It has been essential to make this work some crystallographic and RMN structures available in PDB database. Thus, we think it would be appropriate a brief extract of each publication that contained this PDB structures, with the main results involved in the interaction between DNA and our studied proteins.
The publications that have been used are:
Wolfe SA, Zhou P, Dotsch V, Chen L, You A, Ho SN, Crabtree GR, Wagner G, Verdine GL.Unusual Rel-like architecture in the DNA-binding domain of the transcription factor NFATc. Nature. 1997 Jan 9;385(6612):172-6
The transcription factors NFAT and AP-1 coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein-protein interactions with NFAT in the ternary NFAT / AP-1 / DNA complex. NFAT orients the two subunits of AP-1, c-Jun and c-Fos, on DNA through direct protein-protein interactions.
The DNA recognition elements of many transcription factors are disordered in the absence of DNA and undergo an induced folding transition upon interaction with DNA. Such behaviour appears to be especially common among transcription factors that contain predominantly alpha-helical DNA recognition domains, such as members of the bZIP and basic helix-loop-helix families. Even though the core DNA recognition domain of NFATC1 does not comprise predominantly this structure, it undergoes an induced folding transition upon interaction with DNA. Specifically, NMR relaxation measurements on unliganded NFAT-DBD revealed that the Ig domain adopts a well-defined three-dimensional structure, with the exception of the betaA-B and betaG'-H loops, which are disordered. In the binary solution structure, both of these loops become ordered upon interaction with DNA, and indeed, both contribute directly to the DNA-contact interface. Especially striking is the structural transition of the insert region from a completely disordered loop to a compact module.
The NFAT DNA-binding region (residues 399-678) and the AP1 bZIP (basic leucine zipper) elements form a tight complex with each other and with the DNA. The Fos-Jun heterodimer is oriented specifically so that Jun binds to the half of the AP1 site close to NFAT. This tight association between the three proteins on DNA creates a continuous groove for the recognition of 15 base pair. The extended interface between NFAT and AP-1 is facilitated by the bending of Fos and DNA.
Previously, it was seen that the solution structure of NFAT2/DNA revealed a common overall fold with the DNA-binding domain (DBD) of Rel family proteins. The overall fold of the protein is very similar in a liganded or an unliganded state, comprising a ten-stranded antiparallel b-barrel which is structurally related to s-type domains of the Ig superfamily. The two primary sheets (beta-IHFCE and beta-ABG) that form the core of the beta-barrel lie remote from the DNA interface and are almost completely unaffected by being bound to DNA. The third sheet (b-DG') which does not itself contact DNA but adjoins multiple segments than do, is also very similar in free protein and binary complex. Thus, the most radical changes that occur upon binding to DNA involve two large surface loops. The betaa-betaB loop, which is only partially ordered in the unliganded protein, adopts a well-defined conformation in the binary complex. The betaG'-betaH loop, which is devoid of secondary structure in absence of DNA is folded in the binary complex and contains a short alpha-helix.
Chen FE, Huang DB, Chen YQ, Ghosh G.Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA. Nature. 1998 Jan 22;391(6665):410-3.The overall structure of p50/p65 heterodimer is coherent with that of other members of NFkB family. This dimmer is the most abundant of Rel/NFkB family. The observed base-specific contacts support the idea that the Ig-B site consists of a 5-base-pair 5'-GGGAC-3' subsite contacted by p50, and a 4-base-pair 5'-TTCC-3' subsite contacted by p65.
Base-specific binding by p50 to the subsite occurs through the residues explained previously in the web page. Briefly, both monomers make extensive contacts with the ribose phosphate backbone of DNA.
p50/p65 has a particular high DNA affinity compared to most eukaryotic transcription factors. It can not be accounted for the number of direct contacts, because its number is frequent but for other reasons:
Phelps CB, Sengchanthalangsy LL, Malek S, Ghosh G.Mechanism of kappa B DNA binding by Rel/NF-kappa B dimers. J Biol Chem. 2000 Aug 11;275(32):24392-9.
NFkB p50/p65 heterodimer is shown by a combination of gel mobility shift and fluorescence anisotropy assays to bind the kB DNA target site of the immunoglobulin kB enhancer with a highest affinity than the p50 or p65 homodimers. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA target. The heterodimer binds in a pH-dependent manner and it is observed a strong salt-dependent interaction between the subunits. On the other hand, binding to DNA is temperature independent in a range from 4C degrees and 42C. In conclusion, this heterodimer form complexes with DNA in an entropically driven manner.
Stroud JC, Lopez-Rodriguez C, Rao A, Chen L. Structure of a TonEBP-DNA complex reveals DNA encircled by a transcription factor. Nat Struct Biol. 2002 Feb;9(2):90-4.This publication, base of our work, describes NFAT5 as a dimeric protein which belongs to NFAT falily of transcription factors, but shares with NFkB its structure, reminding a butterfly. It binds assymetric sites, so on half site the interacions DNA-protein are stronger than on the other half site.
The crystallography allows us to know the residues involved in DNA recognition and binding and, moreover, the ones that form the C-terminal and N-terminal dimer interface.
For concrete residues, look at the publication and our previous explanations