UC San Diego

The NF-[kappa]B pathway is one of the hallmark signal transduction pathways associated with inflammation, innate and adaptive immunity, and regulation of apoptosis. Ever since its discovery in 1986, a plethora of studies have led us to a solid understanding of its function and regulation. One group of proteins that are at the center of its regulation is the I[kappa]B family of transcriptional inhibitory proteins. Of the nine gene products classified as functioning I[kappa]B proteins, five reside mainly in the cytosol, four localize in the nucleus. These nuclear I[kappa]B proteins associate with the NF-[kappa]B inside the nucleus and exert additional regulation over the gene expression profile of NF- [kappa]B. This current study is focused on the nuclear I[kappa]B[zeta] protein. I[kappa]B[zeta] is an NF-[kappa]B -dependent transiently expressed protein that has been linked to the regulation of Csf2, Lcn2, IL-12, IL-18, IL-6, and NGAL genes. In the cases of IL-6 and NGAL, the presence of I[kappa]B[zeta] proved to be indispensable. Mechanistically, I[kappa]B[zeta] preferentially binds to NF-[kappa]B p50̣. This preference is directly contrary to the classical cytoplasmic I[kappa]B proteins that favors p65/RelA subunit. In this study, using X-ray crystallography accompanied by in vitro biochemical assays, we have gained insights into the structure/function relationships of I[kappa]B[zeta] and p50̣. The 2.0 Å crystal structure of I[kappa]B[zeta] in complex with the dimerization domains of the NF-[kappa]B p50 homo-dimer reveals that it adopts a structure and binding interactions that is similar to that of the classical I[kappa]B[alpha] and I[kappa]B[beta] complexes with p65- containing NF-[kappa]B dimers. However, the I[kappa]B[zeta] ankyrin repeats domain contains an amino- terminal helix that caps the top of the ankyrin repeat stack. Circular dichroism (CD) and surface plasmon resonance (SPR) analysis indicate that this capping helix has a stabilizing effect on the folding of the rest of the AR structure. Without it, I[kappa]B[zeta] adopts a disordered conformation, which disrupts its ability to bind to p50. The crystal structure also revealed I[kappa]B[zeta] contains elongated ankyrin repeat 4 that is unique among the I[kappa]B proteins. It is not clear whether or not this plays any role in specific functions of I[kappa]B[zeta]. A close examination of the protein- protein interface showed a cluster of salt bridge contacts focused at the nuclear localization sequence (NLS) region of the p50. GST-pull down and SPR experiments revealed that the removal NLS knocked out the interaction entirely. A closer examination of the region with site directed mutagenesis demonstrated a binding "hot spot" composed of Arg359, Lys360, and Arg361. Simultaneous mutations of these residues to alanine resulted in complete loss of interaction. Furthermore, we established that NLS region contributes majority of protein-protein recognition between the two proteins, so much so that the transfer of the p50 NLS region to p65 is sufficient to cause p65: I[kappa]B[zeta] interaction. In a separate but related project we also solved two structures of p50 homo-dimer bound to IL-6 and NGAL promoters. This allowed us to model I[kappa]B[zeta] onto the p50:DNA structure and give us a theoretical ternary complex structure. In the model the carboxy-terminus of I[kappa]B[zeta] can be seen to collide with the DNA backbone of the promoter. The consequence of this would be collision was examined with SPR and EMSA. Both techniques detected the formation of stable ternary complex on DNA promoters. The result also showed the binding of I[kappa]B[zeta] on p50 does not alter its rate of dissociation by any detectable amount. From these finding we conclude that the stable ankyrin repeat domain of I[kappa]B[zeta] interacts specifically with p50:DNA complexes in the nucleus through a relatively focused portion of the overall interface. This suggests that small molecule that inhibitors this interaction could be developed as potential therapeutics