A Pathway of Structural Changes Produced by Monastrol Binding to Eg5

Zoltan Maliga,Jun Xing,Herbert Cheung,Laura J Juszczak,Joel M Friedman,Steven S Rosenfeld

doi:10.1074/jbc.m511955200

Abstract

Monastrol is a small molecule inhibitor that is specific for Eg5, a member of the kinesin 5 family of mitotic motors. Crystallographic models of Eg5 in the presence and absence of monastrol revealed that drug binding produces a variety of structural changes in the motor, including in loop L5 and the neck linker. What is not clear from static crystallographic models, however, is the sequence of structural changes produced by drug binding. Furthermore, because crystallographic structures can be influenced by the packing forces in the crystal, it also remains unclear whether these drug-induced changes occur in solution, at physiologically active concentrations of monastrol or of other drugs that target this site. We have addressed these issues by using a series of spectroscopic probes to monitor the structural consequences of drug binding. Our results demonstrated that the crystallographic model of an Eg5-ADP-monastrol ternary complex is consistent with several solution-based spectroscopic probes. Furthermore, the kinetics of these spectroscopic signal changes allowed us to determine the temporal sequence of drug-induced structural transitions. These results suggested that L5 may be an element in the pathway that links the state of the nucleotide-binding site to the neck linker in kinesin motors.

Highlights

Insights into the structural basis for these effects of monastrol come from comparisons of crystallographic models of Eg5:ADP in the pres
Two major effects have been reported. These include a folding of loop L5 to form the top of an induced fit, hydrophobic drug-binding cavity and a reorientation of the “switch II cluster” that is associated with docking of the neck linker [19]
Effects of Drug Binding on Neck Linker Position and Dynamics as Monitored by fluorescence resonance energy transfer (FRET) and Fluorescence Anisotropy Decay—Crystallographic models of Eg5:ADP in the presence and absence of monastrol suggest that drug binding induces the neck linker to dock along the switch II complex [19], in a position analogous to what has been seen for the Kif1A-AMPPCP complex [26], conventional kinesin with bound sulfate [27], and to what has been proposed for kinesin:ATP when bound to the microtubule [28]

Summary

A Pathway of Structural Changes Produced by Monastrol Binding to Eg5*

Crystallographic models of Eg5 in the presence and absence of monastrol revealed that drug binding produces a variety of structural changes in the motor, including in loop L5 and the neck linker. Because crystallographic structures can be influenced by the packing forces in the crystal, it remains unclear whether these druginduced changes occur in solution, at physiologically active concentrations of monastrol or of other drugs that target this site. The kinetics of these spectroscopic signal changes allowed us to determine the temporal sequence of drug-induced structural transitions These results suggested that L5 may be an element in the pathway that links the state of the nucleotide-binding site to the neck linker in kinesin motors. We use these and additional spectroscopic methodologies to determine the timing and the pathway of the structural changes produced in Eg5 by binding of monastrol

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION