NF-[kappa]B is a family of dimeric transcription factors made up of five family members, p50 (NF-[kappa]B1), RelA (p65), p52 (NF-[kappa]B2), c-Rel and RelB, that regulate the expression of a large number of genes involved in innate and adaptive immunity. The NF-[kappa]B dimers regulate gene expression by binding to a class of decameric DNA duplexes known as [kappa]B sites. RelB is the most poorly understood and unique member of the NF- [kappa]B family. Unlike other members, RelB does not form a stable detectable homodimer in vivo. In uninduced cells, RelB preferentially associates with p100 and to a lesser extent with p50. Gene knockout studies demonstrated that RelB and p52 are involved in common biological functions such as secondary lymphoid organ development and B-cell maturation. It was subsequently found that these two proteins act together as a heterodimeric transcription factor that activates a set of genes in the developmental programs. The promoters of several of these genes have shown to contain [kappa]B sites that somewhat deviate from the classical [kappa]B sites. There are two important questions that remain to be answered. These questions are 1) how is the RelB/p52 heterodimer generated and 2) how does this heterodimer bind to [kappa]B sites? The purpose of this dissertation was to provide answers to these questions. The RelB/p52 heterodimer is the product of NF- [kappa]B signaling pathways, referred to as the non- canonical pathways, which involve processing of the precursor protein p100 into p52. However, whether the RelB /p52 heterodimer is generated from the p100/RelB complex observed in uninduced cells was unclear. Results shown in this dissertation demonstrate that NF-[kappa]B RelB is an unstable protein that is stabilized by forming complexes with both p100 and p52. The p100/RelB complex involves all functional domains of each protein and is not processing competent. A large body of experimental results shown here clearly suggests that p52 is primarily generated from newly synthesized p100 and not from the pre-existing p100/ RelB complex. Processing of the newly synthesized p100 is facilitated when bound to a newly synthesized NF-[kappa]B subunit, including RelB. However, the binding of newly synthesized p100 with RelB can also lead to the formation of an inactive p100/RelB complex. Therefore a dynamic binding mode between p100 and RelB, where one is processing competent and the other is not, is proposed. To understand the [kappa]B DNA recognition by the RelB/p52 heterodimer, the complex between the heterodimer and [kappa]B DNA has been crystallized and the 3.05 Å crystal structure is reported in this dissertation. The structure has revealed a new mode of DNA interaction by an NF- [kappa]B dimer that has never been observed previously. In addition to the conserved DNA contacts made by all NF- [kappa]B family members, a conserved arginine of RelB contacts the outermost G:C base pair of the RelB DNA half site. At the same time RelB loses one contact with the innermost A:T base pair of the half site normally mediated by a conserved tyrosine residue. Binding affinity measurements demonstrate that the RelB/p52 heterodimer binds to the [kappa]B DNA tested, including the newly identified ones indicated above, with similar affinities. We propose that the RelB/p52 heterodimer binds to different [kappa]B sites with high affinity by altering the DNA contact. The RelB subunit allows for different binding modes by binding to some [kappa]B sequences using the tyrosine contacts and to other [kappa]B sequences by utilizing the arginine contact. Specific DNA sequences at the center and flanking region dictate the RelB binding modes

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.