IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells.

NF-kappaB dimers, inhibitor IkappaB proteins, and NF-kappaB.IkappaB complexes exhibit distinct patterns in partitioning between nuclear and cytoplasmic cellular compartments. IkappaB-dependent modulation of NF-kappaB subcellular localization represents one of the more poorly understood processes in the NF-kappaB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-kappaB regulation exhibited by the inhibitors IkappaBbeta and IkappaBalpha. We show that although both IkappaBalpha and IkappaBbeta bind to NF-kappaB with similar global architecture and stability, significant differences exist that contribute to their unique functional roles. IkappaBbeta derives its high affinity toward NF-kappaB dimers by binding to both NF-kappaB subunit nuclear localization signals. In contrast, IkappaBalpha contacts only one NF-kappaB NLS and employs its carboxyl-terminal proline, glutamic acid, serine, and threonine-rich region for high affinity NF-kappaB binding. We show that the presence of one free NLS in the NF-kappaB.IkappaBalpha complex renders it a dynamic nucleocytoplasmic complex, whereas NF-kappaB.IkappaBbeta complexes are localized to the cytoplasm of resting cells.

Preparation and crystallization of dynamic NF-kappa B.Ikappa B complexes.

The formation of single, well-diffracting crystals is a requirement for any molecular structure determination by x-ray crystallography. Crystallization of biological macromolecules can represent a significant obstacle when the subject exhibits internal flexibility or indiscriminate self-association. In such cases, the removal of inherently flexible regions and the addition of stabilizing ligands can improve the probability of crystal formation and ordered growth. We have applied these principles in order to form crystals of the Rel homology region of transcription factor NF-kappaB in complex with its inhibitors IkappaBalpha and IkappaBbeta. None of these molecules crystallizes in the absence of a binding partner. Recombinant overexpression of truncated IkappaBalpha required selection of the correct start site. NF-kappaB.IkappaBalpha complex crystals formed under relatively stringent conditions. NF-kappaB. IkappaBbeta complex crystals were formed by analogy to NF-kappaB. IkappaBalpha, although some modifications in purification and complex formation were necessary due to differences between the inhibitors.

Mechanism of I kappa B alpha binding to NF-kappa B dimers.

X-ray crystal structures of the NF-kappa B.I kappa B alpha complex revealed an extensive and complex protein-protein interface involving independent structural elements present in both I kappa B alpha and NF-kappa B. In this study, we employ a gel electrophoretic mobility shift assay to assess and quantitate the relative contributions of the observed interactions toward overall complex binding affinity. I kappa B alpha preferentially binds to the p50/p65 heterodimer and p65 homodimer, with binding to p50 homodimer being significantly weaker. Our results indicate that the nuclear localization sequence and the region C-terminal to it of the NF-kappa B p65 subunit is a major contributor to NF-kappa B. I kappa B alpha complex formation. Additionally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and I kappa B alpha. A second set of interactions involving the acidic C-terminal/PEST-like region of I kappa B alpha and the NF-kappa B p65 subunit N-terminal domain also contributes binding energy toward formation of the complex. This interaction is highly dynamic and nonspecific in nature, as shown by oxidative cysteine cross-linking. Phosphorylation of the C-terminal/PEST-like region by casein kinase II further enhances binding.

The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation.

IkappaBalpha regulates the transcription factor NF-kappaB through the formation of stable IkappaBalpha/NF-kappaB complexes. Prior to induction, IkappaBalpha retains NF-kappaB in the cytoplasm until the NF-kappaB activation signal is received. After activation, NF-kappaB is removed from gene promoters through association with nuclear IkappaBalpha, restoring the preinduction state. The 2.3 A crystal structure of IkappaBalpha in complex with the NF-kappaB p50/p65 heterodimer reveals mechanisms of these inhibitory activities. The presence of IkappaBalpha allows large en bloc movement of the NF-kappaB p65 subunit amino-terminal domain. This conformational change induces allosteric inhibition of NF-kappaB DNA binding. Amino acid residues immediately preceding the nuclear localization signals of both NF-kappaB p50 and p65 subunits are tethered to the IkappaBalpha amino-terminal ankyrin repeats, impeding NF-kappaB from nuclear import machinery recognition.

Ikappa Balpha functions through direct contacts with the nuclear localization signals and the DNA binding sequences of NF-kappaB.

We have determined the binding energies of complexes formed between Ikappa Balpha and the wild type and mutational variants of three different Rel/NF-kappaB dimers, namely, the p50/p65 heterodimer and homodimers of p50 and p65. We show that although a common mode of interaction exists between the Rel/NF-kappaB dimers and Ikappa Balpha, IkappaB alpha binds the NF-kappaB p50/p65 heterodimer with 60- and 27-fold higher affinity than the p50 and p65 homodimers, respectively. Each of the three flexibly linked segments of the rel homology region of Rel/NF-kappaB proteins (the nuclear localization sequence, the dimerization domain, and the amino-terminal DNA binding domain) is directly engaged in forming the protein/protein interface with the ankyrin repeats and the carboxyl-terminal acidic tail/PEST sequence of Ikappa Balpha. In the cell, Ikappa Balpha functions to retain NF-kappaB in the cytoplasm and inhibit its DNA binding activity. These properties are a result of the direct involvement of the nuclear localization sequences and of the DNA binding region of NF-kappaB in complex with Ikappa Balpha. A model of the interactions in the complex is proposed based on our observations and the crystal structures of Rel/NF-kappaB dimers and the ankyrin domains of related proteins.

The role of DNA in the mechanism of NFkappaB dimer formation: crystal structures of the dimerization domains of the p50 and p65 subunits.

BACKGROUND: Members of the rel/NFkappaB family of transcription factors play a vital role in the regulation of rapid cellular responses, such as those required to fight infection or react to cellular stress. Members of this family of proteins form homo- and heterodimers with differing affinities for dimerization. They share a structural motif known as the rel homology region (RHR), the C-terminal one third of which mediates protein dimerization. Crystal structures of the rel/NFkappaB family members p50 and p65 in their DNA-bound homodimeric form have been solved. These structures showed that the residues from the dimerization domains of both p50 and p65 participate in DNA binding and that the DNA-protein and protein dimerization surfaces form one continuous overlapping interface. We desired to investigate the contribution of DNA to NFkappaB dimerization and to identify the mechanism for the selective association of rel/NFkappaB family peptides into transcriptionally active dimers. RESULTS: We report here the crystal structures of the dimerization domains of murine p50 and p65 at 2.2 A and 2.0 A resolution, respectively. A comparison of these two structures suggests that conservative amino acid changes at three positions are responsible for the differences in their dimer interfaces. The presence of the target DNA does not change the dimer interface of either protein in any significant manner. CONCLUSIONS: These two structures suggest that the rel/NFkappaB family of transcription factors use only a few conservative changes in their amino acid sequences to form a host of dimers with varying affinities for dimerization. Amino acids at positions corresponding to 254, 267, and 307 of murine p50, function as primary determinants for the observed differences in dimerization affinity. The DNA-contacting charged amino acid sidechains from the dimerization domains are held in a similar conformation in both the DNA-bound and free states, therefore, no major structural rearrangement is required to bring these residues into contact with the DNA.