Light-Induced Movement of the LOV2 Domain in an Asp720Asn Mutant LOV2−Kinase Fragment of Arabidopsis Phototropin 2

Abstract

Phototropin, a blue-light receptor protein of plants, triggers phototropic responses, chloroplast relocation, and opening of stomata to maximize the efficiency of photosynthesis. Phototropin is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) that absorb blue light and a serine/theroine kinase domain responsible for light-dependent autophosphorylation leading to cellular signaling cascades. Although the light-activated LOV2 domain is primarily responsible for subsequent activation of the kinase domain, it is unclear how conformational changes in the former transmit to the latter. To understand this molecular mechanism in Arabidopsis phototropin 2, we performed small-angle X-ray scattering analysis on a fragment composed of the LOV2 and kinase domains, which contained an Asp720Asn mutation that led to an absence of ATP binding activity. The scattering data were collected up to a resolution of 25 Å. The apparent molecular weight of the fragment estimated from scattering intensities demonstrated that the fragment existed in a monomeric form in solution. The fragment exhibited photoreversible changes in the scattering profiles, and the radii of gyration under dark and blue-light irradiation conditions were 32.4 and 34.8 Å, respectively. In the dark, the molecular shape restored from the scattering profile appeared as an elongated shape of 110 Å in length and 45 Å in width. The homology modeled LOV2 and kinase domains could be fitted to the molecular shape and appeared to make slight contact. However, under blue-light irradiation, a more extended molecular shape was observed. The changes in the molecular shape and radius of gyration were interpreted as a light-dependent positional shift of the LOV2 domain of approximately 13 Å from the kinase domain. Because the region connecting the LOV2 and kinase domains was categorized as a naturally unfolded polypeptide, we propose that the light-activated LOV2 domain triggers conformational changes in the linker region to separate the LOV2 and kinase domains.

Light-induced Conformational Changes of LOV1 (Light Oxygen Voltage-sensing Domain 1) and LOV2 Relative to the Kinase Domain and Regulation of Kinase Activity in Chlamydomonas Phototropin

“…For the LOV2-KD fragment of Cr phot (CrPL2K), DNA of the LOV2 ϩ linker ϩ kinase (192-749) was synthesized by PCR with primers (Table 1), and the amplified fragment was inserted into the NdeI/SalI site of the pET28a bacterial expression vector (GE Healthcare). CrPFul and CrPL2K were purified as described in the previous paper (27). The Escherichia coli BL21 (DE3) cells overexpressed the protein, which were suspended in the purification buffer and lysed by sonication.…”

“…Because the LOV2-KDs have BL-regulated kinase activity, structural study of these molecules provides useful information regarding the molecular basis for the BL-dependent regulation of the kinase. In this regard we previously measured the SAXS of a D720N substitute of At phot2 LOV-KD and reported its molecular models, which revealed the topological organization of LOV2 and KD and its BL-induced alteration (27).…”

Photosensitivity of Kinase Activation by Blue Light Involves the Lifetime of a Cysteinyl-Flavin Adduct Intermediate, S390, in the Photoreaction Cycle of the LOV2 Domain in Phototropin, a Plant Blue Light Receptor

“…One of these changes was observed with P2L2K by SAXS, although it lost kinase activity because of the substitution of Asp-720 with Gln. In this case, LOV2 is proposed to move away from the N-terminal lobe of the kinase domain upon BL excitation (20). Furthermore, FTIR detected a BL-induced loss of helicity in the activation loop region of the kinase domain in Chlamydomonas full-length phototropin (38).…”

“…Recently, light-induced movement of the LOV2 domain relative to the kinase domain in At phot2 was visualized by SAXS (20). These conformational changes in the linker region may cancel the inhibition of kinase activity by LOV2 and activate the phot kinase (21) to phosphorylate phot itself (22)(23)(24), as well as artificial substrates (25,26).…”

Mutations in N-terminal Flanking Region of Blue Light-sensing Light-Oxygen and Voltage 2 (LOV2) Domain Disrupt Its Repressive Activity on Kinase Domain in the Chlamydomonas Phototropin

“…These structural changes are presumed to disrupt the inhibitory interaction of LOV2 with the kinase domains. Accordingly, photoreversible contact/ separation between the LOV2 and the kinase domains has been suggested by small angle x-ray scattering in a LOV2 linker kinase fragment of Arabidopsis PHOT2 (29).…”