Real-time structural transitions are coupled to chemical steps in ATP hydrolysis by Eg5 kinesin.
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.
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Bokkyoo
B
Jun
Sunyoung
S
Kim
D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000502:physiology; D010710:Phosphates / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014466:Ultraviolet Rays
C470243:KIF11 protein, human; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins