Kinetics of Nucleotide-Dependent Structural Transitions in the Kinesin-1 Hydrolysis Cycle

Abstract

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. The high spatiotemporal resolution of this method enabled real-time recording of structural changes in the protein as it walked at~100 steps per second. 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 the structural transitions to and from this one-headbound intermediate 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, meaning that there is one rate-limited step in each the one-and two-heads bound states. 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, and show that the mechanism underlying stepping is a two-step process. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.

Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity

“…In kinesin-5, which has a unique L5 latch 45 , the coupling between the switch and neck linker domains is diminished and the structural transition are not as sharp as kinesin-1 17,46 . These differences may lead to the slower observed MT-stimulated ADP release in Eg5 compared to kinesin-1 13,14,39 . One possible inhibition mechanism is that in apo-Eg5, binding of rigor inhibitors to the α4/α6 interface may allosterically close the nucleotide cleft, thereby blocking nucleotide exchange 33,35 .…”

“…Engineered Eg5 dimers have a large stall force, and display minimal processivity 11 , consistent with Eg5 working in teams during spindle formation 2,12 . Additionally, Eg5 walks with a 10-fold slower velocity than kinesin-1 11,13,14 , and it is able to resist large mechanical loads (~10 pN) in either the plus- or minus-end directions 15–17 , which contrasts with the directional dependence of kinesin-1 18 . These properties likely result, at least in part, from the motor spending most of its ATP hydrolysis cycle in a state in which both heads are bound to the microtubule (two-head-bound state), a property not shared by kinesin-1 11,14 .…”

Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots

“…33 Kinesin walks by alternating stepping of its two motor domain heads in a hand-over-hand (HoH) motility. 2 The stepping movement of a single head of a kinesin dimer has been studied extensively by labeling with SA-coated QDs 4 or gold nanoparticles, 34 although these large (>20 nm diameter) probes may interfere with kinesin motility. We reasoned that our compact BG-coated QDs enable higher labeling efficiency with minimal perturbation to kinesin motility.…”

“…The step analysis revealed that a kinesin head takes 16 nm steps at limited ATP concentration, in agreement with previous studies and demonstrating that labeling with these QDs does not measurably alter kinesin motions. 2, 34 …”

Family-specific Kinesin Structures Reveal Neck-linker Length Based on Initiation of the Coiled-coil

“…Subsequently, ADP is released from this new leading head resulting in both heads bound to the microtubule (Fig. 1, E4 and E5) (27).…”