemble-averaged distances between the donor and acceptor dyes measured by FRET are shown for Tpx2 titrations performed with and without ADP (see Online Methods). A single representative experiment is shown, except for the titration end points for which values represent the mean; n=2. (d) Structural model for the conformational change detected by FRET, based on x-ray structures of AurA bound to ADP and Tpx2 (blue, pdb code 1OL5) and to an inhibitory nanobody (gray, pdb code 5L8K). (e) Van ‘t Hoff plots of the DFG equilibrium for each biochemical state. Each data point is derived from the ratios of the DFG-In and DFG-Out populations given by the numerical fits of a single absorbance spectrum. (f) FRET-based distances for each biochemical state are plotted as a function of inverse temperature. Data represent mean values ± s.d.; n=3.
Figure 3
Figure 3. The Q185 spine residue participates in a water-mediated allosteric network
(a) Representative snapshot from one of the molecular dynamics simulations of WT AurA bound to ADP and Tpx2. Water molecules are shown as sticks, and their hydrogen bonding interactions with surrounding residues as dashed lines. (b) Autocorrelation functions calculated for W1 and W2 water site occupancies across all MD simulations of WT AurA. Black lines are fits to the exponential tails quantifying long-lived subpopulations, with populations and residence times for the long-lived populations shown in the legend with 95% confidence intervals (subscripts and superscripts). (c) Long-lived water lifetimes (top) and populations (bottom) for the W1 and W2 water sites in simulations of WT AurA and the Q185M and Q185L mutants, in the presence of Tpx2. Vertical black lines denote 95% confidence intervals. (d) Left panel: Cα RMSD distributions for the C-helix from simulations of WT AurA (colored lines and shading) and Q185L (black lines) with and without Tpx2. Right panels: for the WT simulations performed in the presence of Tpx2, the probability distributions were recalculated for simulation frames with or without long-lived waters (>20 ns). The results are shown for W1 (top panel) and W2 (bottom panel) sites.
Figure 4
Figure 4. The Q185 residue is essential for allosteric activation of AurA
(a) Kinase assays of unphosphorylated Cys-lite AurA and the corresponding Q185M and Q185L mutants measured at 30 nM enzyme concentration with and without Tpx2. (b) Kinase assays of WT phosphorylated AurA and phosphorylated Q185 mutants measured at 2 nM enzyme concentration in the absence of Tpx2. (c) WT phosphorylated AurA and phosphorylated Q185 mutants measured at 2 nM enzyme concentration in the same experiment shown in panel b but in the presence of Tpx2. The activities in the absence of Tpx2 are shown for comparison with fold activation by Tpx2 indicated by arrows. a-c show mean values ± s.d.; n=3. (d) Top panel: Kinase activity of unphosphorylated Cys-lite Q185M, Q185L and Q185CN AurA measured at 1 µM enzyme concentration. The values are the mean± s.d. of the maximal activity derived from fitting three independent Tpx2 titrations to a single binding site model. Bottom panel: Normalized kinase activity of the mutants plotted as a function of Tpx2 concentration. Mean normalized values ± s.d.; n=3. (e) Kinase activity assays of the Cys-lite and Q185CN forms of AurA prepared in phosphorylated form measured at 30 nM enzyme concentration in the presence and absence of Tpx2. Fold activation by Tpx2 is indicated by arrows. Mean values ± s.d.; n=3.
Figure 5
Figure 5. A PKA-like water network supports activation of AurA by Tpx2, but not by phosphorylation
(a) Comparison of the active sites of AurA and PKA (pdb code 1L3R). The geometries of the solvent networks are highlighted by shaded lines, and polar residues that participate in the network are shown. The AurA model is from the molecular dynamics snapshot shown in Figure 3a. (b) Kinase assay showing the activity of the unphosphorylated Q185L L194V A273T triple mutant compared to the other Q185L mutants and the Cys-lite construct. (c) Kinase assays of the phosphorylated triple mutant and the phosphorylated Cys-lite kinase. The left panel shows the activities in the absence of Tpx2, the right panel shows the activities in the presence of Tpx2, with the increase due to Tpx2 indicated by the arrows. b-c show mean values ± s.d.; n=3. (d) The structure of phosphorylated AurA bound to ADP and Tpx2 (pdb code 1OL5) with Q185 and the water network highlighted (DFG motif sidechains not shown). The regulatory spine is in gray, and Tpx2 is magenta. (e) The structure of phosphorylated PKA (pdb code 1L3R) with the equivalent structural elements highlighted as in panel d. (f) Structural comparison of the active sites of the AGC kinases PKA and the MRCKβ (myotonic dystrophy-related Cdc42-binding kinase)(pdb codes 1L3R and 4UAK). Residues participating in the water-mediated hydrogen bond networks are shaded and numbered using the PKA numbering. The inset shows the frequencies of particular amino acid combinations at these two positions in AGC kinases.

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