Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: modes of ligand binding and dimerization

Abstract

In several species, GAF domains, which are widely expressed small-molecule-binding domains that regulate enzyme activity, are known to bind cyclic nucleotides. However, the molecular mechanism by which cyclic nucleotide binding affects enzyme activity is not known for any GAF domain. In the cyanobacterium, Anabaena, the cyaB1 and cyaB2 genes encode adenylyl cyclases that are stimulated by binding of cAMP to their N-terminal GAF domains. Replacement of the tandem GAF-A/B domains in cyaB1 with the mammalian phosphodiesterase 2A GAF-A/B tandem domains allows regulation of the chimeric protein by cGMP, suggesting a highly conserved mechanism of activation. Here, we describe the 1.9-A crystal structure of the tandem GAF-A/B domains of cyaB2 with bound cAMP and compare it to the previously reported structure of the PDE2A GAF-A/B. Unexpectedly, the cyaB2 GAF-A/B dimer is antiparallel, unlike the parallel dimer of PDE2A. Moreover, there is clear electron density for cAMP in both GAF-A and -B, whereas in PDE2A, cGMP is found only in GAF-B. Phosphate and ribose group contacts are similar to those in PDE2A. However, the purine-binding pockets appear very different from that in PDE2A GAF-B. Differences in the beta2-beta3 loop suggest that this loop confers much of the ligand specificity in this and perhaps in many other GAF domains. Finally, a conserved asparagine appears to be a new addition to the signature NKFDE motif, and a mechanism for this motif to stabilize the cNMP-binding pocket is proposed.

Fig. 1.

Ribbon-structure views of cyaB2 tandem GAF A/B domains compared with the PDE2A GAF A/B domains. (a) View of the quaternary structure of the regulatory segment of cyanobacterial Anabaena sp. PCC 7120 AC cyaB2. Each AC monomer (red or green) contains a GAF-A and a GAF-B domain, each with a cAMP-binding site. The cAMP-binding domains, connecting helices, and N- and C-terminal extensions (to GAF-A and -B, respectively) form the dimer interface. GAF-A dimerizes with GAF-B′. The four cAMP molecules are shown in CPK colors. The overall dimensions of the regulatory segment dimer are 111 × 102 × 55 Å. In the monomer, an N-terminal helical extension (residues 58–78) precedes the first GAF domain (residues 79–224, GAF-A). It is followed by a linker (residues 225–230), then a connecting helix (residues 231–263) and another linker (residues 264–270) to GAF-B (residues 271–430). A last helical extension (residues 431–440 or 441) ends in the last ordered density. (b) Same view as a but rotated 90° about the vertical axis. (c) Tandem GAF domains from PDE2A (dimer axis vertical). The figure was prepared with molscript (25) and raster3d (26).

Fig. 2.

Close-up view of the cAMP-binding sites in GAF-A (a) and -B (b) from AC cyaB2, chain A. There are four polar side chains directly contacting cAMP (GAF-A: R103, T105, T176, and Q196; GAF-B: R291, T293, T363, and Q383). In GAF-A, T172 hydroxyl group contacts cAMP through a water. In GAF-B, N359 and D356 side chains contact cAMP through a water molecule. Backbone amides or carbonyls of two or five residues contact cAMP through a water (GAF-A: A161 and A165; GAF-B: T309, K324, G328, D348, and A352). Depending on the GAF domain, there are three to four bound waters. Last are three to four hydrophobic contacts (GAF-A: Ile-131, Leu-198, and Tyr-174; GAF-B: Trp-295, Ile-308, I355, and Tyr-361). Helix α4 is partially (b) and strand β3 is entirely (a and b) depicted as coils for visibility of the cAMP. Water molecules are given arbitrary numbers for purposes of discussion. The figure was prepared with molscript (25) and raster3d (26).

Fig. 3.

Diagram showing all of the close interactions between cAMP and AC cyaB2 GAF-A and -B, chain A. The figure was prepared with ligplot (27).

Fig. 5.

The β2–β3 loop of cNMP-binding GAF domain structures has a variable conformation and length. GAF-A (green) and -B (magenta) from the cyclase, and GAF-B (yellow) from PDE2A, were superimposed. Residues corresponding to PDE2A Phe-438–Asp-439 are shown. The figure was prepared with swiss-pdb viewer (28) and rendered with pov-ray.

Fig. 4.

H-bonds of NKFDE motif in cyaB2 GAF-B. NKFDE residues are orange, cAMP is green, and T363 is magenta.

Fig. 6.

Sequence alignment of GAF domains known to bind cGMP or cAMP. From top to bottom, the sequences are cyaB2 AC (AC) GAF-B and -A in a structural alignment with mouse PDE2A GAF-B, cyaB1 AC GAF-B, human PDE5A GAF-A, chicken cone photoreceptor PDE6C GAF-A, and Trypanosoma brucei PDE2B GAF-A. For the homologous cyaB1, a mutation in the NKFDE motif implicates GAF-B in cAMP binding (11). For the PDE5A (7), PDE6C (9), and T. brucei PDE2B, individually expressed GAF-A domains have been studied with the filter-binding assay. The blue background denotes 50% or more identity. Lower conservation is denoted by the green background. A magenta bar indicates the β2–β3 loop that is the least conserved part of the sequence. The 17 residues that make direct and water-mediated contacts to cAMP in cyaB2 GAF-B are labeled and colored. Orange, polar side chains; purple, hydrophobic side chains; red, backbone amides and carbonyls. Yellow residues denote the NNKFDE motif.