Exploring the multiscale signaling behavior of phototropin1 from Chlamydomonas reinhardtii using a full‐residue space kinetic Monte Carlo molecular dynamics technique

Devising analysis tools for elucidating the regulatory mechanism of complex enzymes has been a challenging task for many decades. It generally requires the determination of the structural‐dynamical information of protein solvent systems far from equilibrium over multiple length and time scales, which is still difficult both theoretically and experimentally. To cope with the problem, we introduce a full‐residue space multiscale simulation method based on a combination of the kinetic Monte Carlo and molecular dynamics techniques, in which the rates of the rate‐determining processes are evaluated from a biomolecular forcefield on the fly during the simulation run by taking into account the full space of residues. To demonstrate its reliability and efficiency, we explore the light‐induced functional behavior of the full‐length phototropin1 from Chlamydomonas reinhardtii (Cr‐phot1) and its various subdomains. Our results demonstrate that in the dark state the light oxygen voltage‐2‐Jα (LOV2‐Jα) photoswitch inhibits the enzymatic activity of the kinase, whereas the LOV1‐Jα photoswitch controls the dimerization with the LOV2 domain. This leads to the repulsion of the LOV1‐LOV2 linker out of the interface region between both LOV domains, which results in a positively charged surface suitable for cell–membrane interaction. By contrast, in the light state, we observe that the distance between both LOV domains is increased and the LOV1‐LOV2 linker forms a helix–turn–helix (HTH) motif, which enables gene control through nucleotide binding. Finally, we find that the kinase is activated through the disruption of the Jα‐helix from the LOV2 domain, which is followed by a stretching of the activation loop (A‐loop) and broadening of the catalytic cleft of the kinase. Proteins 2014; 82:2018–2040. © 2014 Wiley Periodicals, Inc.     

Signaling pathway of a photoactivable Rac1-GTPase in the early stages

In modern life‐ and medical‐sciences major efforts are currently concentrated on creating artificial photoenzymes, consisting of light‐ oxygen‐voltage‐sensitive (LOV) domains fused to a target enzyme. Such protein constructs possess great potential for controlling the cell metabolism as well as gene function upon light stimulus. This has recently been impressively demonstrated by designing a novel artificial fusion protein, connecting the AsLOV2‐Jα‐photosensor from Avena sativa with the Rac1‐GTPase (AsLOV2‐Jα‐Rac1), and by using it, to control the motility of cancer cells from the HeLa‐line. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their signaling pathway after photoexcitation is still in its infancy. Here, we show through computer simulations of the AsLOV2‐Jα‐Rac1‐photoenzyme that the early processes after formation of the Cys450‐FMN‐adduct involve the breakage of a H‐bond between the carbonyl oxygen FMN‐C4?O and the amino group of Gln513, followed by a rotational reorientation of its sidechain. This initial event is followed by successive events including β‐sheet tightening and transmission of torsional stress along the Iβ‐sheet, which leads to the disruption of the Jα‐helix from the N‐terminal end. Finally, this process triggers the detachment of the AsLOV2‐Jα‐photosensor from the Rac1‐GTPase, ultimately enabling the activation of Rac1 via binding of the effector protein PAK1. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

A critical element of the light‐induced quaternary structural changes in YtvA‐LOV

YtvA, a photosensory LOV (light‐oxygen‐voltage) protein from Bacillus subtilis, exists as a dimer that previously appeared to undergo surprisingly small structural changes after light illumination compared with other light‐sensing proteins. However, we now report that light induces significant structural perturbations in a series of YtvA‐LOV domain derivatives in which the Jα helix has been truncated or replaced. Results from native gel analysis showed significant mobility changes in these derivatives after light illumination; YtvA‐LOV without the Jα helix dimerized in the dark state but existed as a monomer in the light state. The absence of the Jα helix also affected the dark regeneration kinetics and the stability of the flavin mononucleotide (FMN) binding to its binding site. Our results demonstrate an alternative way of photo‐induced signal propagation that leads to a bigger functional response through dimer/monomer conversions of the YtvA‐LOV than the local disruption of Jα helix in the As‐LOV domain.     

The amino-terminal helix modulates light-activated conformational changes in AsLOV2

The mechanism of light-triggered conformational change and signaling in light-oxygen-voltage (LOV) domains remains elusive in spite of extensive investigation and their use in optogenetic studies. The LOV2 domain of Avenasativa phototropin 1 (AsLOV2), a member of the Per-Arnt-Sim (PAS) family, contains a flavin mononucleotide chromophore that forms a covalent bond with a cysteine upon illumination. This event leads to the release of the carboxy-terminal Jα helix, the biological output signal. Using mutational analysis, circular dichroism, and NMR, we find that the largely ignored amino-terminal helix is a control element in AsLOV2's light-activated conformational change. We further identify a direct amino-to-carboxy-terminal "input-output" signaling pathway. These findings provide a framework to rationalize the LOV domain architecture, as well as the signaling mechanisms in both isolated and tandem arrangements of PAS domains. This knowledge can be applied in engineering LOV-based photoswitches, opening up new design strategies and improving existing ones.     

Optogenetic Navigation of Routes Leading to Protein Amyloidogenesis in Bacteria

Modulation of liquid–liquid and liquid–hydrogel phase transitions is central to avoid the cytotoxic aggregation of proteins in eukaryotic cells, but knowledge on its relevance in bacteria is limited. Here the power of optogenetics to engineer proteins as light-responsive switches has been used to control the balance between solubility and aggregation for LOV2–WH1, a chimera between the plant blue light-responsive domain LOV2 and the bacterial prion-like protein RepA-WH1. These proteins were first linked by fusing, as a continuous α-helix, the C-terminal photo-transducer Jα helix in LOV2 with the N-terminal domain-closure α1 helix in RepA-WH1, and then improved for light-responsiveness by including mutations in the Jα moiety. In the darkness and in a crowded solution in vitro, LOV2–WH1 nucleates the irreversible assembly of amyloid fibers into a hydrogel. However, under blue light illumination, LOV2–WH1 assembles as soluble oligomers. When expressed in Escherichia coli, LOV2–WH1 forms in the darkness large intracellular amyloid inclusions compatible with bacterial proliferation. Strikingly, under blue light, LOV2–WH1 aggregates decrease in size, while they become detrimental for bacterial growth. LOV2–WH1 optogenetics governs the assembly of mutually exclusive inert amyloid fibers or cytotoxic oligomers, thus enabling the navigation of the conformational landscape of protein amyloidogenesis to generate potential photo-activated anti-bacterial devices (optobiotics).     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Dynamics of light-induced activation in the PAS domain proteins LOV2 and PYP probed by time-resolved tryptophan fluorescence

Light-induced activation of the LOV2-Jα domain of the photoreceptor phototropin from oat is believed to involve the detachment of the Jα helix from the central β-sheet and its subsequent unfolding. The dynamics of these conformational changes were monitored by time-resolved emission spectroscopy with 100 ns time resolution. Three transitions were detected during the LOV2-Jα photocycle with time constants of 3.4 μs, 500 μs, and 4.3 ms. The fastest transition is due to the decay of the flavin phosphorescence in the transition of the triplet LOV(L)(660) state to the singlet LOV(S)(390) signaling state. The 500 μs and 4.3 ms transitions are due to changes in tryptophan fluorescence and may be associated with the dissociation and unfolding of the Jα helix, respectively. They are absent in the transient absorption signal of the flavin chromophore. The tryptophan fluorescence signal monitors structural changes outside the chromophore binding pocket and indicates that there are at least three LOV(S)(390) intermediates. Since the 500 μs and 4.3 ms components are absent in a construct without the Jα helix and in the mutant W557S, the fluorescence signal is mainly due to tryptophan 557. The kinetics of the main 500 μs component is strongly temperature dependent with activation energy of 18.2 kcal/mol suggesting its association with a major structural change. In the structurally related PAS domain protein PYP the N-terminal cap dissociates from the central β-sheet and unfolds upon signaling state formation with a similar time constant of ～1 ms. Using transient fluorescence we obtained a nearly identical activation energy of 18.5 kcal/mol for this transition.     

Structural insights into Rhino‐Deadlock complex for germline piRNA cluster specification

PIWI‐interacting RNAs (piRNAs) silence transposons in germ cells to maintain genome stability and animal fertility. Rhino, a rapidly evolving heterochromatin protein 1 (HP1) family protein, binds Deadlock in a species‐specific manner and so defines the piRNA‐producing loci in the Drosophila genome. Here, we determine the crystal structures of Rhino‐Deadlock complex in Drosophila melanogaster and simulans. In both species, one Rhino binds the N‐terminal helix–hairpin–helix motif of one Deadlock protein through a novel interface formed by the beta‐sheet in the Rhino chromoshadow domain. Disrupting the interface leads to infertility and transposon hyperactivation in flies. Our structural and functional experiments indicate that electrostatic repulsion at the interaction interface causes cross‐species incompatibility between the sibling species. By determining the molecular architecture of this piRNA‐producing machinery, we discover a novel HP1‐partner interacting mode that is crucial to piRNA biogenesis and transposon silencing. We thus explain the cross‐species incompatibility of two sibling species at the molecular level.     

Modulating LOV domain photodynamics with a residue alteration outside the chromophore binding site

Phototropins, a class of light-activated protein kinases, are essential for several blue light responses in plants and algae, including phototropism. These proteins contain two internal light, oxygen, and voltage sensitive (LOV) domains, which bind flavin chromophores and undergo a reversible photochemical formation of a cysteinyl-flavin adduct as part of the light sensing process. While the photodynamic properties of such photosensory domains are dictated by interactions between the chromophore and surrounding protein, more distant residues can play a significant role as well. Here we explore the role of the Phe434 residue in the photosensory response of the second LOV domain of Avena sativa phototropin 1 (AsLOV2), a model photochemical system for these LOV domains. Phe434 is more than 6 ? from the FMN chromophore in AsLOV2; nevertheless, an F434Y point mutation is likely to change several structural features of the chromophore binding site, as we demonstrate using molecular dynamics simulations. Transient absorption signals spanning 15 decades in time were compared for wild-type AsLOV2 and the F434Y mutant, showing that the latter has significantly altered photodynamics, including (i) a faster intersystem crossing leading to triplet formation on a nanosecond time scale, (ii) biphasic formation of adduct-state kinetics on the microsecond time scale, and (iii) greatly accelerated ground-state recovery kinetics on a second time scale. We present mechanistic models that link these spectroscopic differences to changes in the configuration of the critical cysteine residue and in the chromophore's accessibility to solvent and oxygen according to MD trajectories and purging experiments. Taken together, these results demonstrate the importance of residues outside the chromophore-binding pocket in modulating LOV domain photodynamics.     

Aromatic interactions with naphthylalanine in a β‐hairpin peptide

Stable peptides have been explored as epitope mimics for protein–protein and protein–nucleic acid interactions; however, presentation of a regular structure is critical. Aromatic interactions are ubiquitous and are competent at stabilizing a β‐hairpin fold. The greatest stabilization has been reported from pairs of tryptophan side chains. Naphthylalanine residues are often used as tryptophan replacements, but it is not clear if 1‐naphthylalanine or 2‐naphthylalanine is adequate at replicating the geometry and stability observed with tryptophan aromatic interactions. Herein, a 12‐residue peptide has been constructed with laterally disposed aromatic amino acids. A direct comparison is made between tryptophan and other bicyclic, unnatural amino acids. Significant stabilization is gained from all bicyclic amino acids; however, geometric analysis shows that only 1‐naphthylalanine adopts a similar edge to face geometry as tryptophan, whereas the 2‐naphthylalanine appears most similar to a substituted phenylalanine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion independent of its topoisomerase II inhibition

The macrolide compound MFTZ‐1 has been identified as a novel topoisomerase II (Top2) inhibitor with potent in vitro and in vivo anti‐tumour activities. In this study, we further examined the effects of MFTZ‐1 on hypoxia‐inducible factor‐1α (HIF‐1α) accumulation, vascular endothelial growth factor (VEGF) secretion and angiogenesis. MFTZ‐1 reduced HIF‐1α accumulation driven by hypoxia or growth factors in human cancer cells. Mechanistic studies revealed that MFTZ‐1 did not affect the degradation of HIF‐1α protein or the level of HIF‐1α mRNA. By contrast, MFTZ‐1 apparently inhibited constitutive and inducible activation of both phosphatidylinositol‐3‐kinase (PI3K)‐Akt and p42/p44 mitogen‐activated protein kinase (MAPK) pathways. Further studies revealed that MFTZ‐1 abrogated the HIF‐1α‐driven increase in VEGF mRNA and protein secretion. MFTZ‐1 also lowered the basal level of VEGF secretion. The results reveal an important feature that MFTZ‐1 can reduce constitutive, HIF‐1α‐independent VEGF secretion and concurrently antagonize inducible, HIF‐1α‐dependent VEGF secretion. Moreover, MFTZ‐1 disrupted tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by hypoxia with low‐concentration serum or by serum at normoxia, and inhibited HUVECs migration at normoxia. MFTZ‐1 also prevented microvessel outgrowth from rat aortic ring. These data reflect the potent anti‐angiogenesis of MFTZ‐1 under different conditions. Furthermore, using specific small interfering RNA targeting Top2α or Top2‐defective HL60/MX2 cells, we showed that MFTZ‐1 affected HIF‐1α accumulation and HUVECs tube formation irrelevant to its Top2 inhibition. Taken together, our data collectively reveal that MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion possibly via PI3K‐Akt and MAPK pathways, eliciting anti‐angiogenesis independently of its Top2 inhibition.     

Amyloid precursor protein expression is induced by tumor necrosis factor α in 3T3‐L1 adipocytes

Amyloid precursor protein (APP) has been characterized as an adipocyte‐secreted protein that might contribute to obesity‐related insulin resistance, inflammation, and dementia. In the current study, regulation of APP by the proinflammatory and insulin resistance‐inducing cytokine tumor necrosis factor (TNF) α was determined in 3T3‐L1 adipocytes. Interestingly, APP protein synthesis and mRNA expression were significantly increased by TNFα in a time‐dependent manner with maximal induction observed after 24 h of treatment. Furthermore, TNFα induced APP mRNA expression dose‐dependently with maximal 6.4‐fold upregulation seen at 100 ng/ml effector. Moreover, inhibitor experiments suggested that TNFα‐induced APP expression was mediated by nuclear factor κ B. Taken together, we show for the first time a potent upregulation of APP by TNFα suggesting a potential role of this adipocyte‐secreted protein in TNFα‐induced insulin resistance and inflammatory disease. J. Cell. Biochem. 108: 1418–1422, 2009. © 2009 Wiley‐Liss, Inc.     

Fluorescence Imaging-Based High-Throughput Screening of Fast- and Slow-Cycling LOV Proteins

Light-oxygen-voltage (LOV) domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2). The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3×10³ s (78-fold slower than wild-type AsLOV2). The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.     

Novel blue light-sensitive proteins from a metagenomic approach

A microarray‐based approach was used to screen a soil metagenome for the presence of blue light (BL) photoreceptor‐encoding genes. The microarray carried 149 different 54‐mer oligonucleotides, derived from consensus sequences of light, oxygen and voltage (LOV) domain BL photoreceptor genes. Calibration of the microarrays allowed the detection of minimally 50 ng of genomic DNA against a background of 2–5 μg of genomic DNA. Identification of a positive cosmid clone was still possible for an amount of 0.25 ng against a background of 10 μg of labelled DNA clones. The array could readily identify targets carrying 4% sequence mismatch. Using the LOV microarray, up to 1200 library clones in concentrations of c. 20 ng each with a c. 40 kb insert size could be screened in a single batch. After calibration and reliability controls, the microarray was probed with cosmid‐cloned DNA from the thermophilic fraction of a soil sample. From this approach, a novel gene was isolated that encodes a protein consisting of several Per‐Arnt‐Sim domains, a LOV domain associated to a histidine kinase and a response regulator domain. The novel gene showed highest similarity to a known sequence from Kineococcus radiotolerans SRS30216 (58% identity for the LOV domain only) and to a gene from Methylibium petroleiphilum PM1 (57% identity). The gene, designated as ht‐met1 (Hamburg Thermophile Metagenome 1), was isolated and fully sequenced (3615 bp). ht‐met1 is followed by a second open reading frame encoding a Fe‐chelatase, an arrangement quite frequent for BL photoreceptors. The LOV domain region of ht‐met1 was subcloned and expressed yielding a fully functional, flavin‐containing LOV domain. Irradiation generated the typical LOV photochemistry, with the transient formation of a flavin‐protein photoadduct. The dark recovery lifetime was found as τ_REC = 120 s (20°C) and is among the fastest ones determined so far for bacterial LOV domains.     

Furanodienone inhibits cell proliferation and survival by suppressing ERα signaling in human breast cancer MCF-7 cells

Estrogen receptor alpha (ERα) plays an important role in the development and progression of breast cancer and thus the attenuation of ERα activities is a promising treatment strategy. Furanodienone is one of the main bioactive chemical components of Rhizoma Curcumae which is commonly used in Chinese medicine for the treatment of cancer. In this study, we investigated the effects of furanodienone on human breast cancer MCF‐7, T47D, and MDA‐MB‐231 cells. Our results showed that furanodienone could inhibit MCF‐7, T47D, and MDA‐MB‐231 cells proliferation in a dose (10–160 µM) dependent manner. ERα‐negative MDA‐MB‐231 cells were less sensitive to furanodienone than ERα‐positive MCF‐7 and T47D cells. Furanodienone could effectively block 17β‐estradiol (E2)‐stimulated MCF‐7 cell proliferation and cell cycle progression and induce apoptosis evidenced by the flow cytometric detection of sub‐G1 DNA content and the appearance of apoptotic nuclei after DAPI staining. Furanodienone specifically down‐regulated ERα protein and mRNA expression levels without altering ERβ expression. Furanodienone treatment inhibited E2‐stimulation of estrogen response element (ERE)‐driven reporter plasmid activity and ablated E2‐targeted gene (e.g., c‐Myc, Bcl‐2, and cyclin D1) expression which resulted in the inhibition of cell cycle progression and cell proliferation, and in the induction of apoptosis. Knockdown of ERα in MCF‐7 cells by ERα‐specific siRNA decreased the cell growth inhibitory effect of furanodienone. These findings suggest that effects of furanodienone on MCF‐7 cells are mediated, at least in part, by inhibiting ERα signaling. J. Cell. Biochem. 112: 217–224, 2011. © 2010 Wiley‐Liss, Inc.     

Illuminating the early signaling pathway of a fungal light‐oxygen‐voltage photoreceptor

Circadian clocks are molecular timekeepers encountered in a wide variety of organisms, which allow to adapt the cell's metabolism and behavior to the daily and seasonal periods. Their function is regulated by light‐sensing proteins, among which Vivid, a light‐oxygen‐voltage (LOV) sensitive domain of the fungus Neurospora crassa, constitutes one of the most prominent examples. Although the major photochemical and structural changes during the photocycle of this photosensor have been elucidated through experimental means, its signal transduction pathway is still poorly resolved at the molecular level. In this article, we show through molecular dynamics simulation that the primary steps after adduct formation involve a switch of Gln182 in vicinity of the chromophore FAD (flavin–adenine–dinucleotide), followed by a coupling between the Iβ‐ and Hβ‐strands through H‐bond formation between Gln182 and Asn161 as well as subsequent weakening of the H‐bonding interaction between the Iβ‐ and Aβ‐strands. These processes then induce a reorientation of the Aβ‐Bβ‐loop with respect to the protein core as well as a simultaneous contraction of the partially unfolded α‐helix onto the α‐Aβ‐linker at the Ncap. Finally, we demonstrate through additional dimer simulations that the light‐induced conformational changes, observed in the monomeric case, play a decisive role in controlling the dimerization tendency of Vivid with its partner domains and that the light‐state homodimer shows a much larger affinity for aggregation than the dark state. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Generation and characterization of PDGFRα‐GFPCreERt2 knock‐In mouse line

Platelet‐derived growth factor (PDGF) and its receptor play an important role in embryogenesis. PDGF receptor α (PDGFRα) is expressed specifically in the embryonic day 7.5 (E7.5) mesoderm and in the E9.5 neural crest among other tissues. PDGFRα‐expressing cells and their descendants are involved in the formation of various tissues. To trace PDGFRα‐expressing cells in vivo, we generated a knock‐in mouse line that expressed a fusion protein of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand‐binding domain (ER^T2) under the control of the PDGFRα promoter. In these mice, Cre activity in PDGFRα‐expressing cells could be induced by tamoxifen treatment. Taken together, our results suggest that the knock‐in mouse line generated here could be useful for studying PDGFRα‐expressing cells and their descendants in vivo at various stages of development. genesis 53:329–336, 2015. © 2015 Wiley Periodicals, Inc.