Loop L5 Acts as a Conformational Latch in the Mitotic Kinesin Eg5

Abstract

All members of the kinesin superfamily of molecular motors contain an unusual structural motif consisting of an ␣-helix that is interrupted by a flexible loop, referred to as L5. We have examined the function of L5 in the mitotic kinesin Eg5 by combining site-directed mutagenesis of L5 with transient state kinetics, molecular dynamics simulations, and docking using cryo electron microscopy density. We find that mutation of a proline residue located at a turn within this loop profoundly slows nucleotideinduced structural changes both at the catalytic site as well as at the microtubule binding domain and the neck linker. Molecular dynamics simulations reveal that this mutation affects the dynamics not only of L5 itself but also of the switch I structural elements that sense ATP binding to the catalytic site. Our results lead us to propose that L5 regulates the rate of conformational change in key elements of the nucleotide binding site through its interactions with ␣3 and in so doing controls the speed of movement and force generation in kinesin motors.The kinesin superfamily of molecular motors share several evolutionarily conserved structural features with myosins and G-proteins. These include domains that coordinate the ␤ phosphate of bound nucleotide (P loop), that sense the ␥ phosphate (switch I), and that induce conformational changes in response to phosphate release (switch II) (1-3). However, kinesins also contain an unusual structural element not found in these other motors and switches. This consists of an ␣-helix (␣2) on the carboxyl terminal end of the P loop, which is interrupted by a stem and loop motif referred to as L5 (Ref. 4 and Fig. 1). The length of L5 varies considerably between different kinesin superfamily members, from short in kinesin 1 and CENP-E to longest in the mitotic kinesin Eg5 (Fig. 2).The function of L5 remains unclear, although three lines of evidence suggest that it may play important roles in regulating nucleotide and microtubule binding. First, several small molecules induce L5 in Eg5 to fold over, generating a hydrophobic pocket bounded by L5, ␣2, and ␣3 (5, 6). In this conformation both ADP release and microtubule binding are prevented (7,8). The analogous site in CENP-E is also the binding site for a novel inhibitor stabilizing the microtubule-motor complex, which appears to stabilize this motor in a strong microtubule binding conformation (9). Second, labeling of L5 with an EPR spin probe demonstrates that its mobility is affected by nucleotide binding, and in turn, deletion of L5 affects nucleotide binding (10). Third, cryo-EM reconstructions of Eg5-decorated microtubules in the presence of AMPPNP suggests that L5 can interact with ␣3, when the Eg5 motor is bound to the microtubule (11). Because the ␣3 helix is on the amino-terminal side of switch I, changes in the L5-␣3 interaction might have downstream effects on nucleotide affinity and, secondarily, on microtubule binding. However, this study generated a docking model using crystallographic structures of Eg5 in the p...

Analysis of the interaction of the Eg5 Loop5 with the nucleotide site

“…Multiple attempts at deforming the ΔL5-domain into a configuration that would stably interact with the nucleotide failed. Interaction of L5 with the nucleotide site is further supported by studies suggesting that mutations of L5 perturb the Eg5 kinetic cycle [1, 12, 15–18, 22]. …”

“…EM reconstructions of microtubule-bound Drosophila kinesin-5 motor KLP61F likewise indicated that L5 undergoes a microtubule-binding dependent interaction with the α3-helix that was postulated to be involved in the transition between force-generating and diffusional modes of MT binding [45]. Spectroscopic and biochemical data have additionally linked L5 to the force-generating cycle through changes in the conformation of the neck-linker [1, 15, 46] that have been proposed to be modulated via interaction with the α3-helix and concomitant perturbations of Switch 1 by the α3-helix [15]. We observed only minimal interaction of L5 with the α3-helix in our simulations (a single interaction with Arg221 in two of the six simulations).…”

“…This is in part due to the fact that the L5-domain is in approximately the same conformation in the X-ray structures in both the presence and the absence of inhibitors. Point mutations of the L5-domain, replacement of the Eg5 L5-domain with a kinesin-1 L5-domain, or deletion of 7 residues in the human Eg5 L5 all decrease the ATPase activity of Eg5 [12, 15, 16]. Replacement of the Eg5 L5 with the comparable domain from Neurospora eliminates STLC inhibition [17, 18].…”

“…EPR spectroscopy can also very easily resolve multiple protein conformations in equilibrium as is the case noted above for Eg5. Computational simulation has proven to be a powerful tool for atomic-level examinations of different conformations of flexible kinesin-family structural elements in X-ray structures [15, 23–27] and their relationship to observations from other experimental techniques. Here we have used the human Eg5•ADP X-ray structure [10] as our starting point for molecular mechanics and molecular dynamics modeling of an Eg5 structure with a deformed L5 loop in order to study possible interactions of L5 with the nucleotide site.…”

Synthesis and characterization of tritylthioethanamine derivatives with potent KSP inhibitory activity

“…16, 23, 30 Loop 5 undergoes dynamic conformational shifts during the ATP binding and hydrolysis cycle, and it is thought that interactions with small molecules such as monastrol “lock” this loop into an ADP bound-like conformation. 1, 4, 31, 32 While monastrol has not demonstrated efficacy at pharmacologically relevant concentrations, several compounds that exhibit anticancer activity have advanced to clinical trials. 22, 33–38 However, recent characterization of drug-resistant variants of KSP raises concern that tumor cells may develop resistance over time.…”