Photoexcited Structure of a Plant Photoreceptor Domain Reveals a Light-Driven Molecular Switch

Abstract

The phototropins are flavoprotein kinases that control phototropic bending, light-induced chloroplast movement, and stomatal opening in plants. Two flavin mononucleotide binding light, oxygen, or voltage (LOV) domains are the sites for initial photochemistry in these blue light photoreceptors. We have determined the steady state, photoexcited crystal structure of a flavin-bound LOV domain. The structure reveals a unique photochemical switch in the flavin binding pocket in which the absorption of light drives the formation of a reversible covalent bond between a highly conserved Cys residue and the flavin cofactor. This provides a molecular picture of a cysteinyl-flavin covalent adduct, the presumed signaling species that leads to phototropin kinase activation and subsequent signal transduction. We identify closely related LOV domains in two eubacterial proteins that suggests the light-induced conformational change evident in this structure is an ancient biomolecular response to light, arising before the appearance of plants. INTRODUCTIONAt least nine photoreceptors that control plant growth and development have been identified in Arabidopsis (Arabidopsis Genome Initiative, 2000), and many genetic and biochemical interactions between these photoreceptors and their downstream signaling partners have been established (reviewed by Singhal et al., 1999). However, the question of how a light signal is transduced by plant photoreceptors at the structural level remains unexplored. Light, oxygen, or voltage (LOV) domains, a subset of the Per-ARNT-Sim (PAS) domain superfamily (Lagarias et al., 1995;Taylor and Zhulin, 1999), bind a single molecule of flavin mononucleotide (FMN) (Christie et al., 1999) and function as sites for blue light absorption and initial photochemistry in the phototropin family of photoreceptors (phot1 and phot2) (Huala et al., 1997;Christie et al., 1998). The phototropins are Ser/Thr kinases that contain two N-terminal LOV domains (LOV1 and LOV2) (Huala et al., 1997), autophosphorylate in response to the absorption of blue light (Christie et al., 1998), and control phototropism, light-induced chloroplast movement, and stomatal opening (Huala et al., 1997;Christie et al., 1998;Jarillo et al., 2001;Kagawa et al., 2001;Kinoshita et al., 2001). The highly homologous phot1 and phot2 photoreceptors exhibit fluence-dependent functional overlap in controlling these physiologically distinct processes , as shown in Figure 1.Classic experiments by Briggs and colleagues (1957) demonstrated that phototropism is a direct result of lateral auxin transport away from the source of light. Chloroplast movement is mediated by microtubules and microfilaments of the cytoskeleton (Sato et al., 2001), and stomatal opening is controlled by the activation of a membrane-bound H ϩ -ATPase and subsequent transport of K ϩ into the guard cells (Schroeder et al., 2001). Thus, phot1 and phot2 affect at least three distinct cellular processes: auxin transport, cytoskeleton-mediated organelle motility, and ion transport. However, the...

Phototropin Receptor Kinase Activation by Blue Light

“…7,8 Moreover, this mutation has been used successfully to probe the roles of LOV1 and LOV2 in regulating phototropin activity and function. [9][10][11][12] As reported previously (ref. 13), the Cys → Ala mutation within LOV2 of Arabidopsis thaliana phot1 (C517A) results in a loss of light-induced receptor autophosphorylation when expressed in insect cells (Fig.…”

“…21,22 Specifically, the side chain of a conserved glutamine residue within LOV2 (Gln 575 in Arabidopsis phot1) which forms hydrogen bonds with the FMN chromophore flips by 180° upon cysteinyl adduct formation 11,23,24 causing protein changes in the central b-sheet scaffold that forms contacts with the Ja-helix. 19,20 We have recently shown that mutation of the conserved glutamine to leucine (Q575L) attenuates light-induced autophosphorylation of Arabidopsis phot1 expressed in insect cells, 13 suggesting that this residue plays a role in transmitting the signal generated upon light-driven cysteinyl adduct formation from within LOV2 to protein changes at the LOV2 surface.…”

Mechanism of signal transduction of the LOV2-Jα photosensor from Avena sativa

“…In this context, it is worth mentioning that the glutamine residue adjacent to the FMN chromophore is highly conserved and has previously been suggested to be crucial for the signalling process of the LOV domains. Several early experimental studies on the isolated LOV2 domain suggested that the primary process after adduct formation involves the breakage of an H-bond between the glutamine residue and FMN-O4 10 , possibly followed through rotation of its sidechain by the formation of a new H-bond with FMN-N5 14,20 . In a later crystallographic study on the AsLOV2-Jα system, Halavaty and Moffat 13 proposed a local reorientation of the conserved glutamine, associated with a disruption of an H-bond between an asparagine and aspartic acid on the surface of the protein.…”

“…Although these studies clearly identified the C-terminal Jα helix to be responsible for communicating the signal to a downstream effector domain, it remained unclear how covalent adduct formation in the core triggers the α-helical unfolding on the surface of the domain. In a series of experimental and theoretical studies with LOV2 domains, a conserved glutamine residue, located on the Iβ strand in the immediate vicinity of the FMN-binding site, was found to change its H-bonding pattern with the FMN upon photoexcitation and was suspected to be involved in the transmission of the local stress to the surface of the LOV domain 4,10,14,15 . By designing point mutations of this glutamine residue and monitoring the effects on the LOV2 domain using ultraviolet-visible absorbance and circular dichroism spectroscopy, Nash et al 16 recently confirmed that it plays a central role in both spectral tuning and signal propagation from the LOV2 core to the peripheral Jα helix.…”

A novel computer simulation method for simulating the multiscale transduction dynamics of signal proteins

“…8 While the in vivo functionality of the LOV1 domain within the protein complex still remains unclear, the photochemical reactivity of the LOV2 domain has been found to be essential for the activation of the kinase. 9 As demonstrated in a series of experimental investigations, [10][11][12][13] the LOV2 domain releases upon illumination its inhibitory effect on the kinase by detaching a peripheral α-helix, the so-called Jα-helix, from the LOV core. The early mechanism of activation was recently elucidated by us at atomistic resolution using MD simulation.…”