Residue alterations within a conserved hydrophobic pocket influence light, oxygen, voltage photoreceptor dark recovery.

Light, oxygen, voltage (LOV) photoreceptors are widely distributed throughout all kingdoms of life, and have in recent years, due to their modular nature, been broadly used as sensor domains for the construction of optogenetic tools. For understanding photoreceptor function as well as for optogenetic tool design and fine-tuning, a detailed knowledge of the photophysics, photochemistry, and structural changes underlying the LOV signaling paradigm is instrumental. Mutations that alter the lifetime of the photo-adduct signaling state represent a convenient handle to tune LOV sensor on/off kinetics and, thus, steady-state on/off equilibria of the photoreceptor (or optogenetic switch). Such mutations, however, should ideally only influence sensor kinetics, while being benign with regard to the nature of the structural changes that are induced by illumination, i.e., they should not result in a disruption of signal transduction. In the present study, we identify a conserved hydrophobic pocket for which mutations have a strong impact on the adduct-state lifetime across different LOV photoreceptor families. Using the slow cycling bacterial short LOV photoreceptor PpSB1-LOV, we show that the I48T mutation within this pocket, which accelerates adduct rupture, is otherwise structurally and mechanistically benign, i.e., light-induced structural changes, as probed by NMR spectroscopy and X-ray crystallography, are not altered in the variant. Additional mutations within the pocket of PpSB1-LOV and the introduction of homologous mutations in the LOV photoreceptor YtvA of Bacillus subtilis and the Avena sativa LOV2 domain result in similarly altered kinetics. Given the conserved nature of the corresponding structural region, the here identified mutations should find application in dark-recovery tuning of optogenetic tools and LOV photoreceptors, alike.

The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins.

Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plant-derived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a red-shifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences.

Mechanistic Basis of the Fast Dark Recovery of the Short LOV Protein DsLOV from Dinoroseobacter shibae.

Light, oxygen, voltage (LOV) proteins, a ubiquitously distributed class of photoreceptors, regulate a wide variety of light-dependent physiological responses. Because of their modular architecture, LOV domains, i.e., the sensory domains of LOV photoreceptors, have been widely used for the construction of optogenetic tools. We recently described the structure and function of a short LOV protein (DsLOV) from the marine phototropic bacterium Dinoroseobacter shibae, for which, in contrast to other LOV photoreceptors, the dark state represents the physiologically relevant signaling state. Among bacterial LOV photoreceptors, DsLOV possesses an exceptionally fast dark recovery, corroborating its function as a "dark" sensor. To address the mechanistic basis of this unusual characteristic, we performed a comprehensive mutational, kinetic, thermodynamic, and structural characterization of DsLOV. The mechanistic basis of the fast dark recovery of the protein was revealed by mutation of the previously noted uncommon residue substitution at position 49 found in DsLOV. The substitution of M49 with different residues that are naturally conserved in LOV domains tuned the dark-recovery time of DsLOV over 3 orders of magnitude, without grossly affecting its overall structure or the light-dependent structural change observed for the wild-type protein. Our study thus provides a striking example of how nature can achieve LOV photocycle tuning by subtle structural alterations in the LOV domain active site, highlighting the easy evolutionary adaptability of the light sensory function. At the same time, our data provide guidance for the mutational photocycle tuning of LOV domains, with relevance for the growing field of optogenetics.

Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae.

BACKGROUND: Light, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which flank the LOV core domain, as well as in the lifetime of the adduct state. RESULTS: Here, we report the photochemical, structural and functional characterization of DsLOV, a LOV protein from the photoheterotrophic marine α-proteobacterium Dinoroseobacter shibae which exhibits an average adduct state lifetime of 9.6 s at 20°C, and thus represents the fastest reverting bacterial LOV protein reported so far. Mutational analysis in D. shibae revealed a unique role of DsLOV in controlling the induction of photopigment synthesis in the absence of blue-light. The dark state crystal structure of DsLOV determined at 1.5 Å resolution reveals a conserved core domain with an extended N-terminal cap. The dimer interface in the crystal structure forms a unique network of hydrogen bonds involving residues of the N-terminus and the ß-scaffold of the core domain. The structure of photoexcited DsLOV suggests increased flexibility in the N-cap region and a significant shift in the Cα backbone of ß strands in the N- and C-terminal ends of the LOV core domain. CONCLUSIONS: The results presented here cover the characterization of the unusual short LOV protein DsLOV from Dinoroseobacter shibae including its regulatory function, extremely fast dark recovery and an N-terminus mediated dimer interface. Due to its unique photophysical, structural and regulatory properties, DsLOV might thus serve as an alternative model system for studying light perception by LOV proteins and physiological responses in bacteria.

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process.

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.

Conserved Signal Transduction Mechanisms and Dark Recovery Kinetic Tuning in the Pseudomonadaceae Short Light, Oxygen, Voltage (LOV) Protein Family.

Light-Oxygen-Voltage (LOV) flavoproteins transduce a light signal into variable signaling outputs via a structural rearrangement in the sensory core domain, which is then relayed to fused effector domains via α-helical linker elements. Short LOV proteins from Pseudomonadaceae consist of a LOV sensory core and N- and C-terminal α-helices of variable length, providing a simple model system to study the molecular mechanism of allosteric activation. Here we report the crystal structures of two LOV proteins from Pseudomonas fluorescens - SBW25-LOV in the fully light-adapted state and Pf5-LOV in the dark-state. In a comparative analysis of the Pseudomonadaceae short LOVs, the structures demonstrate light-induced rotation of the core domains and splaying of the proximal A'α and Jα helices in the N and C-termini, highlighting evidence for a conserved signal transduction mechanism. Another distinguishing feature of the Pseudomonadaceae short LOV protein family is their highly variable dark recovery, ranging from seconds to days. Understanding this variability is crucial for tuning the signaling behavior of LOV-based optogenetic tools. At 37 °C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. To investigate this remarkable difference in dark recovery rates, we targeted three residues lining the solvent channel entrance to the chromophore pocket where we introduced mutations by exchanging the non-conserved amino acids from SBW25-LOV into Pf5-LOV and vice versa. Dark recovery kinetics of the resulting mutants, as well as MD simulations and solvent cavity calculations on the crystal structures suggest a correlation between solvent accessibility and adduct lifetime.

Three-dimensional structure of a schistosome serpin revealing an unusual configuration of the helical subdomain.

Parasitic organisms are constantly challenged by the defence mechanisms of their respective hosts, which often depend on serine protease activities. Consequently, protease inhibitors such as those belonging to the serpin superfamily have emerged as protective elements that support the survival of the parasites. This report describes the crystal structure of ShSPI, a serpin from the trematode Schistosoma haematobium. The protein is exposed on the surface of invading cercaria as well as of adult worms, suggesting its involvement in the parasite-host interaction. While generally conforming to the well established serpin fold, the structure reveals several distinctive features, mostly concerning the helical subdomain of the protein. It is proposed that these peculiarities are related to the unique biological properties of a small serpin subfamily which is conserved among pathogenic schistosomes.

Structural, mechanistic, and physiological insights into phospholipase A-mediated membrane phospholipid degradation in Pseudomonas aeruginosa.

Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF-mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.

Structural determinants underlying the adduct lifetime in the LOV proteins of Pseudomonas putida.

The primary photochemistry is similar among the flavin-bound sensory domains of light-oxygen-voltage (LOV) photoreceptors, where upon blue-light illumination a covalent adduct is formed on the microseconds time scale between the flavin chromophore and a strictly conserved cysteine residue. In contrast, the adduct-state decay kinetics vary from seconds to days or longer. The molecular basis for this variation among structurally conserved LOV domains is not fully understood. Here, we selected PpSB2-LOV, a fast-cycling (τrec 3.5 min, 20 °C) short LOV protein from Pseudomonas putida that shares 67% sequence identity with a slow-cycling (τrec 2467 min, 20 °C) homologous protein PpSB1-LOV. Based on the crystal structure of the PpSB2-LOV in the dark state reported here, we used a comparative approach, in which we combined structure and sequence information with molecular dynamic (MD) simulations to address the mechanistic basis for the vastly different adduct-state lifetimes in the two homologous proteins. MD simulations pointed toward dynamically distinct structural region, which were subsequently targeted by site-directed mutagenesis of PpSB2-LOV, where we introduced single- and multisite substitutions exchanging them with the corresponding residues from PpSB1-LOV. Collectively, the data presented identify key amino acids on the Aß-Bß, Eα-Fα loops, and the Fα helix, such as E27 and I66, that play a decisive role in determining the adduct lifetime. Our results additionally suggest a correlation between the solvent accessibility of the chromophore pocket and adduct-state lifetime. The presented results add to our understanding of LOV signaling and will have important implications in tuning the signaling behavior (on/off kinetics) of LOV-based optogenetic tools.

Structural studies of the phosphatidylinositol 3-kinase (PI3K) SH3 domain in complex with a peptide ligand: role of the anchor residue in ligand binding.

Src homology 3 (SH3) domains are mediators of protein-protein interactions. They comprise approximately 60 amino acid residues and are found in many intracellular signaling proteins. Here, we present the crystal structure of the SH3 domain from phosphatidylinositol 3-kinase (PI3K) in complex with the 12-residue proline-rich peptide PD1R (HSKRPLPPLPSL). The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 A reveals type I ligand orientation of the bound peptide with an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of the SH3 domain shows minimal changes on ligand binding. In addition, we also attempted crystallization with another peptide ligand (PD1) where the residue at anchor position P(-3) is a tyrosine. The crystals obtained did not contain the PD1 ligand; instead, the ligand binding site is partially occupied by residues Arg18 and Trp55 from the symmetry-related PI3K SH3 molecule. Considering these crystal structures of PI3K SH3 together with published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify the individual residues which play an important role in defining target specificity.

Phosphorylated peptide of G protein-coupled receptor induces dimerization in activated arrestin.

Termination of the G-protein-coupled receptor signaling involves phosphorylation of its C-terminus and subsequent binding of the regulatory protein arrestin. In the visual system, arrestin-1 preferentially binds to photoactivated and phosphorylated rhodopsin and inactivates phototransduction. Here, we have investigated binding of a synthetic phosphopeptide of bovine rhodopsin (residues 323-348) to the active variants of visual arrestin-1: splice variant p44, and the mutant R175E. Unlike the wild type arrestin-1, both these arrestins are monomeric in solution. Solution structure analysis using small angle X-ray scattering supported by size exclusion chromatography results reveal dimerization in both the arrestins in the presence of phosphopeptide. Our results are the first report, to our knowledge, on receptor-induced oligomerization in arrestin, suggesting possible roles for the cellular function of arrestin oligomers. Given high structural homology and the similarities in their activation mechanism, these results are expected to have implications for all arrestin isoforms.

Helix formation in arrestin accompanies recognition of photoactivated rhodopsin.

Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed.

Pseudomonas aeruginosa esterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization.

The human membrane-bound α/ß-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence of n-octyl ß-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.

Structure of the SLy1 SAM homodimer reveals a new interface for SAM domain self-association.

Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface. The interface consists primarily of the two long C-terminal helices, α5 and α5', of the domains packing against each other. The dimerization is characterized by a dissociation constant in the lower micromolar range. A SLy1 SAM domain construct with an extended N-terminus containing five additional amino acids of the SLy1 sequence further increases the stability of the homodimer, making the SLy1 SAM dimer two orders of magnitude more stable than previously studied SAM homodimers, suggesting that the SLy1 SAM dimerization is of functional significance. The SLy1 SAM homodimer contains an exposed mid-loop surface on each monomer, which may provide a scaffold for mediating interactions with other SAM domain-containing proteins via a typical mid-loop-end-helix interface.

Small-angle X-ray scattering study of the kinetics of light-dark transition in a LOV protein.

Light, oxygen, voltage (LOV) photoreceptors consist of conserved photo-responsive domains in bacteria, archaea, plants and fungi, and detect blue-light via a flavin cofactor. We investigated the blue-light induced conformational transition of the dimeric photoreceptor PpSB1-LOV-R66I from Pseudomonas putida in solution by using small-angle X-ray scattering (SAXS). SAXS experiments of the fully populated light- and dark-states under steady-state conditions revealed significant structural differences between the two states that are in agreement with the known structures determined by crystallography. We followed the transition from the light- to the dark-state by using SAXS measurements in real-time. A two-state model based on the light- and dark-state conformations could describe the measured time-course SAXS data with a relaxation time τREC of ~ 34 to 35 min being larger than the recovery time found with UV/vis spectroscopy. Unlike the flavin chromophore-based UV/vis method that is sensitive to the local chromophore environment in flavoproteins, SAXS-based assay depends on protein conformational changes and provides with an alternative to measure the recovery kinetics.

Crystal structure of a multi-domain immunophilin from Arabidopsis thaliana: a paradigm for regulation of plant ABC transporters.

FKBP42 is a membrane-anchored immunophilin playing a critical role in morphogenesis and development of higher plants. We present the X-ray structure of the cytoplasmic portion of FKBP42 comprising both the FKBP-like domain and the TPR domain at 2.85 A resolution. The data shed light on the probable binding modes of key interaction partners, including HSP90 and two classes of ABC transporters. The resulting models provide a structural background for further investigation of the unique biological properties of this protein.

N-terminal and C-terminal domains of arrestin both contribute in binding to rhodopsin.

Visual arrestin terminates the signal amplification cascade in photoreceptor cells by blocking the interaction of light activated phosphorylated rhodopsin with the G-protein transducin. Although crystal structures of arrestin and rhodopsin are available, it is still unknown how the complex of the two proteins is formed. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores (Alexa 633). The binding was recorded by time-resolved light scattering. To accomplish site-specific shielding of protein regions, in a first step all three wild-type cysteines were replaced by alanines. Nevertheless, regarding the magnitude and specificity of rhodopsin binding, the protein is still fully active. In a second step, new cysteines were introduced at selected sites to allow covalent binding of fluorophores. Upon attachment of Alexa 633 to the recombinant cysteines we observed that these bulky labels residing in the concave area of either the N- or the C-terminal domain do not perturb the activity of arrestin. By simultaneously modifying both domains with one Alexa 633 the binding capacity was reduced. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. This observation indicates that both concave sites participate in binding.

Expression and purification of arrestin in yeast Saccharomyces cerevisiae.

Protein purity and yield are two critical parameters for successful protein characterization using structural techniques such as X-ray crystallography, NMR, and several other biophysical methods. The yeast Saccharomyces cerevisiae is one of the popular eukaryotic model systems for overexpression and subsequent purification of recombinant proteins. Here, we describe a protocol for cloning, overexpression, purification, and crystallization of arrestin-1 and its splice variant p44 from yeast. The purification protocol involves a single-affinity chromatography step on a Strep-Tactin column. Highly purified arrestins can be concentrated up to 15mg/mL using ultrafiltration and can be stored in the frozen state for several months without any loss of functionality.

Structure of a LOV protein in apo-state and implications for construction of LOV-based optical tools.

Unique features of Light-Oxygen-Voltage (LOV) proteins like relatively small size (~12-19 kDa), inherent modularity, highly-tunable photocycle and oxygen-independent fluorescence have lately been exploited for the generation of optical tools. Structures of LOV domains reported so far contain a flavin chromophore per protein molecule. Here we report two new findings on the short LOV protein W619_1-LOV from Pseudomonas putida. First, the apo-state crystal structure of W619_1-LOV at 2.5 Å resolution reveals conformational rearrangements in the secondary structure elements lining the chromophore pocket including elongation of the Fα helix, shortening of the Eα-Fα loop and partial unfolding of the Eα helix. Second, the apo W619_1-LOV protein binds both natural and structurally modified flavin chromophores. Remarkably different photophysical and photochemical properties of W619_1-LOV bound to 7-methyl-8-chloro-riboflavin (8-Cl-RF) and lumichrome imply application of these variants as novel optical tools as they offer advantages such as no adduct state formation, and a broader choice of wavelengths for in vitro studies.

Crystal structure of a plant immunophilin domain involved in regulation of MDR-type ABC transporters.

We present the three-dimensional structure of the N-terminal FK506-binding protein (FKBP)-like domain of the immunophilin FKBP42 from Arabidopsis thaliana. The data provide the structural background for the explanation of key functional properties reported previously.