Crystal Structure of HsEg5 in Complex with S-trityl-L-cysteine

Abstract

Eg5 is a motor kinesin involved in the formation of the bipolar mitotic spindle which is essential for the completion of mitosis. The discovery of a class of allosteric Eg5 inhibitors has raised the possibility of a novel approach for the treatment of cancer. Monastrol and S-trityl-L-cysteine (STC) are two well-characterized inhibitors of Eg5 known to prevent ADP release by the motor domain, with the latter compound being a more potent inhibitor of Eg5 than the former. We have determined the 2.5 Å resolution crystal structure of the Eg5 motor domain in complex with STC and Mg•ADP. STC interacts with Eg5 via a pocket formed by helices α2, α3 and loop L5, and induces conformational changes within the Eg5 motor domain similar to those seen with bound monastrol. The necklinker is positioned in the “docked” conformation seen in the monastrol-bound Eg5 structures, and the switch I and II regions also adopt conformations similar to those observed for bound monastrol. Moreover, STC contacts with Eg5 differ from those seen in monastrol-bound crystal structures. Monastrol contacts with Eg5 are largely mediated by nonpolar surfaces of the drug. 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 approximately 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 examine whether these residues involved in polar interactions between STC and Eg5, and residues altered in solvent accessibility upon STC binding to Eg5, were critical in the increased efficacy of Eg5 inhibition by this small molecule.