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 Aβ‐Bβ, 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.
The photocycle of light–oxygen–voltage (LOV) domains involves blue‐light‐triggered adduct formation between the bound flavin chromophore and a cysteine residue. LOV proteins show considerable variation in the lifetime of the adduct state. Here, we used a comparative approach selecting two homologous LOV proteins originating from Pseudomonas putida: a fast‐cycling PpSB2‐LOV (~ 3.5 min) and a slow‐cycling PpSB1‐LOV (~ 42 h) to investigate the mechanistic basis for the very different lifetimes of the adduct states.
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.
Abstract
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.
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 A
(PlaF) from
, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the
Δ
mutant showed strongly attenuated virulence in
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.
TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a ...multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids.
Light–Oxygen–Voltage (LOV) domains represent the photo-responsive domains of various blue-light photoreceptor proteins and are widely distributed in plants, algae, fungi, and bacteria. Here, we ...report the dark-state crystal structure of PpSB1-LOV, a slow-reverting short LOV protein from Pseudomonas putida that is remarkably different from our previously published “fully light-adapted” structure 1. A direct comparison of the two structures provides insight into the light-activated signaling mechanism. Major structural differences involve a~11Å movement of the C terminus in helix Jα, ~4Å movement of Hβ–Iβ loop, disruption of hydrogen bonds in the dimer interface, and a~29° rotation of chain-B relative to chain-A as compared to the light-state dimer. Both crystal structures and solution NMR data are suggestive of the key roles of a conserved glutamine Q116 and the N-cap region consisting of A′α–Aβ loop and the A′α helix in controlling the light-activated conformational changes. The activation mechanism proposed here for the PpSB1-LOV supports a rotary switch mechanism and provides insights into the signal propagation mechanism in naturally existing and artificial LOV-based, two-component systems and regulators.
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•Comparison of crystal structures of PpSB1-LOV in dark and light states•Dimer interface and the C-terminal Jα-helix show major structural rearrangements.•A~29° rotation between the two protein chains gated by light•Extensive NMR solution studies reveal light-induced conformational changes.•We propose a rotary switch mechanism for the activation.
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.
Blue-light photoreceptors containing light–oxygen–voltage (LOV) domains regulate a myriad of different physiological responses in both eukaryotes and prokaryotes. Their light sensitivity is ...intricately linked to the photochemistry of the non-covalently bound flavin mononucleotide (FMN) chromophore that forms a covalent adduct with a conserved cysteine residue in the LOV domain upon illumination with blue light. All LOV domains undergo the same primary photochemistry leading to adduct formation; however, considerable variation is found in the lifetime of the adduct state that varies from seconds to several hours. The molecular mechanism underlying this variation among the structurally conserved LOV protein family is not well understood. Here, we describe the structural characterization of PpSB1-LOV, a very slow cycling full-length LOV protein from the Gram-negative bacterium Pseudomonas putida KT2440. Its crystal structure reveals a novel dimer interface that is mediated by N- and C-terminal auxiliary structural elements and a unique cluster of four arginine residues coordinating with the FMN-phosphate moiety. Site-directed mutagenesis of two arginines (R61 and R66) in PpSB1-LOV resulted in acceleration of the dark recovery reaction approximately by a factor of 280. The presented structural and biochemical data suggest a direct link between structural features and the slow dark recovery observed for PpSB1-LOV. The overall structural arrangement of PpSB1-LOV, together with a complementary phylogenetic analysis, highlights a common ancestry of bacterial LOV photoreceptors and Per-ARNT-Sim chemosensors.
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► Crystal structure of the full-length LOV photoreceptor protein PpSB1-LOV in its light state. ► Novel dimer interface, which is mediated by N- and C-terminal auxiliary structural elements. ► Biochemical and structural data provide a mechanistic understanding of the very slow dark recovery reaction of the PpSB1-LOV protein. ► Structural and complementary phylogenetic analyses highlight an evolutionary relationship between bacterial LOV photoreceptors and oxygen/redox-sensing Per-ARNT-Sim chemosensors.
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.
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.
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