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

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 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 N414side 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.

Resonance energy transfer sensitises and monitors in situ switching of LOV2-based optogenetic actuators

“…RET to LOV2 should fall, resulting in measurable dequench of mTurquoise2 ( Fig. 2ai, right on both C-terminal Jα and N-terminal A' α helix 14 . Slower (250 µs 12 ) and complete undocking of Jα follows 12,15 , resulting in the order-disorder transition used to generate actuation in optogenetic tools 1 .…”

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors

“…Specifically, the side chain of the conserved glutamine may trigger -through a 1801 rotation after the H-transfer step -a process of structural changes by altering the hydrogen bond map of the protein. [5][6][7][8][9][10][11][12] The photocycle of LOV domains commences typically via a blue light trigger that excites the ground state flavin (S 0 ) rapidly to the singlet state (S 1 ) within femtoseconds (Fig. 2a).…”

Lighting the way: Recent insights into the structure and regulation of phototropin blue light receptors

“…Concomitantly, the isoalloxazine N5 position becomes protonated, triggering hydrogen bonding changes to an adjacent glutamine residue (Q575) ( 37 , 38 ). This change is thought to propagate from this glutamine within the flavin-binding pocket to the LOV domain surface; while details of subsequent steps diverge among LOV proteins ( 1 ), for phot LOV2 domains this leads to unfolding of the A′α and Jα helices and activation of kinase activity ( 37 , 38 , 39 ). After illumination ends, the cysteinyl photoadduct decays on the timescale of seconds to hours ( e.g.…”