The structure of a CAP-DNA complex having two cAMP molecules bound to each monomer

Abstract

The 2.2 A resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with cAMP and a 46-bp DNA fragment reveals a second cAMP molecule bound to each protein monomer. The second cAMP is in the syn conformation and is located on the DNA binding domain interacting with the helix-turn-helix, a beta-hairpin from the regulatory domain and the DNA (via water molecules). The presence of this second cAMP site resolves the apparent discrepancy between the NMR and x-ray data on the conformation of cAMP, and explains the cAMP concentration-dependent behaviors of the protein. In addition, this site's close proximity to mutations affecting transcriptional activation and its water-mediated interactions with a DNA recognition residue (E181) and DNA raise the possibility that this site has biological relevance.

Figure 1

A molscript (4) ribbon drawing of the CAP dimer bound to DNA and the two cAMP molecules (magenta) per monomer, one labeled SYN and the other, ANTI. In one monomer, the larger N-terminal domain is yellow, and the smaller C-terminal domain is blue, while the DNA half-site bound to it is light gray. The other subunit is green and the DNA bound to it is dark gray. The syn-cAMP lies on the helix-turn-helix and close to the DNA and a loop from the N-terminal domain. The DNA sequence of the half-site is 5′-ATGTCACATTAATTGCGTTGCGC-3′.

Figure 2

Stereo view of the 2F_o-F_c “omit” electron density map at 2.2 Å resolution from which the syn-cAMP (ball-and-stick) was first identified. The density, contoured at 1.2 σ, was computed using experimentally determined phases from 25 to 8 Å and phases from 8 to 2.2 Å derived from the protein and DNA coordinates alone before any coordinates of the syn-cAMP were included in the phase and amplitude calculation.

Figure 3

(A) Close-up stereo view of the syn-cAMP binding site. The syn-cAMP and an α-carbon trace of the helix-turn-helix and β-hairpin loop with which it interacts in one subunit (gray) are shown, as well as a region from the other subunit (red) that interacts with the cAMP; backbone atoms are included where interactions with the cAMP occur. The three DNA nucleotides nearest to the cAMP molecule are shown. The Cys-178 side chain, which has a lower reactivity at higher cAMP concentrations, has been included. In green are four protein side chains and two water molecules. Lys-52 and Asp-53 have been demonstrated by mutational analysis to be important for transcriptional activation (–24), while the Glu-181 carboxylate is making both important sequence-specific DNA interactions and water-mediated interactions to the phosphates of cAMP and of DNA. (B) Diagram of the syn-cAMP and the hydrogen-bond interactions that are made to it. The hydrogen-bond distances given are measured between the donor and acceptor atoms in the structure. Glu-181 makes a water-mediated interaction with the cAMP phosphate. Residue 135 is from the other CAP subunit.

Figure 3

(A) Close-up stereo view of the syn-cAMP binding site. The syn-cAMP and an α-carbon trace of the helix-turn-helix and β-hairpin loop with which it interacts in one subunit (gray) are shown, as well as a region from the other subunit (red) that interacts with the cAMP; backbone atoms are included where interactions with the cAMP occur. The three DNA nucleotides nearest to the cAMP molecule are shown. The Cys-178 side chain, which has a lower reactivity at higher cAMP concentrations, has been included. In green are four protein side chains and two water molecules. Lys-52 and Asp-53 have been demonstrated by mutational analysis to be important for transcriptional activation (–24), while the Glu-181 carboxylate is making both important sequence-specific DNA interactions and water-mediated interactions to the phosphates of cAMP and of DNA. (B) Diagram of the syn-cAMP and the hydrogen-bond interactions that are made to it. The hydrogen-bond distances given are measured between the donor and acceptor atoms in the structure. Glu-181 makes a water-mediated interaction with the cAMP phosphate. Residue 135 is from the other CAP subunit.

Figure 4

Comparison of part of the DNA binding domain (A) and the β-hairpin loop (B) structures of CAP complexed with only one cAMP per subunit (magenta) with that of CAP complexed with DNA and two cAMP molecules per subunit (cyan). (A) α-Carbon backbones of the small domains were superimposed. In the structure of CAP complexed with only one cAMP per subunit and without DNA, Arg-180 could hydrogen-bond with the phosphate of the syn-cAMP (yellow); whereas in the structure of CAP complexed with DNA and two cAMP molecules per subunit, Arg-180 hydrogen bonds to guanosine-7. (B) α-Carbon backbones of the large domains, excluding the β-hairpin loop, were superimposed, and the side chains for Lys-52, Asp-53, and Lys-57 are shown.