Crystal structure of triple-BRCT-domain of ECT2 and insights into the binding characteristics to CYK-4

• Published in: FEBS letters 2014-08, Vol.588 (17), p.2911-2920
• Main Authors: Zou, Yang, Shao, Zhenhua, Peng, Junhui, Li, Fudong, Gong, Deshun, Wang, Chongyuan, Zuo, Xiaobing, Zhang, Zhiyong, Wu, Jihui, Shi, Yunyu, Gong, Qingguo
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Auto-inhibition; Binding pocket; Cattle; circular dichroism; Crystallography, X-Ray; Dbl homology domain and Pleckstrin homology domain; DH–PH; ECT2; GTPase-Activating Proteins - metabolism; Humans; IPTG; isopropyl β-d-1-thiogalactopyranoside; isothermal titration calorimetry; ITC; Mice; mitotic kinesin-like protein 1; MKLP1; molecular dynamic; Molecular Dynamics Simulation; Molecular Sequence Data; Phospho-peptide; phosphorylated serine; Phosphorylation; Phosphoserine - metabolism; Plk1; Polo-like kinase 1; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins - metabolism; RMSD; root mean square deviation; SAXS; small angle X-ray scattering; TEV; The epithelial cell transforming protein 2; tobacco etch virus; Triple-BRCT-domain
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2014.07.019

•We solved the crystal structure of triple-BRCT-domain of ECT2.•We show that phosphorylation of Ser164 in CYK-4 is crucial for the interaction with ECT2.•A canonical binding pocket in the second BRCT domain of ECT2 is proved to bind to pS164 in CYK-4.•The interactions between ECT2 and CYK-4 can relieve the auto-inhibition of ECT2. Homo sapiens ECT2 is a cell cycle regulator that plays critical roles in cytokinesis. ECT2 activity is restrained during interphase via intra-molecular interactions that involve its N-terminal triple-BRCT-domain and its C-terminal DH–PH domain. At anaphase, this self-inhibitory mechanism is relieved by Plk1-phosphorylated CYK-4, which directly engages the ECT2 BRCT domain. To provide a structural perspective for this auto-inhibitory property, we solved the crystal structure of the ECT2 triple-BRCT-domain. In addition, we systematically analyzed the interaction between the ECT2 BRCT domains with phospho-peptides derived from its binding partner CYK-4, and have identified Ser164 as the major phospho-residue that links CYK-4 to the second ECT2 BRCT domain. ECT2 and CYK-4bind by isothermal titration calorimetry (1, 2, 3, 4, 5, 6, 7)