Abstract
Light-oxygen-voltage (LOV) domain-containing proteins function as small light-activated modules capable of imparting blue light control of biological processes. Their small modular nature has made them model proteins for allosteric signal transduction and optogenetic devices. Despite intense research, key aspects of their signal transduction mechanisms and photochemistry remain poorly understood. In particular, ordered water has been identified as a possible key mediator of photocycle kinetics, despite the lack of ordered water in the LOV active site. Herein, we use recent crystal structures of a fungal LOV protein ENVOY to interrogate the role of Thr(101) in recruiting water to the flavin active site where it can function as an intrinsic base to accelerate photocycle kinetics. Kinetic and molecular dynamic simulations confirm a role in solvent recruitment to the active site and identify structural changes that correlate with solvent recruitment. In vivo analysis of T101I indicates a direct role of the Thr(101) position in mediating adaptation to osmotic stress, thereby verifying biological relevance of ordered water in LOV signaling. The combined studies identify position 101 as a mediator of both allostery and photocycle catalysis that can impact organism physiology.
Highlights
Light-oxygen-voltage (LOV)3 domain-containing photoreceptors are widely distributed in nature, where they couple blue light absorption to regulation of a diverse array of signal transduction pathways [1]
LOV proteins can be divided into two subclasses: 1) short LOV proteins that exist as the isolated LOV domain with short ancillary N- or C-terminal caps [2,3,4] and 2) modular LOV proteins that couple blue light activation to allosteric regulation of effector domains [1]
Depending on purification conditions, the FMN cofactor purifies either as a mixture of oxidized FMN and reduced neutral semiquinone or as oxidized FMN (Fig. 1, A and B). Such behavior suggests that the oxidation potential of the FMN cofactor may reside within physiologically relevant ranges as has been observed previously in LOV protein variants [18, 34] and has recently been shown of being competent for signal transduction [35]
Summary
In LOV proteins most residues near the N5 position are hydrophobic, with the exception of a conserved Gln residue important for signal transduction [28]. Recent FTIR studies indicate that these ordered water molecules play a key role in regulating LOV lifetime [26]; whether the effect is direct or indirect remains to be determined. Thr101 directly regulates base catalysis and solvent access to the N5 position, but in a manner opposite to that observed in McLOVn. In ENV1, kinetic studies and computational analysis of ordered water indicate that Thr101 affects both solvent access to the active site and ordering of water molecules adjacent to the N5 position. In vivo analysis verifies that Thr101 plays a fundamental role in ENV1-mediated adaptation to osmotic stress In these regards, ENV1 validates a role of ordered water in functioning as an intrinsic base to tune LOV chemistry and function
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