NF-kB p52 homodimer-DNA complex

Kyle Packer '08 and Josh Mitchell '08

 

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Contents:

I. Introduction

The nuclear factor-kappa B (NF-kB) p52 protein is a eukaryotic transcription factor and belongs to the NF-kB/Rel family of proteins. This family of structurally-related proteins is involved with the transcription of several genes, including those encoding pro-inflammatory cytokines, interferones, major histocompatability complex proteins, growth factors, and cell adhesion molecules, as well as viruses like HIV and herpes. This family of transcription factors are also active in a number of disease states, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease. The NF-kB p52 homodimer is believed to act as a transcriptional repressor by competitively binding DNA with various co-transcription factors. The broad array of genes that NF-kB p52 represses suggests combinatorial control with other transcription factors. In fact, NF-kB p52 can form as a heterodimer with other members of the NF-kB/Rel family, as well as forming a homodimer. The NF-kB p52 homodimer commonly binds two distinct DNA sites, the MHC H-2 site (5'-GGGGGATTCC CC-3') and the Ig/HIV site (5'-GGGGACTT TCC-3'). The NF-kB p52 homodimer can also interact with other proteins of the IkB family, specifically Bcl-3. This interaction creates a ternary complex with DNA and allows for transcriptional activation.

II. General Structure

NF-kB p52 is an asymmetric dimer that wraps around the DNA duplex giving the appearance of a butterfly. Each monomer has three domains, the C-terminal domain, N-terminal domain, and the insert region. <> There is a short linker region between the N-terminal domain and the insert region which makes important DNA contacts. The two monomers, despite their symmetrical appearance, are slightly different due to reorientation of the N-terminal domain of monomer II. The slight rearrangement of the N-terminal domain causes DNA bending. Binding of NF-kB p52 unwinds DNA slightly, with an overall twist of 10.7 bp/turn. The overall DNA has a 20° bend toward the C-terminal domain of monomer II. <> Also, some DNA bases have large propeller twists which allow for unusual hydrogen bonding between bases. These bonds stabilize the non-symmetric DNA distortions and decrease the flexibility of the DNA duplex.

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III. Insert Region

The insert region consists of two alpha helices packed tightly at an angle of 50° and connected by a short loop. The recognition helix (alpha A) points with its N terminal end towards the phosphate backbone through the minor groove. < > The p52 recognition helix is connected to the N-terminal domain by polar contacts of only one amino acid, Lys-153. < > The insert region is positioned away from the core domain surface of the protein and thus it may interact with other DNA binding proteins in a combinatorial manner to control gene expression. Helix alpha B is easily accessible to factors bound to a neighoboring DNA site. < > In addition, the partially charged side chains of Gln-176, Lys-179, Glu-180, and Lys-183 can interact with other proteins potentially bound to nearby DNA sites. < > NF-kB p52 can also interact with proteins containing a basic leucine zipper region through the C-terminal domain.

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IV. Dimer Interface

The dimer interface between the two monomers occurs within the C-terminal domain and is formed by hydrophobic interactions and intermolecular hydrogen bonds. The important inter-dimer hydrogen bonds are made by residues Arg-232, Glu-245, Cys-250, Asp-251, Asp-280, and His-282. < > In, addition inter-dimer water mediated hydrogen bonds are made by residues Ser-231, Asp-234, and Arg-290 at the edge of the dimer. < > The most important hydrophobic interactions in the dimer interface occur with the Tyr-285 residue, which packs tightly between adjacent aliphatic side chains. < > This residue is crucial to NF -kB p52 dimerization specificity by ensuring proper homodimer formation.

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V. DNA Binding

Hydrogen bonding to both DNA bases and the backbone is the basis for DNA recognition. However there are more hydrogen bonds to the backbone of DNA rather than to the DNA bases. NF-kB p52 binding is supposedly sequence specific, but with the majority of DNA contacts being made with the backbone, there must be another way of creating sequence specificity. NF-kB p52 only directly recoginzes four guanine nucleotides with side chains from His-62, Arg-54, Arg-52, and Lys-221. <> (Both monomers make four base contacts, although only one monomer is shown here). His-62, Arg-54, Arg-52 are inserted into the major groove and form a strong base stacking interaction. Additionally, Glu-58 binds to the opposing cytosine creating more base interactions which supports the two argenines. <>

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VI. The Role of Water and Sequence Specifity

Much of DNA backbone recognition is water-mediated, as opposed to base recognition, which is caused by direct hydrogen bonding. 20 water molecules are located in the protein-DNA interface. 14 of these engage in polar interactions between amino acids and the DNA, the other 6 contact either polar amino acid atoms or polar DNA atoms and stabilize the nearby waters interacting in the protein-DNA interface.<> The unique way in which water-mediated bonds form suggest that many of the water-mediated interactions are specific between the two monomers. The NF-kB p52:DNA interface also forms a large water cavity above the major groove in the central part of the DNA. Water molecules form an extensive network of interwoven hydrogen bonds between the DNA base pairs, the linker, and the dimer interface. <> Such unique and extensive water interactions imply that water-mediated hydrogen bonds between NF-kB p52 and DNA may contribute to the specificity of DNA binding. Thus both direct DNA base contacts and the unique water network interactions contribute to NF-kB p52 binding in a sequence specific manner. Tyr-285, seen in the dimer interface above, is the core component of the C-terminal domain water-mediated DNA recognition loop. The important role of Tyr-285 in both dimerization and the water-mediated complex strengthens the idea that the dimer and protein:DNA interfaces are one continuous recognition surface.<>

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VII. References

Gregory L., and Muller, Christoph W., 1997. Structure of the human NF-kB p5Cramer, Patrick, Larson, Christopher J., Verdine, 2 homodimer-DNA complex at 2.1 A resolution. The EMBO Journal 16: 7078-7090.

Baeuerle, P.A. and Henkel,T 1994. Function and activation of NF-kB in the immune system. Annual Review Immunology 12: 141-179.

Gilmore, Thomas. The Rel/NF-kappaB Signal Transduction Pathway. http://people.bu.edu/gilmore/nf-kb/

Thanos, D. and Maniatis, T., 1995. NF-kB: a lesson in family values. Cell 80: 529-532.

Baeuerle, P.A. and Baltimore, D., 1996. NF-kB: ten years after. Cell 87: 13-20.

Baldwin, A.,1996. The NF-kB and IkB proteins: new discoveries and insights. Annual Review Immunology 14: 649-681.

Chytil, M and Verdine, G.L., 1996. The Rel family of eukaryotic transcription factors. Current Opinions in Structural Biology 6: 91-100.


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