Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Oct 26;373(3):723-34.
doi: 10.1016/j.jmb.2007.08.039. Epub 2007 Aug 22.

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

Anu K Moorthy  1 , De-Bin Huang, Vivien Ya-Fan Wang, Don Vu, Gourisankar Ghosh
Affiliations

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

Anu K Moorthy et al. J Mol Biol. .

Abstract

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Primary and secondary structures of NF-κB and κB DNA. (a) A schematic representation of the domain architectures of three of the NF-κB family members. (b) Primary sequences alignment of the RHR. The secondary structures are mapped onto the primary sequence. Subunit and DNA contacting amino acids are denoted by red (base-specific) and blue (backbone) squares. The crystal contact residues are colored. Similar color denotes interaction pairs from two dimers. (c) κB sequence used for crystallization.
Figure 2
Figure 2
Overall structure and the subunit interface of the p50/RelB. (a) The ribbon representation of the complex viewed along the long DNA axis. Throughout the paper the same color code will be maintained for the p50 (green) and RelB (red) subunits. (b) Hydrogen bonding network at the subunit interface. (c) Detailed view of the core of the subunit interface.
Figure 3
Figure 3
Protein–DNA contacts. (a) Schematic representation of base-specific and non-specific contacts between the heterodimer and κB DNA. (b) Detailed sequence specific interactions between amino acids from p50 and RelB (in (c)). The DNA non-contacting residues in this complex are highlighted in grey color. The homologous residues make contact in other complexes.
Figure 4
Figure 4
Comparison of the loop 3 conformations. (a) Overlay of the dimerization domains of RelB and p50 reveals difference in their loop 3 conformations. The RelB loop 3 is more helical, which allows more intra-subunit hydrogen bonds between side-chains. (b) A proline at the center of loop 3 breaks the helical structure in p50. (c) Helical conformation in RelB is stabilized by main-chain hydrogen bonds.
Figure 5
Figure 5
Crystal packing contacts between multiple complexes. (a) Overall view of the packing between RelB and p50 subunits from two different complexes translated one unit cell along the b-axis. (b) Interaction between the two complexes related by a 2-fold axis. A close up view of the same contact surface is shown in two parts. (c) Overall view of the packing interactions along the 2-fold axis. The dimerization domains are shown in grey. (d) Close up view of the same contact surface.
Figure 6
Figure 6
EMSA analysis using human immunodeficiency virus-long terminal repeat (HIV-LTR) as probes. (a) HIV-LTR DNA probes sequences. (b) EMSA analysis of recombinant protein using HIV-LTR probes with different spacing as indicated. Single and double arrowheads denote binary and ternary complexes, respectively. (c)–(e) a EMSA analysis of cell lysates using HIV2HIV, HIV4HIV, and HIVMut probes, respectively. For supershift analysis, RelB antibody from Snata Cruz Biotechnology (sc-226) against the RelB C terminus peptide was used. (f) Protein expression check visualized by Western blotting with Flag antibody.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Baldwin AS., Jr The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol. 1996;14:649–683. - PubMed
    1. Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol. 1998;16:225–260. - PubMed
    1. Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell. 2002;109:S81–S96. - PubMed
    1. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol. 2000;18:621–663. - PubMed
    1. Leung TH, Hoffmann A, Baltimore D. One nucleotide in a kappaB site can determine cofactor specificity for NF-kappaB dimers. Cell. 2004;118:453–464. - PubMed

Publication types

MeSH terms

Associated data