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. 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 onehead-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 | iSCAT | microscopy | structural kinetics | structure-function K inesin-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 handover-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)(15)(16)(17)(18)(19)(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 sin...

Interferometric Scattering Microscopy for the Study of Molecular Motors

“…Prepare a solution of 1.6 mg/mL free tubulin, 4 m M MgCl 2 , 1m M GTP, and 1:25 DMSO in BRB80 buffer, and incubate at 37°C for 30 min.Stabilize by adding taxol to 10 µ M .Dilute to working concentration (~1:80) in BRB80 plus 10 µ M taxol and 0.5 mg/mL casein. Recent work has shown that a rigor form of kinesin-1 (full-length Drosophila KHCR210A) is highly effective in microtubule immobilization (Mickolajczyk et al, 2015). The tail domain of full-length kinesin binds strongly to casein-treated surfaces, while the head domains bind tightly to microtubules.…”

“…Attachment of nanoparticle labels to kinesin, myosin, and dynein motors has to date been achieved via biotin–streptavidin chemistry (Andrecka et al, 2015; Dunn & Spudich, 2007; Isojima, Iino, Niitani, Noji, & Tomishige, 2016; Mickolajczyk et al, 2015; Schneider, Glaser, Berndt, & Diez, 2013; Schneider, Korten, Walter, & Diez, 2015). One method of introducing biotin into kinesin is to create a cysteine-lite mutant, in which solvent-exposed cysteines are mutated out of the motor domain and a single cysteine is kept to take advantage of sulfhydryl chemistry for attaching a maleimide-functionalized label.…”

“…Kinesin-1 ( Drosophila KHC truncated at 560) was prepared with an N-terminal Avi-Tag and coexpressed with BirA in BL21(DE3) bacteria, which resulted in in vivo biotinylation of the motor (Mickolajczyk et al, 2015). Motors were purified by affinity chromatography using a C-terminal His-tag.…”

“…28005). Motors were diluted in imaging solution (0.5 mg/mL casein, 10 µ M taxol, 20 m M glucose, 20 µg/mL glucose oxidase, 8 µg/mL catalase, 0.2 mg/mL BSA, 1:200 β-mercaptoethanol, 2 m M ATP-MgCl 2 in BRB80) and mixed with streptavidin-coated 30 nm gold nanoparticles (Mickolajczyk et al, 2015). …”

“…When controlled appropriately, iSCAT has been shown to detect and image single, unlabeled proteins (Ortega Arroyo et al, 2014; Piliarik & Sandoghdar, 2014) and to track 20 nm gold labels at up to 500 kHz frame rates with nm precision (Andrecka et al, 2015; Lin, Chang, & Hsieh, 2014; Mickolajczyk et al, 2015). Interferometric detection leads to a lesser drop in scattering contrast with decreasing particle size compared to dark-field microscopy, simplifying the detection of very weak scatterers.…”