Solid-State NMR Spectroscopic Study of Chromophore-Protein Interactions in the Pr Ground State of Plant Phytochrome A

Despite extensive study, the molecular structure of the chromophore-binding pocket of phytochrome A (phyA), the principal photoreceptor controlling photomorphogenesis in plants, has not yet been successfully resolved. Here, we report a series of two-dimensional (2-D) magic-angle spinning solid-state NMR experiments on the recombinant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat (Avena sativa), assembled with uniformly C-13- and N-15-labeled phycocyanobilin (u-[C-13,N-15]-PCB-As.phyA3). The Pr state of this protein was studied regarding the electronic structure of the chromophore and its interactions with the proximal amino acids. Using 2-D C-13-C-13 and H-1-N-15 experiments, a complete set of C-13 and N-15 assignments for the chromophore were obtained. Also, a large number of H-1-C-13 distance restraints between the chromophore and its binding pocket were revealed by interfacial heteronuclear correlation spectroscopy. C-13 doublings of the chromophore A-ring region and the C-ring carboxylate moiety, together with the observation of two Pr isoforms, Pr-I and Pr-II, demonstrate the local mobility of the chromophore and the plasticity of its protein environment. It appears that the interactions and dynamics in the binding pocket of phyA in the Pr state are remarkably similar to those of cyanobacterial phytochrome (Cph1). The N-terminus of the region modeled (residues 56-66 of phyA) is highly mobile. Differences in the regulatory processes involved in plant and Cph1 phytochromes are discussed.