Stability of dimer and domain-domain interaction of Arabidopsis phototropin 1 LOV2

Transient grating signals after photoexcitation of Arabidopsis phototropin 1 light-oxygen-voltage 2 (phot1LOV2) domain without the linker were found to be very sensitive to temperature. In particular, the diffusion signal drastically increased with rising temperature. The signal was consistently explained by the superposition of the photo-induced dissociation and association reactions. This observation indicated the presence of an equilibrium between the monomer and dimer forms of the phot1LOV2 domain in the dark. The equilibrium was confirmed by a gel chromatographic technique. The equilibrium constants at various temperatures were calculated from the fraction of the dimer, and the stabilization enthalpy and entropy were determined. Interestingly, the transient grating signal of phot1LOV2 with the linker (phot1LOV2-linker), which exists as the monomer form, was also temperature dependent; the diffusion signal intensity decreased with increasing temperature. Because the diffusion signal reflects a conformation change of the linker upon photoexcitation, this temperature dependence indicated that there were two forms of the phot1LOV2-linker. One form exhibited a conformational change upon photoexcitation whereas the other form showed no change. These two forms are not distinguishable spectroscopically. The fraction of these species depended on the temperature. Considering the monomer-dimer equilibrium of the phot1LOV2 domain, we suggest that the nonreactive form possesses the linker region that is dissociated from the LOV2 domain. Because the dissociation of the linker region from the LOV2 domain is a key step for the conformation change of the phot1LOV2-linker to induce biological activity, we proposed that the phototropins could have a role as a temperature sensor.     

Dynamics of conformational changes of Arabidopsis phototropin 1 LOV2 with the linker domain

Conformational changes of Arabidopsis phot1-LOV2 with the linker (phot1-LOV2-linker) were investigated from the viewpoint of the changes in molecular volume and molecular diffusion coefficient (D) by time-resolved transient grating (TG) and transient lens (TrL) methods. Although the absorption spectrum change completes within a few microseconds, the D-value detected by the TG method decreased drastically with a time constant of 1.0 ms from 9.2(+/-0.4)x10(-11) m(2)/s to 5.0(+/-0.3)x10(-11) m(2)/s. This time-dependent D was interpreted in terms of the unfolding of alpha-helices in the linker region. The change of the alpha-helices was confirmed by observing the recovery of the circular dichroism intensity. The TrL signal showed that the molecular volume decreases with two time constants; 300 micros and 1.0 ms. The former time constant is close to the previously observed photo-dissociation reaction rate of the phot1-LOV2 (without the linker) dimer, and the latter one agrees well with the rate of the D-change. Considering a similar time constant of the dissociation reaction of the LOV2 dimer, we interpreted these kinetics in terms of the dissociation step of the linker region from the LOV2 domain (T(390)(pre) state). After this step, the protein volume and D are decreased significantly with the lifetime of 1.0 ms. The D decrease indicates the increase of the intermolecular interaction between the protein and water molecules. On the basis of these observations, a two-step mechanism of the linker unfolding is proposed.     

Design and Signaling Mechanism of Light-Regulated Histidine Kinases

Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by ∼ 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40°-60° rotational movement within an α-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by α-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.     

Addition at the Molecular Level: Signal Integration in Designed Per-ARNT-Sim Receptor Proteins

Survival of organisms in dynamic environments requires accurate perception and integration of signals. At the molecular level, signal detection is mediated by signal receptor proteins that largely are of modular composition. Sensor modules, such as the widespread Per-ARNT-Sim (PAS) domains, detect signals and, in response, regulate the biological activity of effector modules. Here, we exploit the modularity of signal receptors to design and engineer synthetic receptors that comprise two PAS sensor domains responsive to different signals, and we use these signals to control the activity of a histidine kinase effector. Designed two-input PAS receptors detected oxygen and blue light in a positive cooperative manner. The extent of the response to the signals was dictated by domain topology: the dominant regulatory effect was exerted by the PAS domain proximal to the effector domain. The presence of one sensor domain modulated the signal response function of the other. Sequence and structural data on natural receptors with tandem PAS domains show that these are predominantly linked by short amphipathic α-helices. Signals from multiple sensor domains could be integrated and propagated to the effector domain as torques. Our results inform the rational design of receptors that integrate multiple signals to modulate cellular behavior.     

Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2

Phototropin (phot) is a blue-light receptor protein that triggers phototropic responses, chloroplast relocation, and stomata opening to maximize the efficiency of photosynthesis in higher plants. Phot is composed of three functional domains. The N-terminal half folds into two light-oxygen-voltage-sensing domains called LOV1 and LOV2, each binding a flavin mononucleotide to absorb blue light. The C-terminal half is a serine/threonine kinase domain that causes light-dependent autophosphorylation leading to cellular signaling cascades. LOV2 domain is primarily responsible for activation of the kinase, and LOV1 domain is thought to act as a dimerization site and to regulate sensitivity to activation by blue light. Here we show the crystal structures of LOV1 domains of Arabidopsis phot1 and phot2 in the dark at resolutions of 2.1 Å and 2.0 Å, respectively. Either LOV1 domain forms a dimer through face-to-face association of β-scaffolds in the crystallographic asymmetric unit. Three types of interactions stabilizing the dimer structures found are as follows: contacts of side chains in their β-scaffolds, hydrophobic interactions of a short helix found in the N-terminus of a subunit with the β-scaffolds of both subunits, and hydrogen bonds mediated by hydration water molecules filling the dimer interface. The critical residues for dimerization are Cys261, forming a disulfide bridge between subunits in phot1-LOV1 domain, and Thr217 and Met232 in phot2-LOV1. The topology in homodimeric associations of the LOV1 domains is discussed when referring to those of homodimers or heterodimers of light-oxygen-voltage-sensing or Per-ARNT-Sim domains. The present results also provide clues to understanding structural basis in dimeric interactions of Per-ARNT-Sim protein modules in cellular signaling.     

Effect of computational methodology on the conformational dynamics of the protein photosensor LOV1 from Chlamydomonas reinhardtii

LOV domains are the light-sensitive protein domains of plant phototropins and bacteria. They photochemically form a covalent bond between a flavin mononucleotide (FMN) chromophore and a cysteine, attached to the apo-protein, upon irradiation with blue light, which triggers a signal in the adjacent kinase. Although their signaling state has been well characterized through experimental means, their signal transduction pathway as well as dark-state activity are generally only poorly understood. Here we show results from molecular dynamics simulations where we investigated the effect of thermostating and long-range electrostatics on the solution structure and dynamical behavior of the wild-type LOV1 domain from the green algae Chlamydomonas reinhardtii in the dark. We demonstrate that these computational issues can dramatically affect the conformational fluctuations of such protein domains by suppressing configurations far from equilibrium or destabilizing local configurations, leading to artificial changes of the protein secondary structure as well as the H-bond network formed by the amino acids and the FMN. By comparing our calculation results with recent experimental data, we show that the non-invasive thermostating strategy, where the protein solute is only indirectly coupled to the thermostat via the solvent, in conjunction with the particle-mesh Ewald technique, provides dark-state conformers, which are in consistency with experimental observations. Moreover, our calculations indicate that the LOV1 domains can alter the intersystem crossing rate and rate of adduct formation by adjusting the population distribution of these dark-state conformers. This might permit them to function as a modulator of the signal intensity under low light conditions.     

The Switch that Does Not Flip: The Blue-Light Receptor YtvA from Bacillus subtilis Adopts an Elongated Dimer Conformation Independent of the Activation State as Revealed by a Combined AUC and SAXS Study

Photoreceptors play an important role in plants and bacteria by converting extracellular stimuli into intracellular signals. One distinct class are the blue-light-sensitive phototropins harboring a light-oxygen-voltage (LOV) domain coupled to various effector domains. Photon absorption by the chromophore within the LOV domain results in an activation of the output domain via mechanisms that are hitherto not well understood. The photoreceptor YtvA from Bacillus subtilis is a bacterial analog of phototropins, consists of an LOV and a sulfate transporter/anti-sigma factor antagonist domain, and is involved in the response of the bacterium to environmental stress. We present here analytical ultracentrifugation studies and small-angle X-ray scattering experiments, showing that YtvA is a dimer. On the basis of these results, we present a low-resolution model of the dimer in the dark and the lit state of the protein. In addition, we show that YtvA does not change its oligomerization state or its overall shape upon light activation.     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.     

NTP-binding properties of the blue-light receptor YtvA and effects of the E105L mutation

YtvA is a blue-light-sensing protein from Bacillus subtilis related to plant phototropins. It carries a LOV (light, oxygen and voltage) domain, binding FMN (flavin mononucleotide) as chromophore, and a STAS (sulphate transporters and antisigma-factor antagonists) domain with poorly characterized function. We have recently shown that YtvA binds triphosphate nucleotides (NTP) and highlighted a structural similarity between the STAS domain and small GTP-binding proteins. In this work we further investigated the NTP-binding properties of YtvA, employing a fluorescent derivative of GTP (GTP_TR) and mutagenesis experiments. The main results are as follows: (a) competition experiments indicate that the affinity of YtvA for GTP is much higher than that for GDP and GMP. (b) Blue-light-induced structural changes are transmitted from the LOV core to the NTP-binding cavity, establishing a possible intraprotein signal-transduction pathway. (c) A mutation in the central β-scaffold of the LOV core, E105L, impairs the light-driven spectroscopic changes of bound GTP_TR. This result is supported by circular dichroism data, in that YtvA-E105L does not show the light-induced conformational change in the turn fraction that characterizes YtvA, implying that E105 is functionally important. (d) In the structural model of the LOV-STAS complex, based on docking algorithms, the interface includes the Iβ–Hβ loop on the LOV core, as well as parts of the central β-scaffold. E105 is predicted to interact with the LOV-STAS linker region, suggested to play a role in phototropin signaling. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Structural basis for polyproline recognition by the FE65 WW domain

The neuronal protein FE65 functions in brain development and amyloid precursor protein (APP) signaling through its interaction with the mammalian enabled (Mena) protein and APP, respectively. The recognition of short polyproline sequences in Mena by the FE65 WW domain has a central role in axon guidance and neuronal positioning in the developing brain. We have determined the crystal structures of the human FE65 WW domain (residues 253-289) in the apo form and bound to the peptides PPPPPPLPP and PPPPPPPPPL, which correspond to human Mena residues 313-321 and 347-356, respectively. The FE65 WW domain contains two parallel ligand-binding grooves, XP (formed by residues Y269 and W280) and XP2 (formed by Y269 and W271). Both Mena peptides adopt a polyproline helical II conformation and bind to the WW domain in a forward (N-C) orientation through selection of the PPPPP motif by the XP and XP2 grooves. This mode of ligand recognition is strikingly similar to polyproline interaction with SH3 domains. Importantly, comparison of the FE65 WW structures in the apo and liganded forms shows that the XP2 groove is formed by an induced-fit mechanism that involves movements of the W271 and Y269 side-chains upon ligand binding. These structures elucidate the molecular determinants underlying polyproline ligand selection by the FE65 WW domain and provide a framework for the design of small molecules that would interfere with FE65 WW-ligand interaction and modulate neuronal development and APP signaling.     

Structural basis of the collagen-binding mode of discoidin domain receptor 2

Discoidin domain receptor (DDR) is a cell-surface receptor tyrosine kinase activated by the binding of its discoidin (DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 (DDR2-DS domain), and identified the binding site to fibrillar collagen by transferred cross-saturation experiments. The DDR2-DS domain structure adopts a distorted jellyroll fold, consisting of eight beta-strands. The collagen-binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen-binding site suggests that the DDR2-DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen-binding mode of the DDR2-DS domain.     

On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal, FAD-binding BLUF photoreceptor domain. Upon illumination, the AppA BLUF domain forms a signaling state that is characterized by red-shifted absorbance by 10 nm, a state known as AppA_RED. We have applied ultrafast spectroscopy on the photoaccumulated AppA_RED state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppA_RED, the FAD singlet excited state decays monoexponentially in 7 ps to form the neutral semiquinone radical FADH^•, which subsequently decays to the original AppA_RED molecular ground state in 60 ps. Thus, is deactivated rapidly via electron and proton transfer, probably from the conserved tyrosine Tyr-21 to FAD, followed by radical-pair recombination. We conclude that, in contrast to many other photoreceptors, the AppA BLUF domain is not photoreversible and does not enter alternative reaction pathways upon absorption of a second photon. To explain these properties, we propose that a molecular configuration is formed upon excitation of AppA_RED that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppA_RED, the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppA_RED ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppA_RED is significantly more rigid than in dark-state AppA. Steady-state fluorescence experiments on wild-type, W104F, and W64F mutant BLUF domains show tryptophan fluorescence maxima that correspond with a buried conformation of Trp-104 in dark and light states. We conclude that Trp-104 does not become exposed to solvent during the BLUF photocycle.     

Structural basis for recognition of ubiquitinated cargo by Tom1-GAT domain

Tom1 (Target of Myb1) is suggested to be involved in the transport of ubiquitinated proteins, through the interaction of its GAT (GGA and Tom1) domain with ubiquitin. Here, we demonstrate that the three-helix bundle of Tom1-GAT has two ubiquitin-binding sites recognizing the hydrophobic Ile44 surface of ubiquitin. The complex crystal structure demonstrates that the first site is a hydrophobic patch on helices alpha1 and alpha2. NMR and biochemical data revealed that the N-terminal half of helix alpha3 of Tom1-GAT constitutes the second, stronger binding site. The double-sided ubiquitin binding enhances the efficiency of recognition of ubiquitinated proteins by Tom1.     

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

Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin.     

A conserved isoleucine in the LOV1 domain of a novel phototropin from the marine alga Ostreococcus tauri modulates the dark state recovery of the domain

Background

Phototropins are UV-A/blue light receptor proteins with two LOV (Light-Oxygen-Voltage) sensor domains at their N terminus and a kinase domain at the C-terminus in photoautotrophic organisms. This is the first research report of a canonical phototropin from marine algae Ostreococcus tauri.

Methods

We synthesized core LOV1 (OtLOV1) domain-encoding portion of the phototropin gene of O. tauri, the domain was heterologously expressed, purified and assessed for its spectral properties and dark recovery kinetics by UV–Visible, fluorescence spectroscopy and mutational studies. Quaternary structure characteristics were studied by SEC and glutaraldehyde crosslinking.

Results

The absorption spectrum of OtLOV1 lacks the characteristic 361 nm peak shown by other LOV1 domains. It undergoes a photocycle with a dark state recovery time of approximately 30 min (τ = 300.35 s). Native OtLOV1 stayed as dimer in aqueous solution and the dimer formation was light and concentration independent. Mutating isoleucine at 43rd position to valine accelerated the dark recovery time by more than 10-fold. Mutating it to serine reduced sensitivity to blue light, but the dark recovery time remained unaltered. I43S mutation also destabilized the FMN binding to a great extent.

Conclusion

The OtLOV1 domain of the newly identified OtPhot is functional and the isoleucine at position 43 of OtLOV1 is the key residue responsible for fine-tuning the domain properties.

General significance

This is the first characterized LOV1 domain of a canonical phototropin from a marine alga and spectral properties of the domain are similar to that of the LOV1 domain of higher plants.