Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.Eg5, a Kinesin-5 (formerly known as BimC) family member, has an unusual homotetrameric protein structure that is formed by the anti-parallel arrangement of heavy chains, with pairs of heads situated at opposite ends of a common stalk 1 . In dividing cells, Eg5 is essential for the organization and maintenance of mitotic and meiotic spindles. Organized bipolar spindles contain microtubule arrays emanating from each of two focused poles, with interdigitated, antiparallel microtubules occupying the mid-zone. Immunodepletion of Eg5 leads to monoastral spindles with disorganized poles and to the disruption of preformed bipolar spindles 2 , and several small-molecule inhibitors of Eg5 have been identified 3, 4 .However, some clues about the function of Eg5 in spindle organization are emerging. Microtubule gliding-filament assays have shown that this motor is plus-end-directed and slow, with reported velocities ranging from 10-40 nm s −1 (refs 2 , 5-7 ). These velocities are similar 6 Correspondence should be addressed to S.M.B. (e-mail: sblock@stanford.edu). 5 These authors contributed equally to this work.Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONS P.M.F. and M.T.V. designed, performed and analysed the single-molecule experiments and co-wrote the manuscript. T.C.K. and S.P.G. generated the Eg5 constructs and conducted the biochemical measurements. S.M.B. and S.P.G. supplied guidance, designed research and edited the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. Despite recent advances, fundamental questions persist, particularly with regard to Eg5 processivity (the average number of sucessive enzymatic cycles carried out while bound to the microtubule substrate). Conventional kinesin is processive and single motors can transport cargo over large distances. In contrast, Eg5 is thought to work in small ensembles and need not be processive. A previous study used solution biochemical measur...

Eg5 or KSP is a homotetrameric Kinesin-5 involved in centrosome separation and assembly of the bipolar mitotic spindle. Analytical gel filtration of purified protein and cryo-electron microscopy (cryo-EM) of unidirectional shadowed microtubule-Eg5 complexes have been used to identify the stable dimer Eg5-513. The motility assays show that Eg5-513 promotes robust plus-end-directed microtubule gliding at a rate similar to that of homotetrameric Eg5 in vitro. Eg5-513 exhibits slow ATP turnover, high affinity for ATP, and a weakened affinity for microtubules when compared to monomeric Eg5. We show here that the Eg5-513 dimer binds microtubules with both heads to two adjacent tubulin heterodimers along the same microtubule protofilament. Under all nucleotide conditions tested, there were no visible structural changes in the monomeric Eg5-microtubule complexes with monastrol treatment. In contrast, there was a substantial monastrol effect on dimeric Eg5-513, which reduced microtubule lattice decoration. Comparisons between the X-ray structures of Eg5-ADP and Eg5-ADP-monastrol with rat kinesin-ADP after docking them into cryo-EM 3-D scaffolds revealed structural evidence for the weaker microtubule-Eg5 interaction in the presence of monastrol.

Summary Current models of SIRT1 enzymatic regulation primarily consider the effects of fluctuating levels of its co-substrate NAD+, which binds to the stably-folded catalytic domain. By contrast, the roles of the sizeable disordered N- and C-terminal regions of SIRT1 are largely unexplored. Here, we identify an insulin-responsive sensor in the SIRT1 N-terminal region (NTR), comprising an acidic cluster (AC) and a 3-helix bundle (3HB), controlling deacetylase activity. The allosteric assistor DBC1 removes a distal N-terminal shield from the 3HB, permitting PACS-2 to engage the AC and the transiently exposed helix 3 of the 3HB, disrupting its structure and inhibiting catalysis. The SIRT1 activator (STAC) SRT1720 binds and stabilizes the 3HB, protecting SIRT1 from inhibition by PACS-2. Identification of the SIRT1 insulin-responsive sensor and its engagement by the DBC1/PACS-2 regulatory hub provides important insight into the roles of disordered regions in enzyme regulation and the mode by which STACs promote metabolic fitness.

Eg5/KSP is a homotetrameric, Kinesin-5 family member whose ability to cross-link microtubules has associated it with mitotic spindle assembly and dynamics for chromosome segregation. Transient-state kinetic methodologies have been used to dissect the mechanochemical cycle of a dimeric motor, Eg5-513, to better understand the cooperative interactions that modulate processive stepping. Microtubule association, ADP release, and ATP binding are all fast steps in the pathway. However, the acidquench analysis of the kinetics of ATP hydrolysis with substrate in excess of motor was unable to resolve a burst of product formation during the first turnover event. In addition, the kinetics of P i release and ATP-promoted microtubule-Eg5 dissociation were observed to be no faster than the rate of ATP hydrolysis. In combination the data suggest that dimeric Eg5 is the first kinesin motor identified to have a rate-limiting ATP hydrolysis step. Furthermore, several lines of evidence implicate alternating-site catalysis as the molecular mechanism underlying dimeric Eg5 processivity. Both mantATP binding and mantADP release transients are biphasic. Analysis of ATP hydrolysis through single turnover assays indicates a surprising substrate concentration dependence, where the observed rate is reduced by half when substrate concentration is sufficiently high to require both motor domains of the dimer to participate in the reaction.Eg5 is a member of the homotetrameric, Bim C/Kinesin-5 family. Members of this family function during mitosis and provide a plus-end directed force that has become associated with bipolar spindle assembly, spindle maintenance, and microtubule (MT) 2 flux (1-10). The function of Eg5 in the mitotic spindle has been shown to be indispensable. Perturbation of its function prior to anaphase B by either antibody (1, 11) or small molecule inhibitors (12-23) causes collapse of the bipolar spindle into a monoaster and leads to apoptosis in cells with intact spindle checkpoint machinery (24, 25). As a result Eg5 has garnered substantial interest as a potential chemotherapeutic target in cancer treatment.Mechanistically, the ATPase cycle of monomeric Eg5 motor domains are fairly well understood both free in solution and bound to . Off the MT, monomeric Eg5 demonstrates weak ATP binding and has a propensity to form a nonproductive Eg5⅐ATP complex (31). 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...

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...