Structure-guided engineering enhances a phytochrome-based infrared fluorescent protein

Abstract

Phytochrome is a multidomain dimeric red light photoreceptor that utilizes a chromophore-binding domain (CBD), a PHY domain, and an output module to induce cellular changes in response to light. A promising biotechnology tool emerged when a structure-based substitution at Asp-207 was shown to be an infrared fluorophore that uses a biologically available tetrapyrrole chromophore. We report multiple crystal structures of this D207H variant of the Deinococcus radiodurans CBD, in which His-207 is observed to form a hydrogen bond with either the tetrapyrrole A-ring oxygen or the Tyr-263 hydroxyl. Based on the implications of this duality for fluorescence properties, Y263F was introduced and shown to have stronger fluorescence than the original D207H template. Our structures are consistent with the model that the Y263F change prevents a red light-induced far-red light absorbing phytochrome chromophore configuration. With the goal of decreasing size and thereby facilitating use as a fluorescent tag in vivo, we also engineered a monomeric form of the CBD. Unexpectedly, photoconversion was observed in the monomer despite the lack of a PHY domain. This observation underscores an interplay between dimerization and the photochemical properties of phytochrome and suggests that the monomeric CBD could be used for further studies of the photocycle. The D207H substitution on its own in the monomer did not result in fluorescence, whereas Y263F did. Combined, the D207H and Y263F substitutions in the monomeric CBD lead to the brightest of our variants, designated Wisconsin infrared phytofluor (Wi-Phy).

FIGURE 1.

Fluorescence and three-dimensional structure of DrCBD-D207H. A, excitation (emission monitored at 728 nm; blue) and emission (excitation monitored at 695 nm; orange) scans. B, with the exception of the poorly ordered connection between the PAS and GAF domains, there are no significant backbone changes between DrCBD-D207H (green) and the DrCBD (Protein Data Bank code 2O9C; brown). C, the BV-binding pocket (yellow) of DrCBD-D207H has the same structure as the DrCBD with the exceptions of the D207H substitution and malleable Tyr-263 position. BV itself differs by a slight tip of the A-ring. In this view, α-facial is defined as above the A/B/C-ring plane. D, a detailed view of the BV-binding pocket highlights distances from position 207 to bonding partners in DrCBD-D207H (green dashes) and the DrCBD (brown dashes or parentheses). This figure contains embedded three-dimensional information that can be accessed with free Adobe Reader software. Interactive content is activated or disabled by clicking on the figure; the models can be manipulated, and separate views are available from a pulldown menu for each protein structure and for zoomed-in views of the chromophore.

FIGURE 2.

DrCBD-D207H H:Y263 has alternate His-207 rotamer. A, a superposition of H:Y263 (orange; with bound BV (yellow) and the pyrrole water (red sphere)) and H:Aring (green; with bound BV (yellow)) highlights two conformations available to His-207. B, in the H:Y263 structure, His-207 interacts with Tyr-263 and a water molecule. A 2F_o − F_c map contoured at 1.0σ is displayed around His-207 (blue mesh).

FIGURE 3.

Three substitutions lead to monomerization of DrCBD. A, the dimer interface of the DrCBD (shown in H:Aring structure; green) consists of a six-helix bundle. To create the DrCBD_mon, substitutions P145S, L311E, and L314E (gray side chains) were introduced. B, analytical ultrasedimentation analysis of the DrCBD_mon confirms loss of dimer in solution.

FIGURE 4.

Loss of photochromicity in fluorescent DrCBD variants. Shown are absorption scans from 250 to 800 nm of dark-adapted phytochromes (Pr; black lines) and phytochromes exposed to 700 nm light (Pfr; dashed lines) followed by 750 nm light (gray lines). A, dimeric wild-type DrCBD bleaches, but the fluorescent variants have no light response. B, the DrCBD_mon and DrCBD_mon-D207H undergo red/far-red photoconversion. DrCBD_mon-Y263F and Wi-Phy (DrCBD_mon-D207H/Y263F) do not photoconvert.

FIGURE 5.

Wi-Phy (DrCBD_mon-D207H/Y263F) structure. A, crystallographic packing of Wi-Phy occurs around a 2-fold symmetry axis distinct from the biological dimer interface. B, His-207 is positioned similarly in the Wi-Phy (buff) and H:Y263 (not shown) structures, with additional nearby waters (red spheres with 2F_o − F_c density contoured at 1σ). The C2¹ methyl group was modeled in the up and down alternative conformations. See supplemental Fig. 2 for embedded three-dimensional content.

FIGURE 6.

Contributions of His-207 and Phe-263 to fluorescence. Left panels, DrCBD-D207H H:Aring (upper panel) and Wi-Phy (DrCBD_mon-D207H/Y263F; lower panel) in their respective 1.7 and 1.8 Å resolution dark-adapted (Pr) structures. Right panels, same structures overlaid with Pfr chromophore (gray) from P. aeruginosa BphP (Protein Data Bank code 3G6O; protein atoms not shown (33)). D207H contributes to fluorescence through loss of the Pfr D-ring interaction and stabilization of the Pr A-ring conformation. Y263F has a greater influence due to destabilization of the Pfr D-ring conformation.