From NMR to function: Mechanistic Insight into the Interaction between Ferredoxin and the Ferredoxin Dependent Bilin Reductase PebS

Ferredoxin-dependent bilin reductases (FDBRs) play a critical role in photosynthetic organisms by catalyzing the reduction of biliverdin (BV) to different open-chain tetrapyrroles, including phytochromobilin (PΦB), phycocyanobilin (PCB), and phycoerythrobilin (PEB)1. These open-chain tetrapyrroles serve as chromophores that facilitate light-depended processes, e.g. in development or regulation of photosynthesis. Different representatives of the class of FDBRs are very distinct in their stereo- and regioselective reduction of BV, yet they all rely on the efficient interaction with the electron donor Ferredoxin (Fd) during catalysis.

In this study we investigated the interaction between the phycoerythrobilin synthase PebS1, encoded by a phage, and its host Fd. To determine the molecular interaction between PebS and Fd, NMR spectroscopy and computational docking were employed. Using [15N]-labelled gallium-substituted Fd from Thermosynechococcus elongatus, chemical shift perturbations were observed, allowing the identification of residues critical for protein-protein interactions2. Using these data, we performed HADDOCK docking simulations to predict the contact interface between both proteins and guide the design of PebS variants potentially disrupting the interaction. These variants lead to inhibition of protein activity and therefore support the location of the proposed interface. Based on a phylogenetic analysis, we expand this to the phage Fd and other Fd-FDBR interaction pairs to obtain a better understanding of this essential protein-protein interaction.

References

[1] Thorben Dammeyer, Sarah C. Bagby, Matthew B. Sullivan, Sallie W. Chisholm, Nicole Frankenberg-Dinkel: Efficient Phage-Mediated Pigment Biosynthesis in Oceanic Cyanobacteria; Current Biology; 2008, 442-448; ISSN 0960-9822; https://doi.org/10.1016/j.cub.2008.02.067.[2] Risa Mutoh, Norifumi Muraki, Kanako Shinmura, Hisako Kubota-Kawai, Young-Ho Lee, Marc M. Nowaczyk, Matthias Rögner, Toshiharu Hase, Takahisa Ikegami, and Genji Kurisu; X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin; Biochemistry2015 54 (39), 6052-6061; DOI:10.1021/acs.biochem.5b00601