Conformational switching in the fungal light sensor Vivid

Abstract

The Neurospora crassa photoreceptor Vivid tunes blue-light responses and modulates gating of the circadian clock. Crystal structures of dark-state and light-state Vivid reveal a light, oxygen, or voltage Per-Arnt-Sim domain with an unusual N-terminal cap region and a loop insertion that accommodates the flavin cofactor. Photoinduced formation of a cystein-flavin adduct drives flavin protonation to induce an N-terminal conformational change. A cysteine-to-serine substitution remote from the flavin adenine dinucleotide binding site decouples conformational switching from the flavin photocycle and prevents Vivid from sending signals in Neurospora. Key elements of this activation mechanism are conserved by other photosensors such as White Collar-1, ZEITLUPE, ENVOY, and flavin-binding, kelch repeat, F-BOX 1 (FKF1).

Fig. 1

VVD structure. (A) Crystallographic dimer of VVD-36, including the PAS core (blue), N-terminal cap (yellow), and FAD insertion loop (red). The N terminus, resolved only in the left molecule, projects toward the solvent-exposed FAD adenosine moiety (orange). (B) Superposition of the PAS domains of VVD (green), PYP (magenta), Drosophila PER (red), and AsLOV2 domain (blue). All proteins share a structurally conserved PAS β scaffold (yellow) and helical regions (gray) that pack with a variable helical element possibly involved in signal transduction. (C) Photocycle of VVD-36 at 25°C. Blue-light illumination of VVD forms a photoadduct between Cys¹⁰⁸ and the C4a position of the flavin ring (inset). Adduct formation bleaches the flavin absorption bands at 428, 450, and 478 nm and produces a single peak at 390 nm. Recovery proceeds with t_1/2 = 10⁴ s and three isosbestic points at 330, 385, and 413 nm. Spectra are displayed at 3000 s increments.

Fig. 2

The VVD light state in crystals. (A) Superposition of VVD (yellow) and Adiantum phy3-LOV2 (1G28, blue-gray) active centers show differences in residue composition beneath the flavin [1.5 σ (aqua) and 3.0 σ (purple), 2F_obs – F_calc electron density]. An alternate conformation of Cys¹⁰⁸ contacts conserved Cys⁷⁶ [3.0 σ (green), F_obs – F_calc electron density]. (B) Structural differences in the light state of VVD. Difference electron density reveals covalent bond formation between Cys¹⁰⁸ and flavin C4a and flipping of the Gln¹⁸² amide in response to N5 protonation. F_obs – F_calc electron density [2.0 σ (aqua), 3.0 σ (blue), −2.0 σ (orange), and −3.0 σ (red)], with F_calc calculated from a model refined with 100% of the dark-state conformation. (C) Expanded view of the structural changes propagating from Gln¹⁸² to aα and bβ in the VVD-36 light state. Pro⁶⁶ undergoes the largest shift (2.0 Å) in the light state (yellow) versus the dark state (orange). Hydrogen bonds (dashed lines) are shown for d < 3.2 Å; except for Cys⁷¹-to-Asp⁶⁸ amide, where d = 3.9 Å. Other key contacts are shown in blue. (D) The hinge region between the PAS core and bβ. In the light state, Gln¹⁸² rotates to improve interactions between the Gln¹⁸² amide and the Ala⁷² carbonyl, Cys⁷¹ swivels to hydrogen-bond with the Asp⁶⁸ amide nitrogen, and bβ shifts 2 Å. F_obs – F_calc omit electron density [1.5 σ (aqua) and 3.0 σ (purple)] calculated with bβ absent from the model.

Fig. 3

Increase of the VVD-36 hydrodynamic radius on light excitation. (A) Elution profiles of VVD variants from a size-exclusion column. Light-state VVD (green) elutes at a much larger apparent molecular weight than does dark-state VVD (black), but smaller than a disulfide cross-linked dimer (purple). Addition of a His₆ tag to the VVD N terminus shifts the elution profile of both the dark state (light gray) and the light state (red). Truncation of six residues does not affect the dark state (dark gray) but significantly shifts the light state (pink). Full-length VVD undergoes a smaller shift in the light state (dark green) relative to the dark state (orange). (B) Model of the coupled chemical and conformational changes caused by VVD light activation.

Fig. 4

Decoupling the VVD photocycle from signal transduction. (A) Light-state elution profile of VVD-36 variants. A C71A mutant (orange) and C71V mutant (green) adopt the expanded state on light excitation, whereas C71S (pink) cannot. Q182L (aqua) elutes at a position slightly larger than VVD-36 dark (black) but does not respond normally to light excitation. (B) VVD C71S is incapable of transmitting blue light signals in Neurospora. (Left) Western blots of cellular extracts with an antibody to VVD. The vvd null mutant contains no VVD protein, whereas the protein is abundant when complemented with WT VVD containing a 6-His tag, C71S, or C76S. (Right) Slant cultures of Neurospora crassa grown under constant light conditions. The vvd null and C71S mutants accumulate large amounts of carotenoids as a result of loss of light adaptation, giving the cells an orange color. In contrast, complementation with C76S yields WT behavior.