Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.