Structure of WspR from P. aeruginosa
(A) Domain organization of WspR. The N-terminal CheY-homology phospho-receiver domain is connected via a helical stalk to the GGDEF domain with diguanylate cyclase activity. In WspR, the active site loop contains the GGEEF motif (residues 251–255).
(B) The crystal structure of WspR. The crystals contain two molecules in the asymmetric unit. Two orthogonal views are shown with coloring for molecule A as shown in (A). Molecule B is colored grey. The GGEEF motif is shown in yellow. Cyclic di-GMP molecules bound to the inhibitory site (I-site) are located distal to the active site and are shown as sticks. Mg2+ ions are shown as brown spheres.
(C) Comparison of the two WspR molecules in the asymmetric unit. Molecules A and B were aligned through superpositioning of their GGDEF domains. The CheY-stalk modules are separated by a rigid body rotation of 16° around residue 172 at the tip of the helical stalk.
(D) Crystallographic tetramer consisting of two symmetry-related dimers representing a biological unit. Two C2-symmetry–related crystallographic dimers of WspR are shown intertwined in a head-to-head orientation. The stalks form a tetrameric structure splaying apart the coiled-coils and physically blocking the active sites. Cyclic di-GMP molecules bound at the I-site bridge the GGDEF domains of neighboring molecules. “S” or “SYM” in the cartoon diagram indicate crystal symmetry-related molecules. The boxed region is shown in (E).
(E) Close-up view of the I-site. In the crystal, two intercalated c-di-GMP molecules are bound at the I-site located at the back of the GGDEF domain distal to the catalytic site. An arginine side chain (R198) contributed by a symmetry-related GGDEF domain completes the I-site. Asterisks indicate residues targeted for site-directed mutagenesis.