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Copyright © 2005 by Eric Martz,
University of Massachusetts, Amherst MA USA.

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The DNA-Binding Domain of Gal4.
A transcriptional regulator from yeast.

The yeast Gal4 protein regulates the transcription of b-galactosidase. Native Gal4 has 881 amino acids, but the DNA-binding domain crystallized for this study had only 64 amino acids (only 57 of which were resolved).

This crystal structure was determined to a resolution of 2.7 Å in 1992 by Ronen Marmorstein and Steven C. Harrision (Nature 356:408).


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Protein, DNA, Water
Hydration of the Gal4:DNA crystal.

51 water molecules were tightly bound to the surfaces of the protein and DNA. These are only about 10% of the water present in the crystal when X-ray diffraction was performed. The high level of hydration of protein crystals may explain the good agreement between crystallographic structures and NMR structures determined in aqueous solution.
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The Gal4 DNA-Binding Domain is Homo-Dimeric.

Two identical Gal4 protein chains of equal lengths (57 amino acids), shown as green and blue backbone traces, are seen binding to the DNA double helix. The chains are folded identically and related by rotational symmetry.
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Charge supports DNA binding.
Hydrophobic
Cationic +/Basic
Anionic -/Acidic
Polar Uncharged
Backbone


The net charge of the DNA-binding surface is positive, supporting its binding to the negative surface of the DNA.
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Secondary Structure:
Alpha Helices, Coils, DNA.

The Gal4 DNA-binding domains have paired alpha helices pointing "up", away from the DNA. There are no beta strands.
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Hydrophobic and Polar amino acids, DNA.

The two alpha helices (pointing away from the DNA, which is shown as dots) contact each other forming a small domain with a hydrophobic core.

The front half of the bottom model is cut away to show the uniformly hydrophobic contact strip between the two helices.
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Zinc-containing protein domains sit in the major groove of DNA.

The paired alpha helices sit over the minor groove.

(Zinc was replaced with cadmium in the protein crystallized for the 1d66 experiment.)

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In this view, one of the pairs of Zinc ions is centered (for rotation and zoom).

To zoom, hold down the Shift key while dragging up or down.
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Six cysteine Sulfur atoms cage the Zinc ion pair.

(If you zoom in too much, rotation will push this view off-center. Zoom out to avoid this. To zoom, hold down the Shift key while dragging up or down.)

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The two Gal-4 protein chains, shown here as molecular surfaces, wrap around the DNA double helix.


A green "finger" can be seen protruding into the major groove, against specific bases.


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The DNA double helix consists of two antiparallel strands, held together by Watson-Crick hydrogen bonds between paired bases.


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The sequence of the top DNA strand (left to right) is identical to the sequence of the bottom strand (right to left), hence palindromic:


CCGGA GGACA GTCCT CCGG
GGCCT CCTGA CAGGA GGCC

With the correct spacing, this allows each end of the protein dimer to bind to the same sequence.

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Contacts:
Protein Chain A contacts protein chain B, and both strands of DNA, chain D and chain E.

The Zinc-finger domain of Gal4 recognizes a specific sequence of bases in the major groove of the DNA.

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Contacts:

Non-Covalent Bonds hold the protein to the DNA. Hydrogen bonds are particularly important in protein recognition of the specific base sequence.


More about hydrogen bonds.
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The surface of Chain A is colored by distances from the other chains. Gray to White is close enough for van der Waals interactions, and magenta is close enough for hydrogen bonds.


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The three bases that are hydrogen bonded to the "finger" of chain A are CGG, the published recognition sequence for Gal4.

This view can be displayed in Protein Explorer's QuickViews with these two clicks on the menus:
  • SELECT Chain A
  • DISPLAY Contacts

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The protein-DNA hydrogen bonds can be examined in detail crossing the transparent protein surface.


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For example, an oxygen in Guanosine 4 is 2.66 Ångstroms from the side-chain nitrogen of Lysine 18, an optimal hydrogen-bonding distance.


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