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Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.

The triterpene glycoside glycyrrhizin is the main active compound in liquorice. It is used as a herbal medicine owing to its anticancer, antiviral and anti-inflammatory properties. Its mode of action, however, remains widely unknown. In the present study, we aimed to elucidate the molecular mechanism of glycyrrhizin in attenuating inflammatory responses in macrophages. Using microarray analysis, we found that glycyrrhizin caused a broad block in the induction of pro-inflammatory mediators induced by the TLR (Toll-like receptor) 9 agonist CpG-DNA in RAW 264.7 cells. Furthermore, we found that glycyrrhizin also strongly attenuated inflammatory responses induced by TLR3 and TLR4 ligands. The inhibition was accompanied by decreased activation not only of the NF-κB (nuclear factor κB) pathway but also of the parallel MAPK (mitogen-activated protein kinase) signalling cascade upon stimulation with TLR9 and TLR4 agonists. Further analysis of upstream events revealed that glycyrrhizin treatment decreased cellular attachment and/or uptake of CpG-DNA and strongly impaired TLR4 internalization. Moreover, we found that the anti-inflammatory effects were specific for membrane-dependent receptor-mediated stimuli, as glycyrrhizin was ineffective in blocking Tnfa (tumour necrosis factor α gene) induction upon stimulation with PMA, a receptor- and membrane-independent stimulus. These observations suggest that the broad anti-inflammatory activity of glycyrrhizin is mediated by the interaction with the lipid bilayer, thereby attenuating receptor-mediated signalling. [ABSTRACT FROM AUTHOR]