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

Abstract

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a "bucket" consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pK_a, whereas they are directly hydrogen bonded in the β-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the "leaky bucket" structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.

Keywords: NMR; crystallography; cyanobacteriochrome; phytochrome.

Fig. 1.

Crystal structure of the chromatic acclimation sensor RcaE in the Pr photoproduct state. (A) Domain organization of RcaE. (B) Ribbon drawings of the structure of the GAF domain of RcaE (cyan). The PCB chromophore is depicted in stick representation (gray). The α-face and β-face that account for each side of PCB are indicated by dotted lines. Arrows indicate the direction viewed in E and F. (C) Topology diagram of the secondary structure of the GAF domain of RcaE (cyan). The regions of TePixJ and Slr1393 corresponding to the S2 through S3 loop of RcaE are shown in pink and green, respectively. (D) F_o-F_c map of the PCB chromophore contoured at 2.5σ. The α-face and β-face are indicated by arrows. (E) Close-up view of the PCB-binding site along the arrow labeled E in B. (F) Close-up view shown along the black arrow labeled F in B. The distances between the oxygen atom of the carboxyl group of the Glu217 sidechain and N_A, N_B, and N_C of the PCB are shown in Å. For clarity, H4 has been omitted from E and F.

Fig. 2.

A unique porous cavity filled with water molecules in the RcaE GAF domain. (A) Superimposition of the crystal structure of the GAF domain of RcaE (cyan) on those of TePixJ (green) and Slr1393 (pink). The H4 of RcaE and the corresponding region of TePixJ and Slr1393 have been omitted for clarity. (B) The unique porous cavity inside the protein shown as surface representations in yellow. Oxygen atoms of the water molecules in the cavity were shown as red balls. The chain A of the GAF domain is represented as a cyan ribbon. The chain B of a neighboring asymmetric unit is represented as brown. The PCB chromophore are shown as stick models colored in gray. (C) Clustered waters (oxygen) inside the S2 through S3 loop of chain A shown as red balls. Electron density and difference electron density maps are also shown as gray and green meshes contoured at 1.0σ and 3.0σ, respectively. The hydrogen bond network is illustrated by yellow dashed lines. (D) Calculated average B-factors per residue. MD values and values for chain A and chain B obtained from the crystal structure are shown in red, blue, and green, respectively.

Fig. 3.

NMR spectra and quantum mechanical calculation of the ¹⁵N chemical shifts of PCB in RcaE. (A and B) Observed spectra after photoconversion at neutral pH. Shown is the ¹⁵N 1D NMR spectrum of PCB in the C15-E Pr state (A) and the C15-Z Pg state (B) pt pH 7.5. (C–E) The QM/MM ¹⁵N NMR chemical shifts calculated by using the GIAO. (C) Simulated spectrum of the C15-E Pr state of RcaE, in which all nitrogen atoms are protonated. (D) Simulated spectrum of the C15-Z Pg state of RcaE, in which N_B is deprotonated and N_A, N_C, and N_D are protonated. (E) Simulated spectrum of the C15-Z Pg state of RcaE, in which N_C is deprotonated and N_A, N_B, and N_D are protonated. (F and G) Observed spectra of the C15-E state at alkaline pH. Shown is the ¹⁵N 1D NMR spectrum of PCB at pH 9.0 (F) and pH 10 (G).

Fig. 4.

A proposed model of the photoconversion of the RcaE GAF domain. In the Pr photoproduct state, PCB adopts C5-Z,syn/C10-Z,syn/C15-E,syn conformation and four pyrrole nitrogen atoms (N_A through N_D) are protonated. The proton of the PCB is provided by clustered water molecules in the porous cavity (dashed blue area) and stabilized by the negatively charged carboxylate of E217. We designate the unique GAF domain structure as “leaky bucket,” in which the large S2-S3 loop (pink) and the core of the antiparallel β-sheet (green) form a bucket, whereas the hydrophobic L249 and F252 in the H4 (yellow) form a lid. In the Pg dark state, the D-ring adopts C15-Z,anti conformation (red) and N_B is deprotonated while the other N_A, N_C, and N_D atoms are protonated. An alternative model with C15-Z,syn (blue) conformation discussed in the main text is also shown. The exact position of E217 in the Pg dark state is unclear.