Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor

Abstract

GAF domains are ubiquitous motifs present in cyclic GMP (cGMP)-regulated cyclic nucleotide phosphodiesterases, certain adenylyl cyclases, the bacterial transcription factor FhlA, and hundreds of other signaling and sensory proteins from all three kingdoms of life. The crystal structure of the Saccharomyces cerevisiae YKG9 protein was determined at 1.9 A resolution. The structure revealed a fold that resembles the PAS domain, another ubiquitous signaling and sensory transducer. YKG9 does not bind cGMP, but the isolated first GAF domain of phosphodiesterase 5 binds with K:(d) = 650 nM. The cGMP binding site of the phosphodiesterase GAF domain was identified by homology modeling and site-directed mutagenesis, and consists of conserved Arg, Asn, Lys and Asp residues. The structural and binding studies taken together show that the cGMP binding GAF domains form a new class of cyclic nucleotide receptors distinct from the regulatory domains of cyclic nucleotide-regulated protein kinases and ion channels.

Fig. 1. Domain structure of mammalian GAF and PAS domain-containing phosphodiesterases and other representative GAF domain proteins. Domain structures were obtained from SMART (Schultz et al., 1998), Pfam (Bateman et al., 1999) and Soderling and Beavo (2000).

Fig. 2. Binding of cGMP to PDE5-GAFa and PDE5-GAFb, and YKG9.

Fig. 3. (A) Electron density from solvent-flipped Br MAD map at 2.5 Å. (B) Overall structure of YKG9 dimer. (C and D) YKG9 monomer in two different views related by a 90° rotation about the y-axis.

Fig. 4. (A) The buried Cys/His/Asp/Glu pocket of YKG9 is compared with (B), the chromophore binding site of photoactive yellow protein. (C and D) The same images as (A) and (B) in a surface representation with basic residues colored blue, acidic red, hydrophobic orange and others gray. In (C) and (D) the surface is removed for a portion of the β3–α4 in YKG9 and the equivalent region of photoactive yellow protein in order to show the underlying cavities.

Fig. 5. Similarity between (A) YKG9 and (B) profilin, and (C) YKG9 and (D) photoactive yellow protein. Regions that can be superimposed within 3.0 Å are colored red.

Fig. 6. Threading and profile-based alignment of representative GAF domain sequences. Residues that are part of the aligned core, and can therefore be positioned in the three-dimensional homology models with good confidence, are shown with upper-case letters. The PDE5A sequence is bovine, the PDE2A and PDE6A sequences are human. Residues important for cGMP binding are highlighted in blue. Residues contributing directly to the YKG9 Cys/His/Asp/Glu cluster are marked in red. The PDE6 residue mutated in congenital night blindness is highlighted in white against a black background (top section). PYP, photoactive yellow protein, sequence aligned based on three-dimensional structure superposition.

Fig. 7. (A) Surface representation of the PDE5-GAFa model and its cGMP binding site. A manually docked cGMP molecule is shown as a guide to the eye only. (B) Close-up view of the cGMP binding site of the PDE5-GAFa domain in the same orientation. The β6–α5 loop that potentially folds over bound cGMP is highlighted in blue. Ile170 is in the location corresponding to the PDE6 His residue mutated in congenital night blindness. The predicted guanine binding site is formed by Ile170, Asn276, Asp289 and their surroundings, while the predicted cyclic phosphate binding site is formed by Arg174, Lys277 and surrounding residues. (C) cGMP binding of Arg174 mutants in PDE5-GAFa, and the corresponding Lys356 mutant in PDE5-GAFb.