Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: photoconversion and signal transduction

Abstract

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria via reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here we report the crystal structure at 2.9 A resolution of a bacteriophytochrome from Pseudomonas aeruginosa with an intact, fully photoactive photosensory core domain in its dark-adapted Pfr state. This structure reveals how unusual interdomain interactions, including a knot and an "arm" structure near the chromophore site, bring together the PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains to achieve Pr/Pfr photoconversion. The PAS, GAF, and PHY domains have topologic elements in common and may have a single evolutionary origin. We identify key interactions that stabilize the chromophore in the Pfr state and provide structural and mutational evidence to support the essential role of the PHY domain in efficient Pr/Pfr photoconversion. We also identify a pair of conserved residues that may undergo concerted conformational changes during photoconversion. Modeling of the full-length bacteriophytochrome structure, including its output histidine kinase domain, suggests how local structural changes originating in the photosensory domain modulate interactions between long, cross-domain signaling helices at the dimer interface and are transmitted to the spatially distant effector domain, thereby regulating its histidine kinase activity.

Fig. 1.

Crystal structure of wild-type PaBphP-PCD. (A) Ribbon diagram of the dimeric PaBphP-PCD structure. The PAS, GAF, and PHY domains of one monomer are highlighted in yellow, green, and blue, respectively. Helices in the GAF and PHY domains are identified by letters (A–E). (B) The PAS, GAF, and PHY domains are integrated via extensive interdomain interactions and converge on the chromophore binding site (cyan). (C) Accessory structure elements (gray) decorate the common cores of the PAS, GAF, and PHY domains and are spatially clustered near the chromophore (cyan) and as helical bundles at the dimer interface. (D) The core of the PAS, GAF, and PHY domains contains an antiparallel β sheet with strands in the spatial order of 2–1–5–4–3 and a variable connector between strands 2 and 3 that contains helix C. (E) Both core and accessory elements are highlighted in a topologic diagram of the PaBphP-PCD structure.

Fig. 2.

Residues and interactions in the chromophore binding pocket. (A) The BV chromophore of PaBphP-PCD in the Pfr state adopts the 15Ea configuration (cyan) as compared with the 15Za configuration (gray) in the Pr state of the RpBphP3-CBD structure. The superposition is based on the structural alignment of the PAS and GAF domains of the PaBphP-PCD and RpBphP3-CBD (PDB ID 2OOL) structures. (B) At the interface between the GAF and PHY domains in PaBphP-PCD, conserved residues Tyr-250, Asp-194, Ser-459, and Arg-453 and the chromophore form extensive hydrogen bond interactions (red dotted lines) to stabilize ring D in the 15Ea configuration. (C) The side chains of Tyr-250 and Gln-188 directly interact with the carbonyl group of ring D. (D) UV-visible absorption spectra of the dark-adapted (solid) and light-illuminated (dash) states in PaBphP-PCD wild type (WT) and mutants. Half-times of dark reversion are indicated in parentheses.

Fig. 3.

Conformational changes in the chromophore binding pocket and structural variation of the helical bundle at the dimer interface. (A) In the chromophore binding pocket of PaBphP-PCD, Tyr-190 and Tyr-163 (green) near ring D adopt side chain conformations distinct from the corresponding residues Phe-212 and Tyr-185 (light gray) of RpBphP3-CBD in the Pr state. As ring D flips, the 2 side chains rearrange. (B) Superposition of the 8 monomers (4 shown in green, 4 in dark gray) in the asymmetric unit shows 2 distinct locations for the GAF-hA helix, resulting in 4 tertiary heterodimers. (C) Quaternary structural variation at the dimer interface, as shown by angles between the GAF-hE helices of one monomer when dimeric structures are aligned according to the PAS and GAF domains of the other monomer. Structures compared are DrBphP-CBD (1ZTU, pink; 2O9C, gray), RpBphP3-CBD (2OOL, yellow), PaBphP-PCD-WT (3C2W, cyan), and PaBphP-PCD-Q188L (G.X. Yang, J.K., and K.M., unpublished results; blue).

Fig. 4.

A domain architecture model of the full-length dimeric PaBphP based on the PaBphP-PCD dimer structure and the sensor HK structure (PDB accession ID 2C2A) (PAS, GAF, and PHY in green; HK in blue; BV in cyan).