Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-魏B Essential Modulator/IKK尾 Protein鈥揚rotein Interface

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• 作者：Mary S. Golden ; Shaun M. Cote ; Marianna Sayeg ; Brandon S. Zerbe ; Elizabeth A. Villar ; Dmitri Beglov ; Stephen L. Sazinsky ; Rosina M. Georgiadis ; Sandor Vajda ; Dima Kozakov ; Adrian Whitty
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：April 24, 2013
• 年：2013
• 卷：135
• 期：16
• 页码：6242-6256
• 全文大小：620K
• 年卷期：v.135,no.16(April 24, 2013)
• ISSN：1520-5126

文摘

We report a comprehensive analysis of binding energy hot spots at the protein鈥損rotein interaction (PPI) interface between nuclear factor kappa B (NF-魏B) essential modulator (NEMO) and I魏B kinase subunit 尾 (IKK尾), an interaction that is critical for NF-魏B pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NEMO binding domain (NBD) region of IKK尾 contains the highest concentration of hot-spot residues, the strongest of which are W739, W741, and L742 (螖螖G = 4.3, 3.5, and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for 16 脜 to additionally include the regions that bind IKK尾 L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot-spot regions centered on IKK尾 residues L708/V709 and L719/I723. The computational approach successfully identified all three hot-spot regions on IKK尾. Moreover, the method was able to accurately quantify the energetic importance of all hot-spot residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small-molecule inhibitors that target the NEMO/IKK尾 interaction. They additionally clarify the structural and energetic complementarity between 鈥減ocket-forming鈥?and 鈥減ocket-occupying鈥?hot-spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.