Two TPX2-Dependent Switches Control the Activity of Aurora A Xue Xu1., Xia Wang1., Zhengtao Xiao1, Yan Li2,3, Yonghua Wang 1 * 1 Center of Bioinformatics, Northwest A&F University, Yangling, China, 2 School of Chemical Engineering, Dalian University of Technology, Dalian, China, 3 Lab of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Abstract Aurora A is an important oncogenic kinase for mitotic spindle assembly and a potentially attractive target for human cancers. Its activation could be regulated by ATP cycle and its activator TPX2. To understand the activation mechanism of Aurora A, a series of 20 ns molecular dynamics (MD) simulations were performed on both the wild-type kinase and its mutants. Analyzing the three dynamic trajectories (Aurora A-ATP, Aurora A-ADP, and Aurora A-ADP-TPX2) at the residue level, for the first time we find two TPX2-dependent switches, i.e., switch-1 (Lys-143) and switch-2 (Arg-180), which are tightly associated with Aurora A activation. In the absence of TPX2, Lys-143 exhibits a ‘‘closed’’ state, and becomes hydrogen-bonded to ADP. Once TPX2 binding occurs, switch-1 is forced to ‘‘open’’ the binding site, thus pulling ADP away from Aurora A. Without facilitation of TPX2, switch-2 exits in an ‘‘open’’ conformation which accompanies the outward- flipping movement of P?Thr288 (in an inactive conformation), leading to the crucial phosphothreonine exposed and accessible for deactivation. However, with the binding of TPX2, switch-2 is forced to undergo a ‘‘closed’’ movement, thus capturing P?Thr288 into a buried position and locking its active conformation. Analysis of two Aurora A (K143A and R180A) mutants for the two switches further verifies their functionality and reliability in controlling Aurora activity. Our systems therefore suggest two switches determining Aurora A activation, which are important for the development of aurora kinase inhibitors. Citation: Xu X, Wang X, Xiao Z, Li Y, Wang Y (2011) Two TPX2-Dependent Switches Control the Activity of Aurora A. PLoS ONE 6(2): e16757. doi:10.1371/ journal.pone.0016757 Editor: Vladimir Uversky, University of South Florida College of Medicine, United States of America Received November 26, 2010; Accepted January 12, 2011; Published February 9, 2011 Copyright: 2011 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is financially supported by the National Natural Science Foundation of China (Grant No. 10801025) and also the Fund of Northwest A & F University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dcpwyh@163.com . These authors contributed equally to this work. Introduction Aurora-A, as a member of the Aurora family of serine-threonine kinases, localizes to centrosomes and proximal mitotic spindles and regulates spindle-associated events during early mitosis [1]. It functions mainly in centrosome separation and maturation, spindle assembly and stability, chromosome condensation, and cytokinesis in mammalian cells [2,3]. This kinase is overexpressed in a wide range of tumor types at high frequency compared with essentially nonproliferating matched normal tissue, which is associated with amplification of the region of chromosome 20 encoding AURKA [4]. Increased Aurora A expression cause increased kinase activity, finally contributing to tumor initiation and progression [5]. This oncogenic role of Aurora A, coupled to its essential role in mitotic progression, thus make inhibition of Aurora’s activity become a prominent strategy in the development of rational cancer therapeutics [6]. The kinase activity of Aurora A is tightly regulated by the ATP cycle in the ATP binding site [7] that is located at the interface of catalytic core, comprised of an N-terminal lobe (residues 123–210) and a large C-terminal lobe (residues 217–387). This region involves a complex kinetic cycle of ATP binding, nucleotide hydrolysis, and sequential release of the products Pi and ADP, respectively [8]. A steady cycle of ATP is so critical that an inhibitor which blocks certain step of this cycle deactivates the kinase in seconds [9]. During the process of ATP hydrolysis, conformational coupling to this step allows functionally important conformational transitions of a contiguous segment of the C- terminal domain known as the ‘‘activation segment’’ (residues 274–299) (see Figure S1). The conformational change within the activation segment involves phosphorylation of a conserved threonine residue (Thr-295 in Xenopus, Thr-288 in human) [7] critical for the activity of Aurora A, although it is unclear as to the regulation mechanism of phosphorylation for the kinase activity. This autocatalytic activity of Aurora-A is facilitated by several partner proteins, such as the microtubule-associated protein TPX2, Ajuba [10], protein phosphatase inhibitor-2 [11], the focal scaffolding protein HEF1[12], and a newly identified Bora [13]. TPX2 is one of the most efficiently phosphorylated Aurora A partner proteins. Upon TPX2 binding, the in vitro autopho- sphorylation activity of Aurora A is increased, and dephosphor- ylation is prevented [14,15]. The coprecipitation assay for the interaction of PP1 (protein phosphatase 1) and Aurora A using a phosphospecific P?Thr-288 antibody shows the band correspond- ing to protected P?Thr-288 is only found in the present of full length human TPX2 or TPX2 1–43, which indicates that TPX2 definitely protects residue Thr-288 from dephosphorylation. The TPX2 fragment binds Aurora-A with two separate stretches [16]. PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e16757