Structure and comparison of the motor domain of centromere-associated protein E

Abstract

Centromere-associated protein E (CENP-E) plays an essential role in mitosis and is a target candidate for anticancer drugs. However, it is difficult to design small-molecule inhibitors of CENP-E kinesin motor ATPase activity owing to a lack of structural information on the CENP-E motor domain in complex with its inhibitors. Here, the CENP-E motor domain was crystallized in the presence of an ATP-competitive inhibitor and the crystal structure was determined at 1.9 Å resolution. In the determined structure, ADP was observed instead of the inhibitor in the nucleotide-binding site, even though no ADP was added during protein preparation. Structural comparison with the structures of previously reported CENP-E and those of other kinesins indicates that the determined structure is nearly identical except for several loop regions. However, the retention of ADP in the nucleotide-binding site of the structure strengthens the biochemical view that the release of ADP is a rate-limiting step in the ATPase cycle of CENP-E. These results will contribute to the development of anticancer drugs targeting CENP-E and to understanding the function of kinesin motor domains.

Crystal structure of the motor domain of centromere‐associated protein E in complex with a non‐hydrolysable ATP analogue

“…The structure of CENP‐E motor domain in complex with MgAMPPNP was successfully determined at a resolution of 1.8 Å, which is higher resolution than the previously reported structures, CENP‐E‐MgADP 6M4I (1.9 Å resolution) [16] and CENP‐E‐MgADP 1T5C [15] (2.5 Å resolution) (Fig. 1A,B).…”

“…In our previous study, the CENP‐E motor domain expressed in bacteria retained ADP in the nucleotide‐binding pocket even after three chromatographic purifications [16]. ADP was not replaced by CIBA, an ATP‐competitive CENP‐E inhibitor, when incubating the purified CENP‐E motor domain with CIBA.…”

“…The CENP‐E motor domain carrying the amino‐terminal 339 amino acids of CENP‐E (hereafter called CENP‐E motor domain) was expressed and purified as described previously [16]. In brief, the recombinant protein was expressed in the extracts of BL21(DE3) CodonPlus RIL bacteria strain (Stratagene) and purified from the bacteria extracts by three steps of chromatography; Ni‐affinity, cation exchange and gel filtration [16].…”

“…The structure was determined by the molecular replacement method with the program molrep [19] in the CCP4 suite [20]. The structure of CENP‐E‐MgADP (PDB ID: 6M4I) was used as an initial model [16]. Structural refinement was performed with REFMAC 5 [21].…”

“…There are no crystal structures of the CENP‐E motor domain in complex with its inhibitors. One of the reasons is that the CENP‐E motor domain expressed in bacteria retains the endogenous ADP derived from the expression system, even after multiple chromatographic purifications [16].…”