LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide

Abstract

Phototropism, the bending response of plant organs to or away from a directional light source, is one of the best studied blue light responses in plants. Although phototropism has been studied for more than a century, recent advances have improved our understanding of the underlying signaling mechanisms involved. The NPH1 gene of Arabidopsis thaliana encodes a blue light-dependent autophosphorylating protein kinase with the properties of a photoreceptor for phototropism. NPH1 apoprotein noncovalently binds FMN to form the holoprotein nph1. The N-terminal region of the protein contains two LOV (light, oxygen, or voltage) domains that share homology with sensor proteins from a diverse group of organisms. These include the bacterial proteins NIFL and AER, both of which bind FAD, and the phy3 photoreceptor from Adiantium capillus-veneris. The LOV domain has therefore been proposed to reflect a flavin-binding site, regulating nph1 kinase activity in response to blue light-induced redox changes. Herein we demonstrate that the LOV domains of two nph1 proteins and phy3 bind stoichiometric amounts of FMN when expressed in Escherichia coli. The spectral properties of the chromopeptides are similar to the action spectrum for phototropism, implying that the LOV domain binds FMN to function as a light sensor. Thus, our findings support the earlier model that nph1 is a dual-chromophoric flavoprotein photoreceptor regulating phototropic responses in higher plants. We therefore propose the name phototropin to designate the nph1 holoprotein.

Figure 1

Purification and spectral analysis of the CBP-LOV fusion proteins. (A) Schematic representation of the CBP-LOV domain fusion proteins. The various regions of Avena sativa nph1, Arabidopsis thaliana nph1, and Adiantum capillus-veneris phy3 used to generate the CBP fusion proteins are shown. Structural features of the nph1 and phy3 proteins are also indicated. LOV domains are shown as shaded boxes and other features are shown as solid boxes. Abbreviations: kinase, serine/threonine kinase domain; phytochrome, phytochrome-related region. The CBP region of the fusion proteins is not shown. (B) SDS/PAGE analysis of the purified LOV domain fusion proteins. Coomassie blue-stained SDS/12.5% polyacrylamide gel showing each of the purified CBP-LOV domain fusion proteins. The molecular masses of the marker proteins (MW) are indicated on the left in kilodaltons (kDa). (C) Spectral analysis of the LOV domain fusions, AsNPH1LOV1, AsNPH1LOV2, AsNPH1LOV1/2, AtNPH1LOV1/2, and AcPHY3LOV2. Absorption and fluorescence excitation spectra obtained for each of the CBP-LOV fusions are shown.

Figure 2

Spectrofluorometric and TLC analysis of the chromophore associated with each of the LOV domain fusion proteins. (A) A typical fluorescence excitation spectrum (Upper) and fluorescence emission spectrum (Lower) of the chromophore released from each of the CBP fusion proteins. (B) Identification of the chromophore as FMN by TLC. The mobility of the chromophore released from each of the CBP-LOV fusions, relative to the solvent front (Rf), is indicated. Rf values for riboflavin, FAD, and FMN standards are also shown.