Crystal structure of HsEg5 in complex with S‐trityl‐L‐cysteine

Abstract

Eg5, a motor kinesin involved in the formation of the bipolar mitotic spindle, is essential for the successful completion of mitosis. Monastrol and S‐trityl‐L‐cysteine (STC) are two well‐characterized inhibitors of Eg5, with STC being the more potent inhibitor. We have determined the 2.5 Åresolution crystal structure of the Eg5 motor domain in complex with STC and Mg ?ADP. STC interacts with Eg5 via a pocket formed by helices a2, a3 and loop L5, and induces conformational changes within the Eg5 necklinker and switch I and II regions similar to those seen with bound monastrol. However, there are two distinctions between the STC‐Eg5 and monastrol‐Eg5 structures: the nature of the interactions between the allosteric ligand and Eg5 and solvent accessibility of residues in the allosteric binding site. Monastrol contacts with Eg5 are largely mediated by nonpolar drug surfaces. During STC binding, contact between the motor domain and polar surfaces of the drug is increased in relation to that of monastrol by over 20 Å2. STC binding occludes ~80 Å2 more of the Eg5 surface from solvent access than does monastrol. Site‐directed mutagenesis and coupled biochemical assays monitoring ATP hydrolysis were used to test the hypothesis that several residues altered in solvent accessibility upon STC binding to Eg5 were critical to the increased efficacy of inhibition by this small molecule.Supported by Louisiana Board of Regents Grant