Crystal structure of a far-red-sensing cyanobacteriochrome reveals an atypical bilin conformation and spectral tuning mechanism

Abstract

Cyanobacteriochromes (CBCRs) are small, linear tetrapyrrole (bilin)-binding photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. More spectrally diverse than canonical red/far-red-sensing phytochromes, CBCRs were thought to be restricted to sensing visible and near UV light until recently when several subfamilies with far-red-sensing representatives (frCBCRs) were discovered. Two of these frCBCRs subfamilies have been shown to incorporate bilin precursors with larger pi-conjugated chromophores, while the third frCBCR subfamily uses the same phycocyanobilin precursor found in the bulk of the known CBCRs. To elucidate the molecular basis of far-red light perception by this third frCBCR subfamily, we determined the crystal structure of the far-red-absorbing dark state of one such frCBCR Anacy_2551g3 from Anabaena cylindrica PCC 7122 which exhibits a reversible far-red/orange photocycle. Determined by room temperature serial crystallography and cryocrystallography, the refined 2.7-Å structure reveals an unusual all-Z,syn configuration of the phycocyanobilin (PCB) chromophore that is considerably less extended than those of previously characterized red-light sensors in the phytochrome superfamily. Based on structural and spectroscopic comparisons with other bilin-binding proteins together with site-directed mutagenesis data, our studies reveal protein-chromophore interactions that are critical for the atypical bathochromic shift. Based on these analyses, we propose that far-red absorption in Anacy_2551g3 is the result of the additive effect of two distinct red-shift mechanisms involving cationic bilin lactim tautomers stabilized by a constrained all-Z,syn conformation and specific interactions with a highly conserved anionic residue.

Keywords: bilin tautomers; bilin-based photoreceptor; far-red-light sensing; optogenetics; serial crystallography.

Fig. 1.

Crystal structure of far-red CBCR 2551g3 in the Pfr state. (A) In a handshake dimer structure of 2551g3, the GAF-α1 helix of one protein molecule (green) extends out to form a three-helix bundle with the GAF-α2 and GAF-α3 helices from the partner molecule (blue). (B) Phycocyanobilin (PCB; cyan) is located in a cleft sandwiched by the β3–β4 and β2–β3 linkers. The electron densities of the 2Fo-Fc map (rendered in transparent cyan surface at the 2.2σ contour) are shown for PCB and the Cys943 anchor. Two structural features unique to the frCBCR family, a disjointed helix at the α-face and tri-loop junction at the β-face, are highlighted in red circles. (C) The PCB chromophore adopts an unusual all-Z,syn conformation.

Fig. 2.

The chromophore binding pocket in the Pfr structure of 2551g3. (A) The 2Fo-Fc map (contoured at 1.7 σ; rendered in gray transparent surface) shows close interactions between the propionates of PCB (cyan) and its protein anchors (Arg930/Lys956, His989/Tyr947; in green sticks). Distances between the carboxylate of Glu914 and four N atoms in pyrrole rings A, B, C, and D are 3.0, 3.4, 2.7, and 3.0 Å, respectively. (B) Viewed from the red arrow in A, Leu944 is located next to the Cys943 anchor at the α-face, whereas both rings A and D point toward the β-face. Red dashed lines highlight the hydrogen bonds between PCB and the acidic side chain of the hallmark residue Glu914. Yellow spheres mark the surrounding aromatic residues. (C) PCB in the Pfr structure of 2551g3 (cyan) assumes a distinct disposition relative to other phytochrome systems: canonical phytochrome Cph1 (2VEA: yellow), red/green CBCRs AnPixJ (3W2Z: magenta), and PPHK (6OAP: light blue). All structures are aligned based on the GAF protein framework (shown in thin wires). (D) The electrostatic surface in the PCB cavity is highly asymmetric between the α- and β-faces.

Fig. 3.

pH titration experiments support a protonated chromophore in the far-red–absorbing state. (A) Absorbance spectra for 15Z 4718g3 are shown at pH 7 (red) and pH 10 (dark blue). (B) Absorbance spectra for 15Z 0468g3 are shown at pH 8 (mauve) and pH 10 (dark blue). (C) Absorbance spectra for 15Z 01757 are shown at pH 7 (red), pH 8 (mauve), pH 9 (periwinkle), and pH 10 (dark blue). (D) Normalized absorbance spectra are shown for samples of 15Z 01757 at standard pH 7.8 (teal), pH 7 (red), and pH 10 (dark blue) that were then denatured. (E) Normalized difference spectra are shown for 15Z frCBCRs upon pH change: 2551g3 (orange, calculated from A), 0468g3 (brick red, calculated from B), and 01757 (green, calculated from extrema in C). (F) Normalized photochemical difference spectra are shown for 4718g3 (orange, calculated from SI Appendix, Fig. S7B, Top), 0468g3 (brick red, calculated from SI Appendix, Fig. S9D), and 01757 (green, calculated from SI Appendix, Fig. S9B).

Fig. 4.

Protein–chromophore interactions around D ring, bilin ring twist, and correlation with red-shifted absorption. (A) Direct interactions between the protein moiety and pyrrole rings within a distance of 3.5 Å are shown in dashed lines for nine representative bilin-binding structures organized according to their bilin conformations. Each panel is annotated with the bilin type (PCB or BV; in cyan) along with the corresponding PDB code and peak wavelength of the Q band absorption. In all FR-conferring structures (red outline), the ring D nitrogen directly interacts with the acidic side chain of Glu or Asp (pink) while the ring D carbonyl is hydrogen bonded to a polar moiety (green dashed lines). The green-absorbing phenotype (blue outline) correlates with the absence of the corresponding acidic residue near ring D lactam. In the red-absorbing Pr state (green outline), the ring D carbonyl is stabilized by a polar moiety while no counterion is found for the ring D nitrogen. (B) Side views of the all,Z-syn chromophores show different ring twists in PcyA and 2551g3. The bilin A and D rings in PcyA (Left) are much more coplanar than those in 2551g3 (Right). (C) A plot of ring tilt angles versus peak wavelength calculated using an in-house script. The sum of three angles between adjacent rings from each methine bridge was plotted against peak wavelength for several XRG CBCRs. The I₈₆D PcyA (purple; linear fit, r² = 0.92) and 2551g3 (green) are also shown for comparison.

Fig. 5.

Protein–chromophore interactions and bilin lactim tautomers in 2551g3. At physiological pHs, PCB in solution is neutral in the all-Z,syn configuration with two ionized propionate side chains. Upon binding to the apoprotein, both propionates form ion pairs with the cationic side chains of R930, K956, and H969 in the chromophore pocket. The A ring tilts toward the β-face as the bound chromophore engages multiple interactions with the β2–β3 loop. These include a strong H bond between the A ring carbonyl and the backbone amide NH of K915 along with protonation of the pyrrole ring system by E914. As a result, the anionic side chain of E914 forms a strong ion pair with the positively charged bilin system (Lactam All Nitrogens Protonated, Far Left structure). Formation of the thioether linkage via C943 may occur concomitantly or soon thereafter. Tautomerization to form Lactim 1 can occur via transient NH deprotonation, e.g., B ring NH, and proton transfer to the A ring carbonyl via the axial E914 carboxylate side chain. We envisage this A ring lactim is stabilized by H bonds with two nearby oxygen atoms, i.e., the backbone amide carbonyl of K915 and the D ring carbonyl, affording two Lactim 1 A ring deprotonated isomers. Additional tautomerization to form Lactim 2 may occur via a similar mechanism, via direct proton transfer from the B ring to the A ring pyrrole nitrogens or directly from the original Lactam. Lactim 3 can be generated by proton transfer via the A ring lactim to the D ring carbonyl. Although the positive charge can be readily delocalized on all four pyrroles, we favor an enrichment of far-red–absorbing Lactim 2 in FR/X CBCRs due to the A ring’s proximity to the negatively charge β-face.