Susan P. Gilbert

Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro

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.

Cik1 Targets the Minus-End Kinesin Depolymerase Kar3 to Microtubule Plus Ends

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.

Modulation of kinesin binding by the C-termini of tubulin.

The flexible tubulin C‐terminal tails (CTTs) have recently been implicated in the walking mechanism of dynein and kinesin. To address their role in the case of conventional kinesin, we examined the structure of kinesin–microtubule (MT) complexes before and after CTT cleavage by subtilisin. Our results show that the CTTs directly modulate the motor–tubulin interface and the binding properties of motors. CTT cleavage increases motor binding stability, and kinesin appears to adopt a binding conformation close to the nucleotide‐free configuration under most nucleotide conditions. Moreover, C‐terminal cleavage results in trapping a transient motor–ADP–MT intermediate. Using SH3‐tagged dimeric and monomeric constructs, we could also show that the position of the kinesin neck is not affected by the C‐terminal segments of tubulin. Overall, our study reveals that the tubulin C‐termini define the stability of the MT–kinesin complex in a nucleotide‐dependent manner, and highlights the involvement of tubulin in the regulation of weak and strong kinesin binding states.

Vik1 Modulates Microtubule-Kar3 Interactions through a Motor Domain that Lacks an Active Site

Conventional kinesin and class V and VI myosins coordinate the mechanochemical cycles of their motor domains for processive movement of cargo along microtubules or actin filaments. It is widely accepted that this coordination is achieved by allosteric communication or mechanical strain between the motor domains, which controls the nucleotide state and interaction with microtubules or actin. However, questions remain about the interplay between the strain and the nucleotide state. We present an analysis of Saccharomyces cerevisiae Kar3/Vik1, a heterodimeric C-terminal Kinesin-14 containing catalytic Kar3 and the nonmotor protein Vik1. The X-ray crystal structure of Vik1 exhibits a similar fold to the kinesin and myosin catalytic head, but lacks an ATP binding site. Vik1 binds more tightly to microtubules than Kar3 and facilitates cooperative microtubule decoration by Kar3/Vik1 heterodimers, and yet allows motility. These results demand communication between Vik1 and Kar3 via a mechanism that coordinates their interactions with microtubules.

A structural model for monastrol inhibition of dimeric kinesin Eg5

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.

Decoration of the microtubule surface by one kinesin head per tubulin heterodimer

KINESIN, a microtubule-dependent ATPase, is believed to be involved in anterograde axonal transport. The kinesin head, which contains both microtubule and ATP binding sites, has the necessary components for the generation of force and motility1. We have used saturation binding and electron microscopy to examine the interaction of the kinesin motor domain with the microtubule surface and found that binding saturated at one kinesin head per tubulin heterodimer. Both negative staining and cryo-electron microscopy revealed a regular pattern of kinesin bound to the microtubule surface, with an axial repeat of 8 nm. Optical diffraction analysis of decorated microtubules showed a strong layer-line at this spacing, confirming that one kinesin head binds per tubulin heterodimer. The addition of Mg–ATP to the microtubule–kinesin complex resulted in the complete dissociation of kinesin from the microtubule surface.

The Role of ATP Hydrolysis for Kinesin Processivity

Conventional kinesin is a highly processive, plus-end-directed microtubule-based motor that drives membranous organelles toward the synapse in neurons. Although recent structural, biochemical, and mechanical measurements are beginning to converge into a common view of how kinesin converts the energy from ATP turnover into motion, it remains difficult to dissect experimentally the intermolecular domain cooperativity required for kinesin processivity. We report here our pre-steady-state kinetic analysis of a kinesin switch I mutant at Arg210 (NXXSSRSH, residues 205–212 in Drosophila kinesin). The results show that the R210A substitution results in a dimeric kinesin that is defective for ATP hydrolysis and a motor that cannot detach from the microtubule although ATP binding and microtubule association occur. We propose a mechanistic model in which ATP binding at head 1 leads to the plus-end-directed motion of the neck linker to position head 2 forward at the next microtubule binding site. However, ATP hydrolysis is required at head 1 to lock head 2 onto the microtubule in a tight binding state before head 1 dissociation from the microtubule. This mechanism optimizes forward movement and processivity by ensuring that one motor domain is tightly bound to the microtubule before the second can detach.

ATP-Dependent Simian Virus 40 T-Antigen–Hsc70 Complex Formation

ABSTRACT Simian virus 40 large T antigen is a multifunctional oncoprotein that is required for numerous viral functions and the induction of cellular transformation. T antigen contains a J domain that is required for many of its activities including viral DNA replication, transformation, and virion assembly. J-domain-containing proteins interact with Hsc70 (a cellular chaperone) to perform multiple biological activities, usually involving a change in the conformation of target substrates. It is thought that Hsc70 associates with T antigen to assist in performing its numerous activities. However, it is not clear if T antigen binds to Hsc70 directly or induces the binding of Hsc70 to other T-antigen binding proteins such as pRb or p53. In this report, we show that T antigen binds Hsc70 directly with a stoichiometry of 1:1 (dissociation constant = 310 nM Hsc70). Furthermore, the T-antigen–Hsc70 complex formation is dependent upon ATP hydrolysis at the active site of Hsc70 (ATP dissociation constant = 0.16 μM), but T-antigen–Hsc70 complex formation does not require nucleotide hydrolysis at the T-antigen ATP binding site. N136, a J domain-containing fragment of T antigen, does not stably associate with Hsc70 but can form a transient complex as assayed by centrifugation analysis. Finally, T antigen does not associate stably with either of two yeast Hsc70 homologues or an amino-terminal fragment of Hsc70 containing the ATPase domain. These results provide direct evidence that the T-antigen–Hsc70 interaction is specific and that this association requires multiple domains of both T antigen and Hsc70. This is the first demonstration of a nucleotide requirement for the association of T antigen and Hsc70 and lays the foundation for future reconstitution studies of chaperone-dependent tumorigenesis induced by T antigen.

Getting in Sync with Dimeric Eg5 INITIATION AND REGULATION OF THE PROCESSIVE RUN

Eg5/KSP is the kinesin-related motor protein that generates the major plus-end directed force for mitotic spindle assembly and dynamics. Recent work using a dimeric form of Eg5 has found it to be a processive motor; however, its mechanochemical cycle is different from that of conventional Kinesin-1. Dimeric Eg5 appears to undergo a conformational change shortly after collision with the microtubule that primes the motor for its characteristically short processive runs. To better understand this conformational change as well as head-head communication during processive stepping, equilibrium and transient kinetic approaches have been used. By contrast to the mechanism of Kinesin-1, microtubule association triggers ADP release from both motor domains of Eg5. One motor domain releases ADP rapidly, whereas ADP release from the other occurs after a slow conformational change at ∼1 s-1. Therefore, dimeric Eg5 begins its processive run with both motor domains associated with the microtubule and in the nucleotide-free state. During processive stepping however, ATP binding and potentially ATP hydrolysis signals rearward head advancement 16 nm forward to the next microtubule-binding site. This alternating cycle of processive stepping is proposed to terminate after a few steps because the head-head communication does not sufficiently control the timing to prevent both motor domains from entering the ADP-bound state simultaneously.

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

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 acid-quench 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 Pi 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.