Structure of a light-activated LOV protein dimer that regulates transcription

Abstract

Light, oxygen, or voltage (LOV) protein domains are present in many signaling proteins in bacteria, archaea, protists, plants, and fungi. The LOV protein VIVID (VVD) of the filamentous fungus Neurospora crassa enables the organism to adapt to constant or increasing amounts of light and facilitates proper entrainment of circadian rhythms. Here, we determined the crystal structure of the fully light-adapted VVD dimer and reveal the mechanism by which light-driven conformational change alters the oligomeric state of the protein. Light-induced formation of a cysteinyl-flavin adduct generated a new hydrogen bond network that released the amino (N) terminus from the protein core and restructured an acceptor pocket for binding of the N terminus on the opposite subunit of the dimer. Substitution of residues critical for the switch between the monomeric and the dimeric states of the protein had profound effects on light adaptation in Neurospora. The mechanism of dimerization of VVD provides molecular details that explain how members of a large family of photoreceptors convert light responses to alterations in protein-protein interactions.

Fig. 1

Light-state structure of VVD and the mechanism of light-induced signal transduction. (A) Electron density corresponding to the light-induced adduct between the sulfur of Cys108 and the C4a carbon of the FAD (F_o-F_c omit maps, blue contours at 2.5σ). (B, C) Adduct formation protonates flavin N5 and in response, Gln182 flips to form two new hydrogen bonds – one with the N5 proton and the other with Ala72 carbonyl oxygen. Ala72 shifts and the hydrogen bond between Cys71 thiol breaks as the thiol rotates the Asp68 carbonyl pivots away from the turn. A new hydrogen bond forms between Asp68 and the main chain nitrogen of Ser70. These systematic changes in the hydrogen bonding pattern of the hinge region, result in a 3.5 Å movement of Pro66. Shifts of aα, destabilize the N-terminal latch against the protein core, and lead to its release, which is further facilitated by the flexibility of Gly44. Inset: Adduct formation in one subunit (yellow) leads to smaller changes in the hinge loop position than the other (cyan), which moves forward over Pro66 despite maintaining a similar internal conformation. (D) In the light state structure, the N-cap (represented in darker shades of grey and yellow) restructure to release the N-terminal latch while the other regions of the protein do not change appreciably compared to the dark-state structure.

Fig. 2

The light state dimer of VVD. (A) The protein crystallizes as a dimer with the N-cap (represented in darker shades of yellow and cyan) composing the dimer interface. Met48 and Ile52 in aα make important hydrophobic interactions within the interface. (B) Interactions within the dimer interface: Tyr40 (cyan) hydrogen bonds with the side chain of Thr164 and the main chains of Phe162 and Phe181 of the other subunit. The main chain carbonyl of Pro66, which shifts by 3.5Å, hydrogen bonds with the main chain nitrogen of Ala41 of the other subunit. Cys71, whose side chain flips in the light state, hydrogen bonds with the main chain of Tyr40 in the opposite subunit. (C) Within the dimer interface Tyr87 hydrogen bonds with the side chain and the backbone of Thr69, whereas Met48 and Asp46 hydrogen bond with the backbone of Val67.

Fig. 3

Ability of VVD variants to undergo dimerization. The % monomer is calculated by areas of respective light and dark state elution peaks on size exclusion chromatography (28). The Y40K, Y40E, Y40I and Y40F variants disrupt important hydrogen bonds at the interface and lose the ability to dimerize in the presence of light. The T69W variant is a dimer in both the dark and the light, presumably because it facilitates intersubunit contacts that overcome light-promoted conformational switching. (* from reference , ** from reference 26)

Fig. 4

Dimerization of VVD is essential for the biological functions of VVD in vivo. (A) Carotenoid accumulation as a measure of photoadaptation defects in various vvd mutants. LL indicates strains were exposed to constant white light stimulus with photon flux of 20μmol/m²/sec for four days; DD indicates strains were kept in constant darkness. WT (74A) represents a wild-type strain without photoadaptation defects. Δwc-1 & Δwc-2 represents a “blind” strain. (B) Photoadaptation defects quantified by the amount of carotenoid extracted from mycelia at LL240 (n=5, biological replicates). Horizontal lines denote means. (C) Repression efficacy of various vvd mutant alleles as determined by the repression of al-3 expression at LL60 with RT-QPCR analysis (n=3, mean values ± standard error). Asterisks indicate statistical significance when compared to the wild-type allele of vvd as determined by unpaired t-test, ***p < 0.001, *p < 0.05.