Mechanism-based tuning of a LOV domain photoreceptor

Abstract

Phototropin-like LOV domains form a cysteinyl-flavin adduct in response to blue light but show considerable variation in output signal and the lifetime of the photo-adduct signaling state. Mechanistic studies of the slow-cycling fungal LOV photoreceptor Vivid (VVD) reveal the importance of reactive cysteine conformation, flavin electronic environment and solvent accessibility for adduct scission and thermal reversion. Proton inventory, pH effects, base catalysis and structural studies implicate flavin N(5) deprotonation as rate-determining for recovery. Substitutions of active site residues Ile74, Ile85, Met135 and Met165 alter photoadduct lifetimes by over four orders of magnitude in VVD, and similar changes in other LOV proteins show analogous effects. Adduct state decay rates also correlate with changes in conformational and oligomeric properties of the protein necessary for signaling. These findings link natural sequence variation of LOV domains to function and provide a means to design broadly reactive light-sensitive probes.

Figure 1

(a) Structure of VVD-36 with the PAS core (blue), N-terminal cap (yellow) and FAD binding loop (red). (b) Thermal reversion mechanism for phototropin-like LOV domains. Adduct scission is catalyzed by a base (B), which may include solvent, a conserved glutamine residue that hydrogen bonds to N5, the active site Cys or exogenous compounds such as imidazole.

Figure 2

VVD Kinetics. (a) Thermal reversion of VVD, successive spectra shown at 1.7 hour intervals. Inset demonstrates exponential growth of signal at 450 nm. (b) Active site superposition of VVD and variants: VVD-36 (light blue), I74V (yellow), and I74V:I85V (orange). Residues targeted for mutation near the solvent channel (water-aqua spheres) and the re-face of the flavin are highlighted. (c) Recovery kinetics from the adduct-state for VVD-36 (black), I74V (green) and I74V:I85V (red) and M135I:M165I (blue). (d) Dark state spectra of Native VVD (red) and M135I:M165I (black). M135I:M165I has more pronounced peaks at 364 and 374 nm, which reflect a change in flavin environment. Spectra are offset to aid comparison.

Figure 3

Sequence alignment of VVD, WC-1, Zeitlupe (ZTL), Flavin-Binding Kelch Repeat F-Box protein (FKF1), Brucella mellitus LOV histidine kinase (Bm-LOV), Arabidopsis thaliana phototropin 1 LOV1 and LOV2 (Atphot1LOV1, AtphotoLOV2), and Chlamydomonas reinhardtii LOV1 and LOV2 (CrLOV1, CrLOV2). Positions that border the VVD solvent channel are highlighted in green, whereas residues at the re-face of the flavin are in red. Additional variant residues that affect recovery rates are shown in blue. Non-variant flavin-interacting residues are shown in grey. The presence of M135 is unique to VVD and WC-1.

Figure 4

Proton Inventory and Base Catalysis. (a) Weight of the slow component (equivalent to 100% D₂O, fast component equivalent to 100% H₂O) in biphasic recovery kinetics as a function of the D₂O percentage. (b) Overall weight averaged rate constant as a function of the D₂O percentage for imidazole catalyzed (red) and uncatalyzed (black) I74V. (c) pH dependence of thermal reversion rate for I74V. (d) Comparison of base catalyzed rate constant (see Table 1) by imidazole for VVD-36 (black), I74V (red), I85V (green) and I74V:I85V (blue). In each case concentration dependence is linear with the exception of I74V:I85V, which is best fit as a saturation function with an additional linear term. (e) Rate constant as a function of counter solvent (H₂O) exchange time in the dark. Error bars are depicted at +/− 1 s.d.

Figure 5

Crystal Structures of VVD Variants. Electron density for alternate conformations of Cys108 in VVD-36 (a), I74V (b) and I74V:I85V (c). 2Fo−Fc maps are contoured at 1 σ (cyan) and 2 σ (purple); Fo−Fc maps are contoured at 3 σ (green). VVD-36 has mostly conf1; I74V:I85V only conf2, and I74V has both. (d) SEC elution profile of I74V:I85V. I74V:I85V (green) elutes intermediate of the light- (red) and dark-adapted (black) states of VVD-36 on a Superdex 75 26/60 preparative column.