Department of Chemistry & Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0357, United States.
Structural Biochemistry Laboratory Department of Chemistry & Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, California 92182-1030, United States.
Biochemistry. 2024 Sep 17;63(18):2323-2334. doi: 10.1021/acs.biochem.4c00202. Epub 2024 Aug 26.
The IκB Kinase (IKK) complex, containing catalytic IKK2 and noncatalytic NEMO subunits, plays essential roles in the induction of transcription factors of the NF-κB family. Catalytic activation of IKK2 via phosphorylation of its activation loop is promoted upon noncovalent association of linear or K63-linked polyubiquitin chains to NEMO within the IKK complex. The mechanisms of this activation remain speculative. To investigate interaction dynamics within the IKK complex during activation of IKK2, we conducted hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on NEMO and IKK2 proteins in their free and complex-bound states. Altered proton exchange profiles were observed in both IKK2 and NEMO upon complex formation, and changes were consistent with the involvement of distinct regions throughout the entire length of both proteins, including previously uncharacterized segments, in direct or allosteric interactions. Association with linear tetraubiquitin (Ub) affected multiple regions of the IKK2:NEMO complex, in addition to previously identified interaction sites on NEMO. Intriguingly, observed enhanced solvent accessibility of the IKK2 activation loop within the IKK2:NEMO:Ub complex, coupled with contrasting protection of surrounding segments of the catalytic subunit, suggests an allosteric role for NEMO:Ub in priming IKK2 for phosphorylation-dependent catalytic activation.
IKK 激酶（IKK）复合物包含催化性 IKK2 和非催化性 NEMO 亚基，在 NF-κB 家族转录因子的诱导中发挥重要作用。IKK 复合物中 NEMO 上线性或 K63 连接的多泛素链与 NEMO 的非共价结合促进 IKK2 的催化活性环的磷酸化。这种激活的机制仍在推测之中。为了研究 IKK2 激活过程中 IKK 复合物内的相互作用动态，我们对游离状态和复合物结合状态下的 NEMO 和 IKK2 蛋白进行了氢氘交换结合质谱（HDX-MS）分析。复合物形成后，IKK2 和 NEMO 中的质子交换轮廓都发生了变化，这些变化与两个蛋白全长上多个不同区域的直接或变构相互作用有关，包括以前未表征的片段。与线性四聚泛素（Ub）的结合除了影响 NEMO 上先前确定的相互作用位点外，还影响了 IKK2:NEMO 复合物的多个区域。有趣的是，在 IKK2:NEMO:Ub 复合物中观察到 IKK2 激活环的溶剂可及性增强，而催化亚基周围的片段则受到保护，这表明 NEMO:Ub 在 IKK2 的磷酸化依赖性催化激活的引发中具有变构作用。
