X-ray structure of a NF-kappaB p50/RelB/DNA complex reveals assembly of multiple dimers on tandem kappaB sites.

We describe here the X-ray crystal structure of NF-kappaB p50/RelB heterodimer bound to a kappaB DNA. Although the global modes of subunit association and kappaB DNA recognition are similar to other NF-kappaB/DNA complexes, this complex reveals distinctive features not observed for non-RelB complexes. For example, Lys274 of RelB is removed from the protein-DNA interface whereas the corresponding residues in all other subunits make base-specific contacts. This mode of binding suggests that RelB may allow the recognition of more diverse kappaB sequences. Complementary surfaces on RelB and p50, as revealed by the crystal contacts, are highly suggestive of assembly of multiple p50/RelB heterodimers on tandem kappaB sites in solution. Consistent with this model our in vitro binding experiments reveal optimal assembly of two wild-type p50/RelB heterodimers on tandem HIV kappaB DNA with 2 bp spacing but not by a mutant heterodimer where one of the RelB packing surface is altered. We suggest that multiple NF-kappaB dimers assemble at diverse kappaB promoters through direct interactions utilizing unique protein-protein interaction surfaces.

The 20S proteasome processes NF-kappaB1 p105 into p50 in a translation-independent manner.

The NF-kappaB p50 is the N-terminal processed product of the precursor, p105. It has been suggested that p50 is generated not from full-length p105 but cotranslationally from incompletely synthesized molecules by the proteasome. We show that the 20S proteasome endoproteolytically cleaves the fully synthesized p105 and selectively degrades the C-terminus of p105, leading to p50 generation in a ubiquitin-independent manner. As small as 25 residues C-terminus to the site of processing are sufficient to promote processing in vivo. However, any p105 mutant that lacks complete ankyrin repeat domain (ARD) is processed aberrantly, suggesting that native processing must occur from a precursor, which extends beyond the ARD. Remarkably, the mutant p105 that lacks the internal region including the glycine-rich region (GRR) is completely degraded by 20S proteasome in vitro. This suggests that the GRR impedes the complete degradation of the p105 precursor, thus contributing to p50 generation.

p105.Ikappa Bgamma and prototypical Ikappa Bs use a similar mechanism to bind but a different mechanism to regulate the subcellular localization of NF-kappa B.

p105, also known as NF-kappaB1, is an atypical IkappaB molecule with a multi-domain organization distinct from other prototypical IkappaBs, like IkappaBalpha and IkappaBbeta. To understand the mechanism by which p105 binds and inhibits NF-kappaB, we have used both p105 and its C-terminal inhibitory segment known as IkappaBgamma for our study. We show here that one IkappaBgamma molecule binds to NF-kappaB dimers wherein at least one NF-kappaB subunit is p50. We suggest that the obligatory p50 subunit in IkappaBgamma.NF-kappaB complexes is equivalent to the N-terminal p50 segment in all p105.NF-kappaB complexes. The nuclear localization signal (NLS) of the obligatory p50 subunit is masked by IkappaBgamma, whereas the NLS of the nonobligatory NF-kappaB subunit is exposed. Thus, the global binding mode of all IkappaB.NF-kappaB complexes seems to be similar where one obligatory (or specific) NF-kappaB subunit makes intimate contact with IkappaB and the nonobligatory (or nonspecific) subunit is bound primarily through its ability to dimerize. In the case of IkappaBalpha and IkappaBbeta, the specific NF-kappaB subunit in the complex is p65. In contrast to IkappaBalpha.NF-kappaB complexes, where the exposed NLS of the nonspecific subunit imports the complex to the nucleus, p105.NF-kappaB and IkappaBgamma.NF-kappaB complexes are cytoplasmic. We show that the death domain of p105 (also of IkappaBgamma) is essential for the cytoplasmic sequestration of NF-kappaB by p105 and IkappaBgamma. However, the death domain does not mask the exposed NLS of the complex. We also demonstrate that the death domain alone is not sufficient for cytoplasmic retention and instead functions only in conjunction with other parts in the three-dimensional scaffold formed by the association of the ankyrin repeat domain (ARD) and NF-kappaB dimer. We speculate that additional cytoplasmic protein(s) may sequester the entire p105.NF-kappaB complex by binding through the death domain and other segments, including the exposed NLS.