Structural-Kinetics of the Switch-1 Loop and Neck-Linker Elements Explains the Distinct Molecular Physiologies of Kinesin-1 and Kinesin-5

Abstract

Microtubule motors control a diverse collection of physiologies, including cell division, organelle traffic, and microtubule dynamics. Kinesins use ATP hydrolysis to power a chemical cycle that performs mechanical work. Recent, high-resolution crystallographic and cryo-EM reconstructions, single molecule mechanics, and solution kinetics studies, led to a general mechanochemical scheme for kinesin motors (Clancy, Nature Str. Mol. Biol., 18, 1020-7, (2011)). Nucleotide binding engages key structural elements, including switch I, switch II, and the P loop. Yet, how conformational changes in these switch elements lead in turn to corresponding changes in the microtubule binding domains and the motor mechanical elements (the neck linker and cover strand) remains enigmatic. Likewise, it remains unclear how the thermodynamics and kinetics of these structural transitions differ between two kinesins with very different physiologic roles. We tested two predictions of the model proposed by Clancy et al: the neck-linker gates nucleotide binding; and, coordination between the neck-linker and switch-1 fine tunes the enzymology of specific kinesin motors. We have used a recently developed technique_transient time-resolved fluorescence resonance energy transfer_which enables us to detect transitions between multiple structural states, measured with time-resolved FRET during a biochemical transient. We engineered probe-pairs to report the structure, kinetics, and equilibrium constants for nucleotide-driven structural transitions in the neck-linker and switch-1. We then compared these transitions in kinesin-1 to those in Eg5 (kinesin-5). Our results show that: 1. neck-linker docking gates nucleotide binding, as predicted by Clancy et al., and in both classes of kinesins, 2. The state of the neck-linker controls the conformation of switch-1, and 3. differences in the equilibrium constants for neck-linker docking during the microtubule bound, ATP stimulated working-stroke, explain the unique force dependence of Kinesin 1 and Eg5.