Abstract
Unlike canonical phytochromes, the GAF domain of cyanobacteriochromes (CBCRs) can bind bilins autonomously and is sufficient for functional photocycles. Despite the astonishing spectral diversity of CBCRs, the GAF1 domain of the three-GAF-domain photoreceptor all2699 from the cyanobacterium Nostoc 7120 is the only CBCR-GAF known that converts from a red-absorbing (Pr) dark state to a far-red-absorbing (Pfr) photoproduct, analogous to the more conservative phytochromes. Here we report a solid-state NMR spectroscopic study of all2699g1 in its Pr state. Conclusive NMR evidence unveils a particular stereochemical heterogeneity at the tetrahedral C31 atom, whereas the crystal structure shows exclusively the R-stereochemistry at this chiral center. Additional NMR experiments were performed on a construct comprising the GAF1 and GAF2 domains of all2699, showing a greater precision in the chromophore–protein interactions in the GAF1-2 construct. A 3D Pr structural model of the all2699g1-2 construct predicts a tongue-like region extending from the GAF2 domain (akin to canonical phytochromes) in the direction of the chromophore, shielding it from the solvent. In addition, this stabilizing element allows exclusively the R-stereochemistry for the chromophore-protein linkage. Site-directed mutagenesis performed on three conserved motifs in the hairpin-like tip confirms the interaction of the tongue region with the GAF1-bound chromophore.
Highlights
Phytochromes constitute a superfamily of photosensory proteins in which plant phytochromes are the best-known members [1,2]
Despite the common use of bilins as chromophores, there are variations about the location of chromophore attachment: Phytochromes from fungi and those from bacteria bind to a cysteine located in the N-terminal extension (NTE), whereas all others carry the instrumental cysteine in the GAF domain
We previously reported the PCB chromophore chemical shifts for canonical phytochromes in boWthepphroetvoisotuastelys rteypoifriteeddbtyhethPeCcBycahnroobmacotperhioalreCcphhe1mtihcatl shheirfetssfeorrvceasnaosnaicarelfpehreyntcoechprroomteeins i[n28b]o. th photostates typified by the cyanobacterial Cph1 that here serves as a reference protein [28]
Summary
Phytochromes constitute a superfamily of photosensory proteins in which plant phytochromes are the best-known members [1,2]. A joint feature of phytochromes is the employment of bilins as chromophores that are bound autocatalytically to the protein via conserved cysteine residues, and the domain architecture of their photosensory module (PSM) comprising three domains, namely PAS (Period/Arnt/Singleminded), GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA), and PHY (phytochrome-specific). Phytochromes exhibiting this tridomain arrangement are considered to be canonical phytochromes. Despite the common use of bilins as chromophores, there are variations about the location of chromophore attachment: Phytochromes from fungi and those from bacteria (except cyanobacteria) bind to a cysteine located in the N-terminal extension (NTE), whereas all others carry the instrumental cysteine in the GAF domain. Double-bond photoisomerization is accompanied by a strong shift of the S0 → S1 bilin absorption maxima in the dark and photoproduct states (photochromicity) with a wavelength difference of >100 nm in some cases [2]
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