5VYD

Crystal structure of phosphodiesterase domain of RhoPDE fusion protein from the Choanoflagellate Salpingoeca rosetta

Summary for 5VYD

• Entry DOI: 10.2210/pdb5vyd/pdb
• Descriptor: Phosphodiesterase, ZINC ION, MAGNESIUM ION, ... (4 entities in total)
• Functional Keywords: pde, cgmp specific, enzyme, alpha helical, metal binding, hydrolase
• Biological source: Salpingoeca rosetta (strain ATCC 50818 / BSB-021)
• Total number of polymer chains: 2
• Total formula weight: 73841.14

Authors

Prem Kumar, R.,Lamarche, L.B.,Oprian, D.D. (deposition date: 2017-05-25, release date: 2017-10-18, Last modification date: 2023-10-04)

Primary citation

Lamarche, L.B.,Kumar, R.P.,Trieu, M.M.,Devine, E.L.,Cohen-Abeles, L.E.,Theobald, D.L.,Oprian, D.D.
Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta.
Biochemistry, 56:5812-5822, 2017

PubMed Abstract: RhoPDE is a type I rhodopsin/phosphodiesterase gene fusion product from the choanoflagellate Salpingoeca rosetta. The gene was discovered around the time that a similar type I rhodopsin/guanylyl cyclase fusion protein, RhoGC, was shown to control phototaxis of an aquatic fungus through a cGMP signaling pathway. RhoPDE has potential as an optogenetic tool catalyzing the hydrolysis of cyclic nucleotides. Here we provide an expression and purification system for RhoPDE, as well as a crystal structure of the C-terminal phosphodiesterase catalytic domain. We show that RhoPDE contains an even number of transmembrane segments, with N- and C-termini both located on the cytoplasmic surface of the cell membrane. The purified protein exhibits an absorption maximum at 490 nm in the dark state, which shifts to 380 nm upon exposure to light. The protein acts as a cGMP-selective phosphodiesterase. However, the activity does not appear to be modulated by light. The protein is also active with cAMP as a substrate, but with a roughly 5-7-fold lower k. A truncation consisting solely of the phosphodiesterase domain is also active with a k for cGMP roughly 6-9-fold lower than that of the full-length protein. The isolated PDE domain was crystallized, and the X-ray structure showed the protein to be a dimer similar to human PDE9. We anticipate that the purification system introduced here will enable further structural and biochemical experiments to improve our understanding of the function and mechanism of this unique fusion protein.

PubMed: 28976747
DOI: 10.1021/acs.biochem.7b00519

Experimental method

X-RAY DIFFRACTION (2.3 Å)