A structural model for monastrol inhibition of dimeric kinesin Eg5

Abstract

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.

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

“…1B). Previous moderate resolution (25-30 Å) cryo-EM studies of K5 showed no such rotation upon binding ATP analogues (27,28). However, at these lower resolutions, it has been demonstrated that it would not technically be possible to visualize such movements (49).…”

“…The first, cryoelectron microscopy (cryo-EM) and image reconstruction, is ideally suited to examine the structure of large complexes and has allowed us to determine the MT-bound structures of human K5 MD at successive steps in its ATPase cycle. Although prior intermediate resolution (ϳ25-30 Å) cryo-EM studies of human K5-MT complexes provided initial insight into the function of these motors (27,28), the movements of individual components of the MD could not been visualized. Our subnanometer resolution reconstructions now enable us to visualize coupled, nucleotide-dependent conformational transitions of human K5 structural elements, including those in L5, the NL, and the N terminus.…”

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

“…However, the affinity of Eg5-513 for AMP is so weak that apyrase treatment effectively generates a nucleotide-free state for Eg5 (data not shown). Apyrase-treated Eg5-513 was fully active based on MT binding (34) and steady-state ATP turnover (data not shown).…”

“…Indeed, previous analysis of dimeric Eg5, Eg5-513, has indicated that two conjoined motor domains exhibit cooperativity in vitro (34,35). In comparison to a single Eg5 motor domain, the steady-state ATPase of Eg5-513 is reduced 10-fold suggesting the physical attachment of two motor domains causes a reciprocal modulation of each enzymatic cycle (34).…”

“…Indeed, previous analysis of dimeric Eg5, Eg5-513, has indicated that two conjoined motor domains exhibit cooperativity in vitro (34,35). In comparison to a single Eg5 motor domain, the steady-state ATPase of Eg5-513 is reduced 10-fold suggesting the physical attachment of two motor domains causes a reciprocal modulation of each enzymatic cycle (34). Also, a His 5 -tagged form of Eg5-513 has displayed processivity in single molecule studies (35), strongly suggesting that dimeric Eg5, like other kinetically characterized dimeric kinesins, is able to maintain the ATPase cycles of its motor domains out of phase to facilitate processive stepping.…”