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Inhibitor of [kappa]B (I[kappa]B) kinase (IKK) phosphorylates I[kappa]B proteins, leading to their degradation and the liberation of nuclear factor [kappa]B for gene transcription. Here we report the crystal structure of IKK[beta] in complex with an inhibitor, at a resolution of 3.6 Å. The structure reveals a trimodular architecture comprising the kinase domain, a ubiquitin-like domain (ULD) and an elongated, [alpha]-helical scaffold/dimerization domain (SDD). Unexpectedly, the predicted leucine zipper and helix-loop-helix motifs do not form these structures but are part of the SDD. The ULD and SDD mediate a critical interaction with I[kappa]B[alpha] that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKK[beta] dimerization, but dimerization per se is not important for maintaining IKK[beta] activity and instead is required for IKK[beta] activation. Other IKK family members, IKK[alpha], TBK1 and IKK-i, may have a similar trimodular architecture and function. Structure of inhibitor of [kappa]B kinase IKK (inhibitor of [kappa]B kinase) is an important element in the activation of nuclear factor [kappa]B (NF-[kappa]B) transcription factors -- master regulators of inflammatory, immune and apoptotic responses. Here Wu and colleagues describe the long awaited X-ray structure of a member of the IKK family in combination with an inhibitor. They show that IKK[beta] has a tri-molecular architecture that contains the kinase domain, a ubiquitin-like domain and an extended scaffolding and dimerization domain, and discuss the functional implications., Author(s): Guozhou Xu [sup.1] , Yu-Chih Lo [sup.1] , Qiubai Li [sup.1] , Gennaro Napolitano [sup.2] , Xuefeng Wu [sup.2] , Xuliang Jiang [sup.3] , Michel Dreano [sup.4] , Michael [...]

Author(s): Guozhou Xu [1]; Maurizio Cirilli [1, 2]; Yihua Huang [3]; Rebecca L. Rich [4]; David G. Myszka [4]; Hao Wu (corresponding author) [1] Apoptosis is a highly regulated process [...]

Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses in Arabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein, Camelina sativa lectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space group C222 or C2221, with unit-cell parameters a = 94.7, b = 191.5, c = 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants. [ABSTRACT FROM AUTHOR]

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the >70 human F box proteins allowing the recognition of a wide range of substrates. The F box protein β-TrCP1 recognizes the doubly phosphorylated DpSGφXpS destruction motif, present in β-catenin and IκB, and directs the SCFβ-TrCP1 to ubiquitinate these proteins at specific lysines. The 3.0 A˚ structure of a β-TrCP1-Skp1-β-catenin complex reveals the basis of substrate recognition by the β-TrCP1 WD40 domain. The structure, together with the previous SCFSkp2 structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant β-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection. [Copyright &y& Elsevier]

R2219 --> Nuclear factor (NF)‐kB transcription factors are the master regulators of a variety of cellular processes, particularly immune and inflammatory responses. Activation of the IkB kinase β (IKKβ) is a central event in the nuclear factor‐kB mediated canonical pro‐inflammatory pathway, which has been indicated by numerous studies to be regulated by oligomerization‐mediated trans auto‐phosphorylation of the IKKβ kinase. The activation of the IKKβ kinase entails phosphorylation on two key serine residues S177 and S181, which are present on the IKKβ activation segment. This confers IKKβ kinase activity to phosphorylate downstream substrates such as the IkB proteins. In addition, its activation requires the interaction with NF‐kB essential modifier (NEMO). Interestingly, some viruses encode proteins that inhibit NF‐kB mediated anti‐viral immune response by targeting the IKKβ kinase. B14 is a vaccinia viral protein that inhibits IKKβ activation through direct interaction. Using GST pull‐down assay, we have mapped the binding interface on both the B14 and IKKβ proteins. B14 binds to both the kinase domain (KD) and the scaffold and dimerization domain (SDD) of IKKβ. Molecular docking modeling studies have identified key interface residues in both IKKβ and B14, which were further confirmed by mutational studies. This interaction causes a steric hindrance that blocks the direct contact between the kinase domains of IKKβ in the IKK complex, which contains both IKKβ and NEMO. During trans auto‐phosphorylation of the IKKβ kinase, the activation segment of a kinase domain needs to interact with the active site of another neighbor kinase domain for trans phosphorylation and activation. As such, the B14‐binding hinders IKKβ trans auto‐phosphorylation and activation, which eventually inhibits NF‐kB signaling. [ABSTRACT FROM AUTHOR]

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the >70 human F box proteins allowing the recognition of a wide range of substrates. The F box protein beta-TrCP1 recognizes the doubly phosphorylated DpSGphiXpS destruction motif, present in beta-catenin and IkappaB, and directs the SCF(beta-TrCP1) to ubiquitinate these proteins at specific lysines. The 3.0 A structure of a beta-TrCP1-Skp1-beta-catenin complex reveals the basis of substrate recognition by the beta-TrCP1 WD40 domain. The structure, together with the previous SCF(Skp2) structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant beta-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection.