Protein Engineering of the N-Terminus of NEMO: Structure Stabilization and Rescue of IKK尾 Binding

详细信息    查看全文

• 作者：Bingqian Guo ; Christopher O. Audu ; Jared C. Cochran ; Dale F. Mierke ; Maria Pellegrini
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：November 4, 2014
• 年：2014
• 卷：53
• 期：43
• 页码：6776-6785
• 全文大小：496K
• ISSN：1520-4995

文摘

NEMO is a scaffolding protein that, together with the catalytic subunits IKK伪 and IKK尾, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-魏B pathway. Rational drug design targeting the IKK-binding site on NEMO would benefit from structural insight, but to date, the determination of the structure of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK-binding domain NEMO(44鈥?11) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled coil at the N-terminus, C-terminus, or both ends of NEMO(44鈥?11) present high thermal stability and cooperative melting and, most importantly, restore IKK尾 binding affinity. We examined the consequences of structural content and stability by circular dichoism and nuclear magnetic resonance (NMR) and measured the binding affinity of each construct for IKK尾(701鈥?45) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method for engineering short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile, the rescuing of the binding affinity implies that a preordered IKK-binding region of NEMO is compatible with IKK binding, and the conformational heterogeneity observed in NEMO(44鈥?11) may be an artifact of the truncation.