Opening Image: A ternary complex containing DNA and two subunits of the GA-binding protein (GABP). The α subunit contains a DNA-binding domain that is a member of the ETS family of transcription factors, whereas the β subunit is bound to the Ets-1 domain via an extensive protein-protein interface.

Note that the β subunit does not make contact with the DNA. However, the Ets-1 domain of the α subunit cannot bind DNA in the absence of a specific protein partner, in this case the β subunit of the GA-binding protein. Binding of a specific partner to the Ets domain induces a conformational change in the Ets domain which allows it to bind to its GGAA recognition motif at the promoters of the target genes.

Protein-DNA Interactions

Recognition of DNA by proteins involves specific interactions of these proteins with specific base sequences. As in protein-protein interactions, protein-nucleic acid interactions are mediated by the complex interplay of noncovalent interactions. These include hydrogen bonds, electrostatic and van der Waals interactions, as well as cation-π and π-π interactions.

A cation-π interaction at a protein-DNA interface involves a side chain of the protein carrying a positive charge, such as Arg or Lys, and the π system of the DNA bases. These specific interactions have been found in many protein-DNA structures from the Protein Data Bank.

Winged-helix Protein-DNA Interfaces

In this tutorial we'll explore cation-π interactions at the protein-DNA interface of two exemplary eukaryotic transcription factors, SAP-1 and Ets-1. Both belong to the winged-helix family of transcription factors, of which there are over 300 members in the human genome. Winged-helix proteins recognize specific base sequences by inserting an α-helix into the major groove of the DNA. Several side chains of the recognition helix interact with four to six bases. There does not appear to be any constraint on the possible 6-base pair sequences that can be recognized by winged-helix proteins.

SAP-1

We'll begin with SAP-1, since only two residues of the recognition helix, Arg 61 and Tyr 65, are involved in specific base recognition via a π-cation-π interaction.

The interface between SAP-1 and DNA is shown. The sequence of DNA – GGAA – that interacts directly with the protein is shown in CPK colors. Note that recognition of the GGAA motif occurs via the major groove of the DNA.

The winged-helix structural motif consists of a helix–turn–helix–loop–β-sheet. The recognition helix is highlighted in red; the helix and beta-sheet "wings" are shown in different shades of yellow.

The recognition helix fits into the major groove.

SAP-1

The π-cation-π Sandwich

In a π-cation-π interaction or sandwich, a cationic side chain at the protein-DNA interface forms a cation-π complex with a aromatic side chain. This locks the cationic side chain in a position where it can simultaneously form a cation-π complex with the π system of a single base in the DNA. Thus, the cation-π interaction in the protein provides a highly complementary interface to several bases in the DNA.

First, let's explore the role of Tyr-65 and Arg-61. Both residues are part of the recognition helix; they interact both with each other and with the DNA.

Tyr-65 interacts with Arg-61 to form a cation-π complex.

The interaction of Tyr-65 with Arg-61 orients Arg-61 so that it simultaneously forms a cation-π complex with G5 of the DNA.

The guanidino group of arginine bears a full positive charge at physiological pH values. The guanine base in nucleic acids is uncharged. However, its electron density is unevenly distributed. If you examine the model carefully, you'll see that the center of positive charge of the guanidino group is directly over the nitrogen atom bearing a -0.5 charge. In other words, the geometry of Arg-61 and G5 results in a strong electrostatic attraction between the cation and the π system.

Here is the complete π-cation-π sandwich.

Arg-61 also forms two hydrogen bonds with G6. Consequently, Arg-61 interacts with a GG sequence with very high specificity.

Further specificity for GGAA comes from hydrogen bonds between Arg-64 and G5.

Finally, we see all direct contacts between SAP-1 and its GGAA binding motif. The –OH of Tyr-65 acts as a hydrogen-bond acceptor with the amino group of A7.

Ets-1

The Ets-1 domain forms two cation-π interactions with CGGAA. The Ets-1 domain is found within the α subunit of the GA-binding protein (GABP), shown here complexed to promoter DNA. Nucleotides in chain D that interact with the recognition helix are shown in purple.

Several water molecules are entrapped in the crystal structure, either at the protein-DNA interface or within the protein itself. To find them, the backbone model of the protein. How many water molecules are located at the protein-DNA interface? Within the protein?

Now that you have a backbone model of the Ets domain, identify the residues that comprise the recognition helix.

Answer: The recognition helix of the Ets domain comprises residues 371-383 of the α subunit of GA-binding protein.

Winged helix transcription factors insert an α-helix into the major groove of the DNA, forming amino-acid-base contacts over a region spanning about five to six bases. These proteins tend to form homodimers, which often contact two consecutive major grooves. Thus, their binding sequences are palindromic repeats of a five to six base-pair sequence.

Arginines 376 and 379 form two cation-π complexes with the GGAA binding motif.

Tyr-380 forms a cation-π complex with Arg-376.

Finally, we see all the interactions between the recognition helix and the GGAA binding motif of the Ets domain.

In summary, this tutorial highlights the importance of sidechain contacts at the protein-DNA interface. In particular, we've seen how cation-π interactions between cationic sidechains and aromatic rings at the protein-DNA interface play an important role in molecular recognition.