Inhibitor of κB (IκB) kinase (IKK) phosphorylates IκB proteins leading to their degradation and liberation of nuclear factor κB (NF-κB) for gene transcription. Here we report the crystal structure of IKKβ in complex with an inhibitor at 3.6 Å resolution. The structure reveals a tri-modular architecture with the kinase domain (KD), a ubiquitin-like domain (ULD) and an elongated, α-helical scaffold/dimerization domain (SDD). Surprisingly, the predicted leucine zipper and helix-loop-helix motifs do not form these structures but are part of SDD. The ULD and SDD mediate a critical interaction with IκBα that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKKβ dimerization, but dimerization per se is not important for maintaining IKKβ activity, and instead is required for IKKβ activation. Other IKK family members IKKα, TBK1 and IKKi may share the similar tri-modular architecture and function.

The lymphocyte activation gene-3 (LAG-3), selectively transcribed in human activated T and NK cells, encodes a ligand for major histocompatibility complex (MHC) class II molecules. Like CD4, LAG-3 ectodomain is composed of four Ig-like domains (D1-D4). Nothing is known about the LAG-3 regions or residues required to form a stable MHC class II binding site. In contrast to CD4, soluble LAG-3 molecules stably interact with MHC class II molecules expressed on the cell surface. In addition, the first two Nterminal domains of soluble LAG-3 (D1 and D2) molecules, alone, are capable of binding MHC class II. From a LAG-3 model structure, we designed mutants and tested their ability to bind MHC class II molecules in an intercellular adhesion assay. We found residues on the membrane-distal, CDR1-2-containing top face of D1 that are essential for either binding or repulsing MHC class II proteins. Most of these residues are clustered at the base of a large extra-loop structure that is a hallmark of the LAG-3 D1 Ig-like domain. In addition, as for CD4, oligomerization of LAG-3 on the cell surface may be required to form a stable MHC binding site because mutation of three residues in the ABED ␤-strands containing side of D1 results in a dominant negative effect (i.e., binding inhibition of coexpressed wild-type LAG-3).

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The predicted models for the structures and mode of hormone binding of the glycoprotein hormone receptors are to a large extent consistent with currently available biochemical and mutational data. Repeated sequences in beta-barrel proteins are shown to have general implications for constraints on structure. Averaging techniques used here to recognize the structural motif in these receptors should also apply to other proteins with repeated sequences.