Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome

Abstract

Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses. Several structures of microbial phytochromes have been determined in their dark-adapted Pr or Pfr states. However, the structural nature of initial photochemical events has not been characterized by crystallography. Here we report the crystal structures of three intermediates in the photoreaction of Pseudomonas aeruginosa bacteriophytochrome (PaBphP). We used cryotrapping crystallography to capture intermediates, and followed structural changes by scanning the temperature at which the photoreaction proceeded. Light-induced conformational changes in PaBphP originate in ring D of the biliverdin (BV) chromophore, and E-to-Z isomerization about the C(15) = C(16) double bond between rings C and D is the initial photochemical event. As the chromophore relaxes, the twist of the C(15) methine bridge about its two dihedral angles is reversed. Structural changes extend further to rings B and A, and to the surrounding protein regions. These data indicate that absorption of a photon by the Pfr state of PaBphP converts a light signal into a structural signal via twisting and untwisting of the methine bridges in the linear tetrapyrrole within the confined protein cavity.

Fig. 1

Trap-pump-trap-probe experiment. (a) Ribbon diagram of the PaBphP-PCM dimer. The BV chromophore is colored in cyan. (b) Experimental difference (F_light-F_dark) map at 130K (contoured at +/-5σ, where σ is the standard deviation of difference densities across the entire map). Strong positive (green) and negative (red) densities with peak signal greater than +/-12σ are clustered near the chromophores of the eight monomers (A-H) in the asymmetric unit. (c) Dark stripes of a mounted crystal correspond to segments from which X-ray datasets were collected. (d) A ball-and-stick representation of the chromophore in the Pfr state (PDB 3NHQ). The α-face of a pyrrole ring is defined when atom numbering follows a clockwise direction, with β defined as the opposite face. The α-face of an entire bilin chromophore is defined according to Rockwell et al..

Fig. 2

Difference maps (F_light-F_dark) at pump temperatures between 100 and 180K (contoured at +/- 4σ). The BV chromophore (cyan) is shown in the Pfr state. Positive (green) and negative (red) densities represent structural changes associated with formation of photoproduct state(s) and loss of the parent Pfr state, respectively. No significant difference densities with signal greater than +/-4σ are detected at longer range, beyond the 5Å radius around the chromophore.

Fig. 3

Light-induced structural changes. (a) Representative difference (F_light-F_dark) maps at 110, 130 and 173K. Arrows indicate viewpoints of Fig. 3c and 3d. (b) Stereoview of the superposition of the chromophore conformations in the Pfr (cyan), L1(magenta), L2(yellow) and L3 (blue) structures. (c) Sideview of difference map at 130K (contoured at +/-3σ) shows a twist in the C₁₅ methine bridge. (d) Sideview of difference map at 173K (contoured at +/-2σ) indicates a β–facial shift of rings B/A. Arrows in Figs. 3c, 3d mark the α-face of the chromophore. (e) Difference densities at 173K (contoured at +/-2σ) near the PXSDIP motif (residues 191-196) at the GAF-PHY interface. (f) Differences densities associated with the side chains of His277, Tyr163 and Tyr190 (map contours: +/-3σ at 173K). (g) Relative concentrations of the L1, L2 and L3 structures as a function of pump temperature.

Fig. 4

Light-induced molecular events in PaBphP. (Upper panel) The chromophores are in ball-and-stick representation, with their surrounding residues shown as van der Waals spheres. Green dotted lines indicate potential interactions with each cryo-trapped structure. (Lower panel) Schematic representation of changes in relative disposition of the four pyrrole rings of the BV chromophore, in which pyrrole rings A, B, C and D (boxes) are linearly connected by methine bridges (circles). The α- and β-faces of the chromophore are denoted by arrows.