Unraveling the mechanism of a LOV domain optogenetic sensor: A glutamine lever induces unfolding of the Jα helix

Iuliano, James N., Collado, Jinnette Tolentino, Gil, Agnieszka A., Ravindran, Pavithran T., Lukacs, Andras, Shin, Seung Youn, Woroniecka, Helena A., Adamczyk, Katrin, Aramini, James M., Edupuganti, Uthama R., Hall, Christopher R., Greetham, Gregory M., Sazanovich, Igor V., Clark, Ian P., Daryaee, Taraneh, Toettcher, Jared E., French, Jarrod B., Gardner, Kevin H., Simmerling, Carlos L., Meech, Stephen R ORCID: https://orcid.org/0000-0001-5561-2782 and Tonge, Peter J. (2020) Unraveling the mechanism of a LOV domain optogenetic sensor: A glutamine lever induces unfolding of the Jα helix. ACS Chemical Biology, 15 (10). pp. 2752-2765. ISSN 1554-8929

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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.

Item Type:	Article
Faculty \ School:	Faculty of Science > School of Chemistry (former - to 2024) Faculty of Medicine and Health Sciences > Norwich Medical School
UEA Research Groups:	Faculty of Science > Research Groups > Chemistry of Light and Energy Faculty of Science > Research Groups > Centre for Photonics and Quantum Science
Depositing User:	LivePure Connector
Date Deposited:	21 Nov 2020 00:50
Last Modified:	30 Sep 2024 00:19
URI:	https://ueaeprints.uea.ac.uk/id/eprint/77740
DOI:	10.1021/acschembio.0c00543

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