Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events: phosphoryl transfer (>105-fold) and lid opening (103-fold). In contrast, mutation of an essential active site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.
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We dedicate this manuscript to Tom Alber, a truly amazing and inspiring scientist, and a close friend who will live in our hearts forever; his creativity, joy and generosity have deeply influenced several generations of scientists. We are grateful to the staff at the Advanced Light Source–Berkeley Center for Structural Biology and the Advanced Photon Source (APS) for support, Y. Xiong, T. Lang, P. Afonine, R. Read and mentors from the Collaborative Computational Project No. 4 School at APS (2011) for advice in the refinement of X-ray data, members of the C. Miller laboratory for handling crystals, the staff at the National Energy Research Scientific Computing Center, and K.A. Johnson for assistance with the KinTek Explorer software and fitting kinetic data. We thank P. Varilly (University of Cambridge) for kindly providing scripts. This work was supported by the Howard Hughes Medical Institute (HHMI), the Office of Basic Energy Sciences, Catalysis Science Program, US Department of Energy (award DE-FG02-05ER15699), the US National Institutes of Health (RO1-GM100966) and the Teragrid (XSEDE) allocation TG-MCB090166 (D.K.). R.O. is supported as an HHMI Fellow of the Damon Runyon Cancer Research Foundation (DRG-2114-12).
The authors declare no competing financial interests.
Integrated supplementary information
a,b. Stereo view of Fig. 1e showing detailed active-site interactions for the Mg2+–ADP–ADP (blue) and Mg2+–ADP–AMP–AlF4– structures (green). c. Superimposition of X-ray structures (PDB 3SR0 and 2RGX), and a representative snapshot from the MD simulation. The positions of the divalent cation, water molecules, and the nucleotide oxygen atoms in the simulation are in full agreement with the corresponding atoms in the crystal structures. The blue wireframe surface represents the isosurface of value 0.99 for the fractional occupancy of the water oxygen atoms during a typical MD simulation. d. Octahedral coordination of the Mg2+ ion in the active site monitored during a 200 ns MD simulation of AAdk bound to Mg2+–ADP–ADP. The coordination partners are depicted on the structure and the corresponding time-dependent Mg2+–O distances during the MD simulation are shown.1. Henzler-Wildman, K.A. et al. Intrinsic motions along an enzymatic reaction trajectory. Nature 450, 838-44 (2007)
Supplementary Figure 2 Mg2+ prevents water molecules trapped in the active site from exchanging with bulk water.
(a–e) Behavior of all water molecules in the active-site cavity for 100 ns MD simulations of AAdk bound to ADP–ADP with and without Mg2+. a. The fraction of the water molecules present in the active site at the beginning of the simulation, that are still inside the cavity at time t is plotted for four simulations in the presence (dark blue to green) and absence (red to yellow) of Mg2+. The cavity considered for the analysis is displayed in the structures in panels b–e. The water oxygen atoms initially present in the cavity are displayed as large red spheres and the four water molecules coordinating Mg2+ are shown in red and white sticks. In the simulations with Mg2+ (b and c), all water molecules stay in the cavity (see also Movie 1). In the simulations without Mg2+ (d and e), the water molecules in the pocket progressively diffuse out, exchanging with bulk water and are substituted by other water molecules (depicted as cyan spheres). The average number of water molecules in the cavity is constant in all simulations.
Supplementary Figure 3 Comparison of AAdk and EAdk sequences and pre-steady-state kinetics with and without Mg2+, and determination of the on-enzyme equilibrium for EAdk.
a. Sequence alignment of adenylate kinase from Aquifex aeolicus (AAdk) and Escherichia coli (EAdk). Identical residues are colored in red and similar residues in purple. To avoid confusion, the residue numbering used in the present manuscript refers always to the AAdk sequence. For example, when we indicate R150 (marked with * in the sequence), we are in fact referring to R150 for AAdk and R156 for EAdk. b. Quench-flow experiments performed in the forward direction with 100 μM AAdk, 4 mM ATP and AMP, and 8 mM Mg2+. c. Quench-flow experiments performed in the forward direction with 25 μM AAdk, 4 mM ATP and AMP, and 50 mM EDTA to remove Mg2+ contamination. Data shows that the rate-limiting step of AAdk turnover, like EAdk (Fig. 3), is lid-opening in the presence of Mg2+ (b) and phosphoryl transfer in the absence of Mg2+ (c). This highlights the mechanistic similarity between the two proteins, which is further bolstered by the nearly identical active site in crystal structures of AAdk (PDB 2RGX) and EAdk (PDB 1AKE) in complex with the bi-substrate inhibitor Ap5A. d. EDTA titration of EAdk + 4 mM ADP complex monitored by a change in kcat for the reverse reaction. Due to the fact that 50 mM EDTA was needed to remove trace elements of divalent cations, all “no divalent cation” kinetics experiments were performed with 50 mM EDTA to ensure that the measured rates were not influenced by minor divalent cation contamination. e. Quench-flow experiments performed at different EAdk concentrations in the forward (25 and 100 μM) and reverse (485 μM) directions with 4 mM ATP and AMP or 4 mM ADP. 50 mM EDTA was present in all experiments to remove residual Mg2+. Data show the same behavior as at low EAdk concentration (Fig. 3), but fitting was less reliable because the back reaction had to be accounted for, due to the increased amount of generated product. f,g. Determination of the on-enzyme equilibrium in the presence of Mg2+ (f, 600 μM of MgADP) and absence of Mg2+ at (g, 600 μM ADP + 50 mM EDTA). The [ADP]/2*[ATP] ratio was measured as a function of increasing concentration of EAdk. At low enzyme concentration most nucleotides are free, and the ADP to ATP ratio mainly reflects equilibrium in solution. When the plateau is reached, all nucleotides are bound and the corresponding [ADP]/2*[ATP] ratio reflects the on-enzyme equilibrium. In the presence of Mg2+, fitting it to a generalized hyperbola (y = a - b/(1 + c*x)^(1/d)) yielded an equilibrium value of 11.6 ± 0.7, which is in good agreement with the number obtained from kinetic experiments (see Fig. 3a). In the absence of Mg2+ the equilibrium was shifted even further, yielding a value of 29 ± 1.1. Henzler-Wildman, K.A. et al. Intrinsic motions along an enzymatic reaction trajectory. Nature 450, 838-44 (2007).2. Muller, C.W. & Schulz, G.E. Structure of the Complex between Adenylate Kinase from Escherichia-Coli and the Inhibitor Ap5a Refined at 1.9 a Resolution - a Model for a Catalytic Transition-State. Journal of Molecular Biology 224, 159-177 (1992)
a. The overlay of [1H-15N]-TROSY-HSQC spectra of EAdk with saturating concentrations of Mg2+ (blue) or Ca2+ (red) and saturating concentrations of nucleotides shows only minor chemical shifts changes indicating that the bound structures are very similar. The NMR experiments were collected with 2 mM EAdk, 20 mM ADP, and either 20 mM Mg2+ or 20 mM Ca2+. b,c. Pre-steady-state kinetics of EAdk measured by quench-flow at 25 °C in the forward (green) and reverse direction (blue) with different divalent ions to obtain the rate constants in Table 2. b. 25 μM EAdk, 4 mM ATP and AMP, and 8 mM CaCl2 in the forward reaction, and 100 μM EAdk and 4 mM ADP and CaCl2 in the reverse direction (blue). c. 100 μM EAdk, 4 mM ATP and AMP, and 8 mM CoCl2 in the forward direction and 100 μM EAdk and 4 mM ADP and CoCl2 in the reverse direction. For Ca2+ and Co2+, P-transfer is rate limiting in the reverse reaction, whereas lid-opening is rate limiting in the forward reaction (burst represents P-transfer).
a. Quench-flow experiments performed with 100 μM EAdk–R150K and 4 mM MgAMP + 4 mM MgATP in the forward direction and 100 μM EAdk–R150K and 4 mM MgADP in the reverse direction. b. 100 μM EAdK–R150K, 4 mM ATP + 4 mM AMP, and 50 mM EDTA in the forward direction and 100 μM EAdK-R150K, 4 mM ADP, and 50 mM EDTA in the reverse direction. For R150K, P-transfer is rate limiting in both directions with and without Mg2+.
Supplementary Figures 1–5, Supplementary Tables 1 and 2, and Supplementary Notes 1–4 (PDF 5442 kb)
Even in the presence of Mg2+, the water molecules trapped inside the active site are highly dynamic and repeatedly swap their positions, although they cannot escape the active site pocket and exchange with the bulk. This can be easily visualized if the oxygen atom of each water molecule is labeled with a different color so that one can follow their individual dance inside the active site pocket during a 100ns MD trajectory. (MP4 5458 kb)
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Kerns, S., Agafonov, R., Cho, Y. et al. The energy landscape of adenylate kinase during catalysis. Nat Struct Mol Biol 22, 124–131 (2015) doi:10.1038/nsmb.2941
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