MAT Homeodomain-DNA Structure

Reference:

Li, 1995. Crystal structure of the MATa1/MATalpha2 homeodomain heterodimer bound to DNA Science 270, 262-269. [Medline abstract]
Click this button to color the structure according to the tutorial below and as for Fig. 1 of the reference.

I. Introduction
The A1/alpha2 - DNA Complex exhibits several properties which are important in DNA protein interactions:

a1 and alpha2 regulate yeast mating type loci:

So MCM1 and a1 can change binding specificity of alpha2.

All are homeodomain proteins- originally discovered in Drosophila homeotic genes, a gene family with a conserved 60 aa sequence near the C-terminus (the homeodomain). Many have been shown to regulate development in various organisms. They belong to the "helix-turn-helix" class of DNA binding proteins and often act in combination with other homodomain proteins. The "helix-turn-helix" proteins have an alpha helix which fits into the major groove of the DNA, backed by 2 alpha helices which lay across the groove.

Both a1 and alpha2 bind DNA by themselves, but with low affinity and specificity. Both binding specificity and affinity are increased 105-fold when they bind as a homeodimer. Binding is dependent on presence of both binding sites at proper spacing.

The sequences above are sites to which the a1/alpha2 heterodimer binds in vivo. The boxed regions are conserved in at least 75% of the yeast binding sites for MATa1/alpha2. Note that the conserved regions are spaced the same distance apart at all sites.

II. The complex
The crystal structure of the a1/alpha2 heterodimer complexed with DNA is shown at left, similar to Fig1a of the reference. To make the coloring match the refernce and the rest of the figures of this tutorial, click this button . The a1 protein is shown in cyan, the alpha2 in red, and the DNA in yellow. In other figures, the DNA will be shown as yellow and magenta strands. Note that the axis of the DNA double helix is bent. This is caused by an increased roll angle of the bases, which in turn cause a narrowing of the minor groove. Click this button to see the distances between phosphates across the minor groove. The distances vary up to 2-fold. A graph of the groove widths and roll angle for each base is shown below.

The helix bends in the direction of the protein to facilitate binding. The combination of an a1 and alpha2 binding site, flanking a region of DNA of the proper length which can bend, leads to the formation of a high affinity complex. The a1 and alpha2 proteins bind to similar sites in the major groove of the DNA. The binding sites are so similar that the authors transposed the figure legends describing the binding sites in figure 6B andD. Red-Blue stereo picture of Fig1.

III. Protein-protein interaction

The a1/alpha2 protein-protein interaction can be seen by clicking here . A hydrophobic patch of the a1 homeodomain interacts with hydrophobic residues of the alpha2 C-terminal tail. Note the way the hyrophobic amino acids interdigitate between the two proteinThis interaction resembles the Oct1/VP16 interaction; these proteins share homology with a1/alpha2 in this region (see alignment above). Mutation of the hydrophobic residues to A results in loss of a1/alpha2 binding activity, based on an in vivo reporter gene assay.  H-bonds, shown as dotted lines here, also contribute to this interaction.

IV. Protein-DNA contacts

A diagram of the DNA-protein contacts is shown below. H-bonds are indicated by arrows, and the proteins are color coded as above. Water molecules involved in H-bonds are shown as blue "w" s. Conserved nucleotides are shown in green.

This button colors the structure as in fig 6B, showing interactions of a1 with the major groove of the DNA. N51 forms 2 H-bonds with A26 of the DNA. This interaction is conserved in all homeodomain proteins, including alpha2 (below). Also conserved, but not shown, is an H-bond between R53 and the phosphate of A4. R55 makes 2 H-bonds with G25, an interaction similar to the N51-A26 interaction. Many other H-bonds are made through ordered water molecules. Since water can act as both an H donor and acceptor, these types of interactions are difficult to predict in the absence of crystallographic data. Red-Blue stereo picture of this view.
This button Shows the interactions of alpha2 with the major groove, as in fig. 6D. N51 of alpha2 makes the conserved interaction with an adenosine residue, in this case A38. R54 makes H-bonds with G6 and to N51 of alpha2 itself. S50 makes 2 water-mediated H-bonds, one each to A4 and T5.
This button shows the interactions alpha2 makes with the minor groove. R4 has several potential H-bond partners. The epsilon N can pair with N3 of A8 or A38. The terminal amine can make water mediated bonds with G6 and C39. It is also close enough to make a direct bond with O2 of C39. The backbone NH of glycine5 donates an H-bond to T37, and R7 donates an H-bond to A35 and T11. Red-Blue stereo picture of this view.

JSTOR link to article.
a1/alpha2 mutagenesis paper (MOLECULAR AND CELLULAR BIOLOGY, Jan. 1999, p. 585Ð593 Vol. 19, No. 1)
Operator sequences and Promoter Assay


DNA mutation figure


DNA binding assay on mutant operators


alpha2 mutant has same binding specificuty as wildtype


a1 mutations affect specificity, alpha2 has effects on residual activity

Table of h3 repression values

 

Science 1999 Jun 11;284(5421):1841-5 Crystal structure of the Zalpha domain of the human editing enzyme ADAR1 bound to left-handed Z-DNA.
PDB Chime link to 1QBJ

Dimer binding figure


DNA contact in major "groove"


Comparison of orientation of HTH motif


Sequence Alignment

Crystal structure of yeast nucleosome core particle

Figure of differences from metazoans

Link to PDB for 1ID3

 

Comments or Suggestions to:Jim Nolan at JNolan@tulane.edu