Real-time structural transitions are coupled to chemical steps in ATP hydrolysis by Eg5 kinesin

Abstract

At the biochemical level, motor proteins are enzymatic molecules that function by converting chemical energy into mechanical motion. The key element for energy transduction and a major unresolved question common for all motor proteins is the coordination between the chemical and conformational steps in ATP hydrolysis. Here we show time-lapse monitoring of an in vitro ATP hydrolysis reaction by the motor domain of a human Kinesin-5 protein (Eg5) using difference Fourier transform infrared spectroscopy and UV photolysis of caged ATP. In this first continuous observation of a biological reaction coordinate from substrate to product, direct spectral markers for two catalytic events are measured: proton abstraction from nucleophilic water by the catalytic base and formation of the inorganic phosphate leaving group. Simultaneous examination of conformational switching in Eg5, in parallel with catalytic steps, shows structural transitions in solution consistent with published crystal structures of the prehydrolytic and ADP-bound states. In addition, we detect structural modifications in the Eg5 motor domain during bond cleavage between the beta- and gamma-phosphates of ATP. This conclusion challenges mechanochemical models for motor proteins that utilize only two stages of the catalytic cycle to drive force and motion.

FIGURE 1.

Time-resolved IR spectroscopy of Eg5 catalysis in solution. a, schematic diagram of the Eg5 ATPase cycle. Free ATP (1) from caged ATP photolysis is bound to Eg5 (2), which undergoes conformational isomerization (3). The biochemical reaction proceeds through the transition state to form products (4). Sequential release of inorganic phosphate (5) and ADP (6) completes the catalytic cycle. b, Difference techniques applied to vibrational spectroscopy. When the sample is triggered to initiate catalysis, infrared modes in the protein can differ in frequency relative to the resting state. Positive (blue) or negative (red) lines in the difference data reflect either acquisition/loss of or chemical changes in functional groups upon reaction. c, three-dimensional representations of difference photolysis spectra for (i) wild-type Eg5 in the presence of caged ATP (Eg5 + cATP; n = 180), (ii) caged ATP alone (n = 40), and (iii) buffer (n = 38). The t = 0 s designates the laser flash applied to the sample. The scale bar represents 1 × 10⁻⁴ absorbance units. d and e, the amide I region (d) and the phosphate regions (e) of the time-resolved hydrolytic spectra. Alterations in absorbance are visualized by color, in which blue is the greatest amplitude and red is the lowest negative amplitude. The bar represents 0.5 × 10⁻⁴ absorbance units. All spectral data have 12-cm⁻¹ resolution.

FIGURE 2.

Comparison of Eg5·ADP and Eg5·AMPPNP crystal structures. In the right inset is the ribbon representation of the Eg5·AMPPNP structure (PDB ID 3HQD (16)), with α3 and switch I highlighted in red and α4 and switch II in purple. Enlargements of these Eg5 regions provide overlays with the Eg5·ADP structure (PDB ID 1II6 (32)) in green. The structures were superpositioned using the P-loop residues.