NF-kappaB p52:RelB heterodimer recognizes two classes of kappaB sites with two distinct modes

Abstract

The X-ray structure of the nuclear factor-kappaB (NF-kappaB) p52:RelB:kappaB DNA complex reveals a new recognition feature not previously seen in other NF-kappaB:kappaB DNA complexes. Arg 125 of RelB is in contact with an additional DNA base pair. Surprisingly, the p52:RelB R125A mutant heterodimer shows defects both in DNA binding and in transcriptional activity only to a subclass of kappaB sites. We found that the Arg 125-sensitive kappaB sites contain more contiguous and centrally located A:T base pairs than do the insensitive sites. A protein-induced kink observed in this complex, which used an AT-rich kappaB site, might allow the DNA contact by Arg 125; such a kink might not be possible in complexes with non-AT-rich kappaB sites. Furthermore, we show that the p52:RelB heterodimer binds to a broader spectrum of kappaB sites when compared with the p50:RelA heterodimer. We suggest that the p52:RelB heterodimer is more adaptable to complement sequence and structural variations in kappaB sites when compared with other NF-kappaB dimers.

Figure 1

The structures of the p52:RelB:κB DNA complex and free DNA. (A) Overall structure of the complex. (Left) Ribbon drawing of the entire complex viewed down the DNA helical axis. The RelB and p52 subunits are shown in purple and blue, respectively; the three DNA strands in the asymmetric unit are shown in yellow, green and grey. (Right) View of the complex after rotating 90° along the vertical axis. (B) Schematic representation of the DNA packing as observed in the structure. The DNA duplex shown in grey is unbound. The crystallographic twofold axis and non-crystallographic twofold axis are indicated by an oval and an arrow, respectively. The RelB subunit binds base pairs at positions +1 to +5, whereas the p52 subunit contacts base pairs at positions −1 to −4. The central base pair ‘0' is at the pseudo twofold axis. The nucleotides out of the continuous DNA helix are written above and below the duplex sequence. The yellow and green colours represent the DNA sequence bound by the p52:RelB heterodimer. The underlined sequences represent symmetry-related DNA. (C) Comparison of the bound (green) and unbound (grey) DNA structures overlaid around the central seven base pairs; the arrow indicates the kinked area.

Figure 2

Detailed contacts between the protein and DNA. (A) Schematic representation of the DNA contacts made by the p52:RelB heterodimer (p52, blue; RelB, purple). Arrows indicate hydrogen bonds; orange circles indicate van der Waals contacts. (B) Hydrogen bonds (red dotted lines) between the complementary functional groups of R54, R52, H62, S61, E58 and K221 of the p52 subunit and the DNA bases are shown; blue and red colours indicate the basic and acidic groups, respectively. (C) Hydrogen bonds between the functional groups in R117, R119, R125 and E123 of the RelB subunit (purple) and DNA bases and a phosphate are shown as red dotted lines. R125 and K274 are shown in grey. (D) Orientations of R125 of RelB in the current complex are compared with the same in the p50:RelB (Moorthy et al, 2007), and with the homologous arginines in RelA and c-Rel in DNA-bound complexes (Huang et al, 2001; Chen-Park et al, 2002). (E) Hydrogen bonds (dotted lines) between RelB side chains (C122, Y120, R209, K210, Q307, K308, R333 and Q334) and the DNA backbone are shown. Figures are generated using PyMOL.

Figure 3

RelB Arg 125 of the p52:RelB complex binds to and promotes transcriptional activation from a subset of promoters. (A) Binding isotherms of ELC-κB (left), BLC-κB (middle) and IFNβ-κB (right) by wild-type and mutant p52:RelB complex using fluorescence-based polarization assay. (B) Reporter assay showing the effect of Arg 125 mutation on luciferase activity. All reporter constructs contained 2 × κB sites in the promoter except MCP-1, which contained a single κB site. (C) EMSA assay using ELC-κB, HIV-κB, cyclin D1-κB, IP-10(P)-κB probes and whole-cell lysates of HEK 293T cells co-transfected with full-length p52 and RelB. ‘NC' and ‘RelB' lanes indicate EMSA using cell extracts prepared from ‘empty vector' and ‘RelB only' transfected cells, respectively. Please note that RelB does not bind to DNA. Specificity of the p52:RelB:DNA complex is indicated by competition (50-fold excess cold κB DNA) and supershift (α-RelB antibody; sc-226 from Santa Cruz Biotechnology, Santa Cruz, CA, USA) experiments. (D) Sequences of two classes of κB sites: the top three (low A:T) and bottom five (high A:T) sequences are insensitive and sensitive, respectively, to R125 interaction. BLC, B lymphocyte chemokine; ELC, Epstein–Barr virus-induced molecule-1-ligand chemokine; EMSA, electrophoretic mobility shift assay; E Sel, E-selectin; HEK, human embryonic kidney; HIV, human immunodeficiency virus; IFN, interferon; IP-10, inducible protein-10; MCP-1, monocyte chemotactic protein 1; wt, wild type.

Figure 4

Binding affinities of NF-κB family members to κB sites. (A) Binding affinities (expressed as equilibrium dissociation constants) are plotted for each κB DNA and NF-κB dimers. (B) Sequences of SDF1, IL-2 CD28RE and IL-8 κB sites. (C) A model of two modes of DNA recognition by the p52:RelB heterodimer. Arg 125 switches its conformation depending on the DNA sequence/conformation. BLC, B lymphocyte chemokine; ELC, Epstein–Barr virus-induced molecule-1-ligand chemokine; HIV, human immunodeficiency virus; IFN, interferon; IL-2, interleukin 2; NF-κB, nuclear factor-κB; SDF1, stromal cell-derived factor 1.