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, 106 (37), 15639-44

Conformational Differences Between the Pfr and Pr States in Pseudomonas Aeruginosa Bacteriophytochrome

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Conformational Differences Between the Pfr and Pr States in Pseudomonas Aeruginosa Bacteriophytochrome

Xiaojing Yang et al. Proc Natl Acad Sci U S A.

Abstract

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria by means of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here, we report the crystal structure of the Q188L mutant of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) photosensory core module, which exhibits altered photoconversion behavior and different crystal packing from wild type. We observe two distinct chromophore conformations in the Q188L crystal structure that we identify with the Pfr and Pr states. The Pr/Pfr compositions, varying from crystal to crystal, seem to correlate with light conditions under which the Q188L crystals are cryoprotected. We also compare all known Pr and Pfr structures. Using site-directed mutagenesis, we identify residues that are involved in stabilizing the 15Ea (Pfr) and 15Za (Pr) configurations of the biliverdin chromophore. Specifically, Ser-261 appears to be essential to form a stable Pr state in PaBphP, possibly by means of its interaction with the propionate group of ring C. We propose a "flip-and-rotate" model that summarizes the major conformational differences between the Pr and Pfr states of the chromophore and its binding pocket.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Crystal structure of the Q188L mutant of PaBphP-PCM. (A) Ribbon diagram colored by the main-chain rmsd values between the Q188L [Protein Data Bank (PDB) ID code 3G6O] and WT (PDB ID code 3C2W) structures. (B) Superposition of the Q188L (gray) and WT (green) structures as a dimer. The structures are aligned based on the left monomer. Same view as in A.
Fig. 2.
Fig. 2.
Stereoviews of the SA-omit maps of the chromophore in the Pr, Pfr, and mixed states. The chromophore models of the Pfr (ZZEssa; cyan) and Pr (ZZZssa, gray) states are from the DrBphP-CBM (2O9C) and PaBphP-PCM WT (3C2W) structures, respectively. The SA-omit maps from DrBphP-CBM, PaBphP-PCM-WT, Q188L-Pa62, and Q188L-Pa125 are aligned based on their GAF core domains. Note that a single model accounts for the SA-omit density in DrBphP-CBM and PaBphP-PCM, but a mixture of the two models is required for both Q188L crystals.
Fig. 3.
Fig. 3.
Conformational differences in the chromophore-binding pocket between the Pfr (cyan) and Pr (gray) states. (A) Interactions between the propionate groups of rings B/C and the protein moiety in the Pfr (red dashed line) and Pr (green dashed line) states (PDB ID code 3G6O). (B and C) The SA-omit maps of the side chains of Tyr-163 and Tyr-190 flanking ring D in Q188L-Pa125 and Q188L-Pa62.
Fig. 4.
Fig. 4.
Absorption properties of the PaBphP-PCM WT and selected mutant proteins in solution. The spectra in solid lines represent the dark-adapted state; spectra in dotted lines are taken immediately after 5-min illumination at a wavelength of 750 or 690 nm; and spectra in dashed lines are recorded after 5-min dark reversion. Estimated half-times of dark reversion are indicated in parentheses.

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