Molecular Insights into Phytochrome Photoactivation and Signaling

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

Intellectual Merit A complex array of photoreceptors coordinates the response of most organisms to their surrounding light environment. One of the most influential is the phytochromes (Phys), a large and diverse group of photoreversible chromoproteins that use a bilin pigment for light detection. These biliproteins sense red (R) and far-red light (FR) through two relatively stable conformational states, an R-absorbing Pr form that typically represents the ground state, and an FR-absorbing Pfr form that typically represents the activated state. By photointerconverting between Pr and Pfr, Phys act as light-regulated switches. Phy-type photoreceptors were first discovered in higher plants by their ability to trigger numerous photoresponses critical for agricultural productivity. More recently, they were found in various microorganisms including bacteria and fungi. Despite their agricultural importance and evolutionary conservation, it is still not fully understood at the molecular level how Phy-type photoreceptors photoconvert between Pr and Pfr nor how this switch tells organisms about the light around them. A major breakthrough was the success in determining the first 3-D structure of the chromophore-binding module as Pr by x-ray crystallography using a Phy from the proteobacterium Deinococcus radiodurans. This structure showed the configuration of the bilin pigment and how it is cradled within its binding pocket, identified a figure-of-eight knot that stabilizes the pocket, discovered a heretofore unknown dimerization domain between sister Phys, and revealed how plant Phys arose from their microbial ancestors. During the prior NSF-funded studies, progress was made in determining the first paired Pr and Pfr solution structures of the chromophore pocket by nuclear magnetic resonance (NMR) spectroscopy using a Phy from the thermotolerant cyanobacterium Synechococcus OSB. Comparison of these structures provided the first glimpse into how Phys photoconvert between their ground and activated states. Contrary to expectations, the A pyrrole ring and not the D ring of the bilin pigment was discovered to rotate during Pr to Pfr photoconversion. This flip induces structural rearrangements within the polypeptide, which then appear to alter the contact between adjacent output domains within the Phy dimer to ultimately modulate signaling. The intellectual merits of this renewal project are to build upon these structural studies to answer key questions, including: is this A ring rotation central to the photoconversion of all Phys? What is the structure of a complete Phy dimer? How does rotation of the pigment followed by structural changes within the binding pocket alter Phy signaling? Significant to this work are the development of recombinant systems that produce large amounts of assembled photoreceptors, and the study of a novel set of Phys that photoconvert between blue- and green-light absorbing forms which should aid in the analysis of the photoactivated state. Specifically, this research plan will: (1) use a combination of NMR spectroscopy and x-ray crystallography to provide further support for the rotation of the A ring during photoconversion, (2) use x-ray crystallography to develop more complete structures of Phys, (3) exploit single particle electron microscopy to determine the architecture of the Phy dimer as Pr and Pfr, and (4) use biochemical methods to further understand how light-driven conformational changes in the Phy dimer regulate signaling. Broader Impact This research will provide an essential framework to better understand the structure, function, and evolution of the Phy superfamily. The anticipated results will ultimately help elucidate how microorganisms and plants sense their light environment, which could have important ramifications for understanding microbial ecosystems, the control of important microbial pathogens, and the development of new strategies to improve the productivity of food and biofuel crops. In addition, the project will enhance scientific infrastructure via a cooperative arrangement for the training of postdoctoral, graduate, undergraduate, and minority students in modern molecular and structure-based approaches in biological research.