Wolter Madita, Santo Domenico Lentini, Herman Petr, Ballone Alice, Centorrino Federica, Obsil Tomas, Ottmann Christian
Department of Biomedical Engineering, Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.
Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague 12843, Czech Republic.
ACS Omega. 2020 Mar 6;5(10):5380-5388. doi: 10.1021/acsomega.9b04413. eCollection 2020 Mar 17.
Inflammatory responses mediated by the transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) play key roles in immunity, autoimmune diseases, and cancer. NF-κB is directly regulated through protein-protein interactions, including those with IκB and 14-3-3 proteins. These two important regulatory proteins have been reported to interact with each other, although little is known about this interaction. We analyzed the inhibitor of nuclear factor kappa B α (IκBα)/14-3-3σ interaction via a peptide/protein-based approach. Structural data were acquired via X-ray crystallography, while binding affinities were measured with fluorescence polarization assays and time-resolved tryptophan fluorescence. A high-resolution crystal structure (1.13 Å) of the uncommon 14-3-3 interaction motif of IκBα (IκBαpS63) in a complex with 14-3-3σ was evaluated. This motif harbors a tryptophan that makes this crystal structure the first one with such a residue visible in the electron density at that position. We used this tryptophan to determine the binding affinity of the unlabeled IκBα peptide to 14-3-3 via tryptophan fluorescence decay measurements.
由转录因子核因子κB（NF-κB）介导的炎症反应在免疫、自身免疫性疾病和癌症中起关键作用。NF-κB通过蛋白质-蛋白质相互作用直接受到调控，包括与IκB和14-3-3蛋白的相互作用。尽管对这两种重要调节蛋白之间的相互作用了解甚少，但已有报道称它们会相互作用。我们通过基于肽/蛋白质的方法分析了核因子κBα抑制剂（IκBα）/14-3-3σ的相互作用。通过X射线晶体学获得结构数据，同时用荧光偏振测定法和时间分辨色氨酸荧光测定结合亲和力。评估了IκBα（IκBαpS63）与14-3-3σ复合物中罕见的14-3-3相互作用基序的高分辨率晶体结构（1.13 Å）。该基序含有一个色氨酸，使得这个晶体结构成为第一个在该位置的电子密度中可见此类残基的结构。我们利用这个色氨酸通过色氨酸荧光衰减测量来确定未标记的IκBα肽与14-3-3的结合亲和力。
