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.

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

“…Similarly, the distances between RcaE and Slr1393 with respect to the representative PCB atoms range from 3.2 to 5.1 Å. Recently, Bandara et al reported the far-red-absorbing state of the 2551g3 of far-red/orange CBCR subfamily (25), whose GAF domain shows high sequence similarity to that of RcaE. The Pfr dark state of 2551g3 adopts all syn conformation and all Z configuration but also shows a clockwise-rotated location of PCB similar to that seen in RcaE (25).…”

“…Recently, Bandara et al reported the far-red-absorbing state of the 2551g3 of far-red/orange CBCR subfamily (25), whose GAF domain shows high sequence similarity to that of RcaE. The Pfr dark state of 2551g3 adopts all syn conformation and all Z configuration but also shows a clockwise-rotated location of PCB similar to that seen in RcaE (25). The key interactions contributing to the location of PCB in the GAF domain of RcaE are hydrogen bonds and/or ion pairs between the carboxylates of the propionic acid of the B-ring and C-ring (hereafter called B-carboxylate and C-carboxylate, respectively).…”

“…(8,(20)(21)(22)(23); this is known as the "trapped-twist photocycle" (24). Recently, Bandara et al reported the structure of a far-red-absorbing state of a CBCR that adopts an atypical C15-Z,syn configuration (25). They proposed a model that its far-red absorption maximum arises from a cationic bilin tautomer in which the A-ring is formally di-protonated while the B-ring remains deprotonated.…”

Structural basis of bilin binding by the chlorophyll biosynthesis regulator GUN4

“…One possible role of GUN4 is to maintain the lowest-energy conformation, and thus preventing photoisomerization as observed in a cyanobacterial photoreceptor that has an atypical PCB chromophore. 31 The function of the N-terminal domain conserved in cyanobacterial and algal GUN4 remains unclear. Its αA-αE arrange like the armadillo-repeats, which are often found to be involved in protein-protein interactions.…”

“…It appears that the ZZZsss conformer fits better into the binding pocket than the more rigid porphyrin (Figures 2c and 4b). One possible role of GUN4 is to maintain the lowest‐energy conformation, and thus preventing photoisomerization as observed in a cyanobacterial photoreceptor that has an atypical PCB chromophore 31 . The function of the N‐terminal domain conserved in cyanobacterial and algal GUN4 remains unclear.…”