Classification of DNA-BPs

Terms of the regulatory proteins, DNA-BPs can be divided into two parts, one is a DNA-binding domain common motif that can recognize the specific DNA sequences, including helix-turn-helix, zinc finger, and homeodomain, the other for the protein-protein interaction domain involve in leucine zipper and helix-loop-helix.

2.1.1 Helix-Turn-Helix

Helix-turn-helix DNA-binding motif consist of two alpha-helices and a loop (Fig.4), it uses the alpha-helices fits into the major groove and makes bases pairing of the double helix DNA , these motifs usually bind as dimers to inverted repeats in the DNA(Fig. 5).

Fig. 4 Helix-turn-helix

5

Fig. 5 (A) Helix-turn-helix protein is a dimer, it binding of Helix-Turn-Helix Motif to DNA. (B) The pairs of α-helices bind into two neighboring major grooves in the DNA.

This motif is also the key to the interaction of many prokaryotic regulatory proteins and some similar motifs occur in eukaryotic regulatory proteins with DNA. The helix-turn-helix motif comprises about 20 amino acids in two short alpha-helical segments, each seven to nine amino acid residues long, separated by a beta turn (Fig. 6).

Fig. 6 Helix-turn-helix (A) DNA-binding domain of Lac repressor (PDB ID 1LCC) (B) entire Lac repressor (PDB ID 1LBG)

Helix-Turn-Helix motif

(A) (B)

(A) (B)

DNA-binding domain

6

One of the two alpha-helical segments is called the recognition helix in this DNA-binding motif, because it generally involve in many of the amino acids that interact with the DNA in a specific sequence way. This alpha-helix is stacked on other segments of the protein structure so that it protrudes from the protein surface. When bound to DNA, the recognition helix is positioned in or nearly in the major groove. The Lac repressor has this DNA-binding motif. [13, 14]

2.1.2 Zinc Finger

Zinc finger about 25-30 amino acid residues form an elongated loop held together at the base by a central zinc atom (Fig. 7), the Zn is bound to two cysteines and two histidines (or four cysteines), the far end of the a-helix protrudes into the major groove of the DNA. The zinc does not itself interact with DNA; so, the coordination of zinc with the amino acid residues stabilizes this small structural motif. Several hydrophobic side chains in the core of the structure also lend stability. It shows the interaction between DNA and three zinc fingers of a single polypeptide from the regulatory protein Zif268 (Fig. 8). To this day, about thousand of zinc finger proteins are known, and many of them have multiple fingers.

Fig. 7 Zinc Finger DNA-Binding Protein a central zinc atom is bound to the sulfurs of cysteine (C) and the nitrogens of histidine (H). Chains of amino acids of varying lengths (x = chain length) extend from these binding regions. The zinc finger forms a component of a much larger protein and binds the protein to DNA.

Fig. 8 Zinc Fingers. Zinc fingers (purple) interaction with DNA (PDB ID 1A1L)

Zinc Finger

7

Each zinc finger unit usually recognizes three bases (lesser four or five) in the DNA. The sequence specificity of each zinc finger depends on the amino acid sequence of the polypeptide chain between the His and Cys residues that bind the zinc. Amino acids in this region make hydrogen bonds with bases in the DNA.[13, 14]

2.1.3 Homeodomain

This DNA-binding domain has been identified in a lot of proteins that function as transcriptional regulators, especially during eukaryotic development. This domain of 60 amino acids—called the homeodomain, because it was discovered in homeotic genes (genes that regulate the development of body patterns)—is highly conserved and has now been identified in proteins from a wide variety of organisms (Fig. 9). The DNA-binding segment of the domain is related to the helix-turn-helix motif. The DNA sequence that encodes this domain is known as the homeobox.[13, 14]

Fig. 9 Homeodomain. This picture is a homeodomain bound to DNA, this is a protein Ultrabithorax (PDB ID 1B81)

2.1.4 Leucine Zipper

The leucine zipper is found in many eukaryotic transcription factors, like the Fos, Jun and Myc proteins that are involved in control of cell division and carcinogenesis. This motif is an amphipathic alpha-helix with a series of hydrophobic amino acid residues concentrated on one side (Fig. 10), with the hydrophobic surface forming the area of contact between the two polypeptides of a dimer, and leucine zipper motif include α-helix with leucine residues every seventh amino acid, forming a straight line along the hydrophobic surface.

Although researchers initially thought the Leu residues interdigitated (hence the name

8

“zipper”), we now know that they line up side by side as the interacting α-helices coil around each other(forming a coiled coil; Fig. 11).Furthermore, the amino acids halfway between the leucines are usually hydrophobic. Because there are 3.6 amino acids per turn, these hydrophobic residues form a strip down the side of the α-helix (Fig. 12).

Fig. 10 Leucine zippers.The Leu (L) residues at every seventh position in the zipper region, and the number of Lys(K) and Arg (R) residues in the DNA-binding region.

Fig. 11 Leucine zipper from the GCN4 (PDB ID 1YSA). Only the “zippered” α-helices (purple) derived from different subunits of the dimeric protein. The two helices wrap around each other in a gently coiled coil. The interacting Leu residues are shown in red line.

9

Fig. 12 Leucine Zipper Protein Binding DNA (A) The leucine zipper consists of two a-helixes that have

hydrophobic zones and basic ends. (B) The helixes of the leucine zipper binds to each other by their hydrophobic regions and to DNA by their basic regions.

Regulatory proteins with leucine zippers often have a separate DNA-binding domain with a high concentration of basic (Lys or Arg with positively charged) residues because it can interact with the negatively charged phosphates of the DNA backbone. Leucine zippers have been found in many eukaryotic and a few prokaryotic proteins.[13, 14]

2.1.5 Basic Helix-Loop-Helix

These proteins share a conserved region of about 50 amino acid residues important in both DNA binding and protein dimerization occurs in some eukaryotic regulatory proteins.

This region can form two short amphipathic alpha-helices linked by a loop of variable length, the helix-loop-helix (distinct from the helix-turn-helix motif associated with DNA binding).

The helix-loop-helix motifs of two polypeptides interact to form dimers (Fig. 13). In these proteins, DNA binding is mediated by an adjacent short amino acid sequence rich in basic residues, similar to the separate DNA-binding region in proteins containing leucine zippers.[13, 14]

Fig. 13 Helix-loop-helix. The protein is dimeric

(A) (B)

10

(purple); The DNA-binding segment (red circle) merges with the first helix of the helix-loop-helix (blue circle).

The second helix merges with the carboxyl-terminal end of the subunit (green circle) (PDB ID 1HLO).