Real-time Structural Transitions Are Coupled to Chemical Steps in ATP Hydrolysis by Eg5 Kinesin

Bokkyoo Jun,Sunyoung Kim

doi:10.1074/jbc.c110.103762

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.

Highlights

Coupling between chemical events and conformational transitions is crucial for the catalytic function of proteins
Notable examples include molecular motors, signaling proteins, and membrane transporters, in which large scale conformational transitions are integral to the chemical steps of ATP hydrolysis to yield the products, ADP and inorganic phosphate, or to phosphorylation in which Pi is covalently attached to a protein and ADP is a leaving group
Open questions in kinesin catalysis include the chemical nature of the catalytic base, timing of proton abstraction from the nucleophilic water in relation with major conformational changes, the nature of the transition state, and understanding of which steps within ATP hydrolysis drive major structural transitions

Summary

EXPERIMENTAL PROCEDURES

The motor domain of wild-type Eg5 monomer, composed of residues 1–367, was expressed in Escherichia coli BL21 CodonPlus(DE3)-RIL-competent cells (Stratagene) and purified as described [2]. The numbers of spectra co-added for the samples were 40 and 180 for caged ATP and Eg5-. Positive (blue) or negative (red) lines in the difference data reflect either acquisition/loss of or chemical changes in functional groups upon reaction. Integration of amide I absorbance allows for a simple estimate of structural changes in the polypeptide backbone during a protein reaction; the FTIR measurements were acquired with previously de- integrated total protein absorbance is the averaged value between scribed methods [2]. Parameters for the spectral acquisition on an IFS66v/S spectrometer (Bruker Optics, Billerica MA) with a water-cooled IR source and liquid nitrogen-cooled HgCdTe measured (via use of experimental measurement of ATP release per laser flash in our samples [2])

RESULTS

DISCUSSION

Methods