Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix.

Autor:	Iuliano JN; Department of Chemistry, Stony Brook University, New York, 11794, United States., Collado JT; Department of Chemistry, Stony Brook University, New York, 11794, United States., Gil AA; Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, United States., Ravindran PT; Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, United States., Lukacs A; School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, United Kingdom.; Department of Biophysics, Medical School, University of Pecs, Szigeti út 12, 7624 Pecs, Hungary., Shin S; Department of Chemistry, Stony Brook University, New York, 11794, United States., Woroniecka HA; Department of Chemistry, Stony Brook University, New York, 11794, United States., Adamczyk K; School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, United Kingdom., Aramini JM; Structural Biology Initiative, CUNY Advanced Science Research Center, 85 St. Nicholas Terrace, New York, New York 10031, United States., Edupuganti UR; Structural Biology Initiative, CUNY Advanced Science Research Center, 85 St. Nicholas Terrace, New York, New York 10031, United States.; Ph.D. Program in Biochemistry, CUNY Graduate Center, New York, New York, United States., Hall CR; School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, United Kingdom., Greetham GM; Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, OX11 0QX, United Kingdom., Sazanovich IV; Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, OX11 0QX, United Kingdom., Clark IP; Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, OX11 0QX, United Kingdom., Daryaee T; Department of Chemistry, Stony Brook University, New York, 11794, United States., Toettcher JE; Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, United States., French JB; Department of Chemistry, Stony Brook University, New York, 11794, United States.; Hormel Institute, University of Minnesota, Austin, Minnesota 55912, United States., Gardner KH; Structural Biology Initiative, CUNY Advanced Science Research Center, 85 St. Nicholas Terrace, New York, New York 10031, United States.; Ph.D. Programs in Biochemistry, Biology, and Chemistry, CUNY Graduate Center, New York, New York, United States.; Department of Chemistry and Biochemistry, City College of New York, New York, New York, United States., Simmerling CL; Department of Chemistry, Stony Brook University, New York, 11794, United States., Meech SR; School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, United Kingdom., Tonge PJ; Department of Chemistry, Stony Brook University, New York, 11794, United States.
Jazyk:	angličtina
Zdroj:	ACS chemical biology [ACS Chem Biol] 2020 Oct 16; Vol. 15 (10), pp. 2752-2765. Date of Electronic Publication: 2020 Sep 18.
DOI:	10.1021/acschembio.0c00543
Abstrakt:	Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light-oxygen-voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output domain. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in a loss of the interaction between the side chain of N414 and the backbone C═O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.
Databáze:	MEDLINE
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