High Resolution Structure of Deinococcus Bacteriophytochrome Yields New Insights into Phytochrome Architecture and Evolution

Abstract

Phytochromes are red/far red light photochromic photoreceptors that direct many photosensory behaviors in the bacterial, fungal, and plant kingdoms. They consist of an N-terminal domain that covalently binds a bilin chromophore and a C-terminal region that transmits the light signal, often through a histidine kinase relay. Using x-ray crystallography, we recently solved the first three-dimensional structure of a phytochrome, using the chromophore-binding domain of Deinococcus radiodurans bacterial phytochrome assembled with its chromophore, biliverdin IX␣. Now, by engineering the crystallization interface, we have achieved a significantly higher resolution model. This 1.45 Å resolution structure helps identify an extensive buried surface between crystal symmetry mates that may promote dimerization in vivo. It also reveals that upon ligation of the C3 2 carbon of biliverdin to Cys 24 , the chromophore A-ring assumes a chiral center at C2, thus becoming 2(R),3(E)-phytochromobilin, a chemistry more similar to that proposed for the attached chromophores of cyanobacterial and plant phytochromes than previously appreciated. The evolution of bacterial phytochromes to those found in cyanobacteria and higher plants must have involved greater fitness using more reduced bilins, such as phycocyanobilin, combined with a switch of the attachment site from a cysteine near the N terminus to one conserved within the cGMP phosphodiesterase/ adenyl cyclase/FhlA domain. From analysis of site-directed mutants in the D. radiodurans phytochrome, we show that this bilin preference was partially driven by the change in binding site, which ultimately may have helped photosynthetic organisms optimize shade detection. Collectively, these three-dimensional structural results better clarify bilin/protein interactions and help explain how higher plant phytochromes evolved from prokaryotic progenitors. Phytochromes (Phys)3 comprise a ubiquitous superfamily of photoreceptors present in the plant, fungal, and bacterial kingdoms. They play critical roles in various light-regulated processes, ranging from phototaxis and pigmentation in bacteria to seed germination, chloroplast development, shade avoidance, and flowering in higher plants (1, 2). Phys are homodimeric complexes with each polypeptide containing an N-terminal chromophore-binding domain (CBD) that autocatalytically attaches via a thioether linkage a single linear tetrapyrrole (or bilin) chromophore, a PHY domain that is important for spectral integrity, and a C-terminal domain that promotes dimerization and often signal transmission (3). Through unique interactions among the bilin and the CBD and PHY domains, Phys can exist as two metastable conformers, a red light (R)-absorbing Pr form and a far red light (FR)-absorbing Pfr form. By photoconverting between Pr and Pfr, Phys act as unique light-regulated switches in various signal transduction cascades. In bacteria and fungi, a canonical histidine kinase (HK) domain is typically present downstream of the PHY domain, thus allowing these P...

Two ground state isoforms and a chromophore D -ring photoflip triggering extensive intramolecular changes in a canonical phytochrome

“…In both states, four protons bound to the tetrapyrrole nitrogens are well resolved and can be assigned straightforwardly according to our previous studies (18) (Datasets S1 and S2). The absence of cross-peaks involving carboxylate protons (C8 3 and C12 3 ) in our data indicates that these side chains are deprotonated, implicated by earlier studies (19)(20)(21)29).…”

“…Early NMR spectroscopic studies on proteolytic phytochrome fragments (12,13) indicated that this isomerization occurs at the C15═C16 double bond (for numbering, see Fig. 1A), a geometrical change in line with vibrational spectroscopic investigations (14)(15)(16) and results from recent 13 C solid-state NMR (17,18) in which the most significant changes during the light-triggered conversions are confined to rings C and D. Exact geometries of the chromophore in the Pr state have been resolved as periplanar ZZZssa configurations in bacteriophytochromes from Deinococcus radiodurans (19) and Rhodopseudomonas palustris (20) as well as in the more plant-phytochrome-like Cph1 from the cyanobacterium Synechocystis 6803 (21). On the other hand, the crystal structure of the unusual bacteriophytochrome PaBphP Pseudomonas aeruginosa (22) whose ground state is Pfr shows a ZZEssa conformation, consistent with the expected primary photochemistry at the C15═C16 double bond (Fig.…”

Crystal structure of the photosensing module from a red/far-red light-absorbing plant phytochrome

“…SEC supported an elongated shape for the crystallographic subunits by estimating a Stokes radius ∼1.3 times that predicted. Analogous to several BphPs (12,13,17), the dimerization interface involved the GAF domain α1/α2/α6-helical bundle ( Fig. 1A and Fig.…”

“…The Arabidopsis PhyB PSM structure (Protein Data Bank ID code 4OUR) shares its core PAS-GAF-PHY domain architecture with canonical BphPs/Cphs (9,(11)(12)(13) with the inclusion of unique features in each domain (Fig. 1A).…”

Evolutionary Studies Illuminate the Structural-Functional Model of Plant Phytochromes

“…Both the branch-site test and the character mapping exercise identify the sequence coordinates of change, and these can be evaluated in the light of crystal structures that have recently been presented (Wagner et al, , 2007Yang et al, 2007Yang et al, , 2008Cornilescu et al, 2008;Essen et al, 2008;Ulijasz et al, 2010) and can be compared with known mutants. Currently, high-resolution structures of the photosensory core of prokaryotic phytochromes are available; this region is homologous with the sensory module of plant phytochromes (Montgomery and Lagarias, 2002;Lamparter, 2004;Karniol et al, 2005) and consists of PAS, GAF, and PHY domains ( Figure 3B).…”