CHEM 4343


DNA-Binding Domains

Proteins can either bind nonspecifically or specifically to DNA. Regulatory proteins generally bind to specific DNA sequences. This requires the protein to have Two structural motifs that play a major role in protein-binding to DNA are the leucine zipper and the zinc finger
Two examples of specific recognition are shown below .

In this example Glutamine or Asparagine form hydrogen bonds with the N6 and N7 nitrogens of adenine, allowing for specific recognition of the T=A base pair

Likewise Arginine can form two specific hydrogen bonds with the N7 and O6 locations of guanine, thus allowing for the recognition of CG base pairs.
Other interactions, such as "protein pockets" that allow for the insertion of the thymine CH3 group, can also be used to achieve sequence specificity.

Nonspecific DNA binding generally involves simple attachment through either of the DNA grooves.

The Leucine Zipper

This motif consists of a dimer of two alpha-helices associated through their hydrophobic surfaces. Leucine residues typically appear on the hydrophobic side of the helix every 7-10 amino acids, hence the name. the DNA-binding domain often contains a high concentration of basic (Lys/Arg) residues.

The yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif. The x-ray crystal structure of a peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4 has been determined at 1.8 angstrom resolution. The peptide forms a parallel, two-stranded coiled coil of alpha helices packed as in the "knobs-into-holes" model proposed by Crick in 1953. Contacts between the helices include ion pairs and an extensive hydrophobic interface that contains a distinctive hydrogen bond. The conserved leucines, like the residues in the alternate hydrophobic repeat, make side-to-side interactions (as in a handshake) in every other layer of the dimer interface. The crystal structure of the GCN4 leucine zipper suggests a key role for the leucine repeat, but also shows how other features of the coiled coil contribute to dimer formation.

The bZIP dimer is a pair of continuous alpha helices that form a parallel coiled coil over their carboxy-terminal 30 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA-binding site.

The coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T. There are no kinks or sharp bends in either bZIP monomer. Numerous contacts to DNA bases and phosphate oxygens are made by basic region residues that are conserved in the bZIP protein family. The details of the bZIP dimer interaction with DNA can explain recognition of the AP-1 site by the GCN4 protein.

Reference

1. Ellenberger TE, Brandl CJ, Struhl K, Harrison SC ,Cell 1992 Dec 24;71(7):1223-37
2. O'Shea EK, Klemm JD, Kim PS, Alber T Science 1991 Oct 25;254(5031):539-44

The Zinc Finger

The zinc finger DNA-binding motif occurs in many proteins that regulate eukaryotic gene expression. The "finger" consists of about 30 amino acids; four of the residues, either 4 Cys or 2 Cys+ 2 His, coordinating a single Zn2+ ion. An example, found in the Zif268 protein, of a Zn2+ ion tetrahedrally coordinated to 2 Cys and 2 His residues is shown below:

Several fingers may be found in a single protein, the highest number found to date being 37. Zinc fingers may contain residues involved in sequence discimination, or may bind the DNA non-specifically

The crystal structure of a complex containing the three zinc fingers from Zif268 (a mouse immediate early protein) and a consensus DNA-binding site has been determined at 2.1 angstroms resolution and refined to a crystallographic R factor of 18.2 percent.

In this complex, the zinc fingers bind in the major groove of B-DNA and wrap part way around the double helix. Each finger has a similar relation to the DNA and makes its primary contacts in a three-base pair subsite.

Residues from the amino-terminal portion of an alpha helix contact the bases, and most of the contracts are made with the guanine-rich strand of the DNA. This structure provides a framework for understanding how zinc fingers recognize DNA and suggests that this motif may provide a useful basis for the design of novel DNA-binding proteins.

Reference

Pavletich NP, Pabo CO,Science 1991 May 10;252(5007):809-17

The protein shown below is a specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4.

. The protein binds as a dimer to a symmetrical 17-base-pair sequence. A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. A short coiled-coil dimerization element imposes 2-fold symmetry.

A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.

Reference

Marmorstein R, Carey M, Ptashne M, Harrison SC, Nature 1992 Apr 2;356(6368):408-14

Acknowledgements

The images for the binding motifs were abtained by searching through the Protein Bank Database. The DNA-protein complexes were obtained from Image Library of Biological Macromolecules in Jena, Germany.