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

James N. Iuliano, Jinnette Tolentino Collado, Agnieszka A. Gil, Pavithran T. Ravindran, Andras Lukacs, Seungyoun Shin, Helena A. Woroniecka, Katrin Adamczyk, James M. Aramini, Uthama R. Edupuganti, Christopher R. Hall, Gregory M. Greetham, Igor V. Sazanovich, Ian P. Clark, Taraneh Daryaee, Jared E. Toettcher, Jarrod B. French, Kevin H. Gardner, Carlos L. Simmerling, Stephen R. MeechPeter J. Tonge

Research output: Contribution to journalArticlepeer-review

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)2752-2765
Number of pages14
JournalACS Chemical Biology
Volume15
Issue number10
DOIs
StatePublished - Oct 16 2020

Bibliographical note

Funding Information:
This study was supported by the National Science Foundation (NSF; MCB-1817837 to P.J.T., MCB-1750637 to J.B.F.) and the EPSRC (EP/N033647/1 to S.R.M.). J.N.I. was supported by a National Institutes of Health Chemistry-Biology Interface Training Grant (T32GM092714). J.T.C. was supported by the IMSD-MERGE Program at Stony Brook University (5R25GM103962-04). J.E.T. was supported by NIH-DP2EB024247. A.A.G. was supported by NIH-F32GM128304. A.L. is a Bolyai Janos Research Fellow and was supported by OTKA NN113090. U.R.E. and K.H.G. were supported by NIH-R01GM106239. J.B.F. would like to acknowledge the Research Corporation for Science Advancement for support from a Cottrell Scholar Award.

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