An allosteric transition trapped in an intermediate state of a new kinesin–inhibitor complex

Abstract

Human kinesin Eg5 plays an essential role in mitosis by separating duplicated centrosomes and establishing the bipolar spindle. Eg5 is an interesting drug target for the development of cancer chemotherapy, with seven inhibitors already in clinical trials. In the present paper, we report the crystal structure of the Eg5 motor domain complexed with a potent antimitotic inhibitor STLC (S-trityl-L-cysteine) to 2.0 A (1 A=0.1 nm) resolution. The Eg5-STLC complex crystallizes in space group P3(2) with three molecules per asymmetric unit. Two of the molecules reveal the final inhibitor-bound state of Eg5, whereby loop L5 has swung downwards to close the inhibitor-binding pocket, helix alpha4 has rotated by approx. 15 degrees and the neck-linker has adopted a docked conformation. The third molecule, however, revealed an unprecedented intermediate state, whereby local changes at the inhibitor-binding pocket have not propagated to structural changes at the switch II cluster and neck-linker. This provides structural evidence for the sequence of drug-induced conformational changes.

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines

“…The same experimental protocol used in the monastrol study to determined whether the Eg5 point mutants could confer resistance to cells exposed to STLC, a chemically distinct inhibitor of Eg5 that is also thought to bind to the same inhibitor binding pocket in the motor domain as monastrol [29,33,34,38].…”

“…Thus, the issue of target specificity is of great importance for inhibitors targeting Eg5. Structural analyses of the interaction between the Eg5 motor domain and various inhibitors such as monastrol, its analogues and other A c c e p t e d M a n u s c r i p t inhibitor scaffolds have been recently carried out [25][26][27][28][29]. These ligands bind in a unique pocket in the Eg5 motor domain formed by the secondary elements helix 2/loop L5 (Tyr125-Glu145) and helix 3 (Ile202-Leu227) (shown in Fig.…”

“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

“…3 and 4). Finally, we note that we have already described an Eg5-ADP-Strityl-L-cysteine structure that is very similar to our new Eg5-ADP-ispinesib "intermediate" (20) (PDB entry 2WOG). However, unlike the present study, this earlier report did not correlate the crystallographic model with kinetics in order to unequivocally place this S-trityl-L-cysteine-induced structure in a temporal sequence of conformational changes.…”

“…Labeling with a 10-fold molar excess of TMR was carried out at 4°C for 24 h, followed by removal of excess probe by gel filtration on Sephadex G25 (PD10, GE Healthcare), yielding a labeling stoichiometry of 1.7-1.9 (12) and a preparation with a MT-activated k cat of 5.1 Ϯ 1.6 s Ϫ1 . Eg5(1-368) (containing residues 1-368) used for crystallization of the Eg5-ispinesib complex was cloned, expressed, and purified as described previously (20).…”

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

“…Structures solved using x-ray crystallography have provided residue-by-residue views of the human K5 MD, including highlighting potential conformational variations in the K5 NL and L5 (11,15,20,21). In parallel, spectroscopic measurements have provided dynamic data concerning the timing and regulation of conformational changes within the MD and have been correlated with K5 enzyme kinetics (12,14,22).…”