Pathway of ATP Hydrolysis by Monomeric Kinesin Eg5

Abstract

Kinesin-5 family members including human Eg5/KSP contribute to the plus-end-directed force necessary for the assembly and maintenance of the bipolar mitotic spindle. We have used monomeric Eg5-367 in the nucleotide-free state to evaluate the role of microtubules at each step in the ATPase cycle. The pre-steady-state kinetic results show that the microtubule-Eg5 complex binds MgATP tightly, followed by rapid ATP hydrolysis with a subsequent slow step that limits steady-state turnover. We show that microtubules accelerate the kinetics of each step in the ATPase pathway, suggesting that microtubules amplify the nucleotide-dependent structural transitions required for force generation. The experimentally determined rate constants for phosphate product release and Eg5 detachment from the microtubule were similar, suggesting that these two steps are coupled with one occurring at the slow rate after ATP hydrolysis followed by the second step occurring more rapidly. The rate of this slow step correlates well with the steady-state k cat , indicative that it is the rate-limiting step of the mechanism.Kinesins are cytoskeletal motor proteins that utilize the energy from the ATPase cycle to perform mechanical work along microtubules. Conversion of the nucleotide state at the motor active site triggers conformational changes in the motor, which dictates the affinity of the motor for its filament and ultimately results in force generation. Although the specific structural events linking the ATP hydrolysis cycle to molecular motion are unknown, a mechanism for the pathway of conformational change that is responsible for amplifying small movements at the nucleotide binding site to large-scale changes in distant regions of the motor is now emerging.Kinesins share an ~350 amino acid motor domain that contains the nucleotide binding site and the microtubule binding region (reviewed in refs 1 and 2 and also see the kinesin homepage: http://www.proweb.org/kinesin/). A third region that is conserved within kinesin subfamilies is the ~14-20 amino acid sequence adjacent to the motor domain called the neck linker. Together, the motor domain and the neck linker make up the core domain, and numerous studies have investigated the ATPase mechanism of different kinesin monomers in the presence of microtubules including Kinesin-1/conventional kinesin (3-8), Kinesin-5/Eg5/BimC (9-11), and Kinesin-14/Ncd/Kar3/(12-15). The structural differences that arise between various kinesin subfamilies have fine-tuned the rate and equilibrium constants that govern the overall mechanochemical cycles. Therefore, each kinesin motor elicits a different work output that is † This work was supported by NIH Grant GM54141 from NIGMS and NIH K02-AR47841 Career Development Award from NIAMS to S.P.G. * Corresponding author. . E-mail: spg1@pitt.edu. ‡ Present address: Department of Chemistry, Dartmouth College, Hanover, NH 03755.1 Abbreviations: Eg5-367, human Eg5/KSP motor domain containing the N-terminal 367 residues followed by a C-terminal His6 tag; Eg5-43...

Dimeric Eg5 Maintains Processivity through Alternating-site Catalysis with Rate-limiting ATP Hydrolysis

“…ATP Hydrolysis Is Rate Limiting for Eg5-513-For monomeric Eg5, the rate-limiting step in the ATPase cycle was proposed to be the coupled step of phosphate release and detachment from the MT (30,33). The data presented here suggest that the rate-limiting step for dimeric Eg5 is altered from that of the monomer.…”

“…However, on the MT, ATP binding is tight, substrate productively proceeds through ATP hydrolysis, and the rates of all the individual steps in the mechanochemical cycle are accelerated (30). The MT-activated ATPase cycle concludes with a conformational change of the motor domain in relation to the MT, termed "rolling" (29), followed by the rate-limiting event in the cycle, the coupled action of phosphate (P i ) release, and motor detachment from the MT (29,30,33).In vivo the individual Eg5 motor domains probably do not function independently; therefore, analysis of a higher order oligomeric structure is necessitated. Indeed, previous analysis of dimeric Eg5, Eg5-513, has indicated that two conjoined motor domains exhibit cooperativity in vitro (34,35).…”

“…However, on the MT, ATP binding is tight, substrate productively proceeds through ATP hydrolysis, and the rates of all the individual steps in the mechanochemical cycle are accelerated (30). The MT-activated ATPase cycle concludes with a conformational change of the motor domain in relation to the MT, termed "rolling" (29), followed by the rate-limiting event in the cycle, the coupled action of phosphate (P i ) release, and motor detachment from the MT (29,30,33).…”

Modulation of the Kinesin ATPase Cycle by Neck Linker Docking and Microtubule Binding

“…Site-directed mutagenesis was performed to generate E358(wt), E368(R234K), and E368(S233C/ R234K) (supplemental Table S2), and these mutations were confirmed by DNA sequencing. Each construct was co-transformed into B834(DE3) cells with pRIL (Stratagene), and the kinesin-5 protein was expressed and purified in the nucleotidefree state as described previously (9,20,21). Briefly, MgATP was excluded from all column chromatography buffers, and the enriched kinesin-5 fractions from the nickel-nitrilotriacetic acid agarose column (Qiagen) were pooled, incubated with 10 mM EDTA, and buffer-exchanged using desalting columns (GE Healthcare).…”

“…This structural arrangement allows the enzyme to slide along adjacent MTs during mitosis, thus contributing to the plus enddirected force that is necessary for forming and maintaining the mitotic spindle (17,18). Previous studies have kinetically characterized the ATPase mechanism of dimeric (19) and monomeric kinesin-5 in the absence (20) and presence of MTs (9,10,21). The results of these kinetic studies have been correlated to changes in the neck linker orientation of kinesin-5 for force generation and processive motility along the MT (10,19,22).…”