A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli

Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of b-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137–His258–Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield

The Structure of the Polar Core Mutant R175E and Its Functional Implications

Mutation of arginine 175 to glutamic acid (R175E), a central residue in the polar core and previously predicted as the ‘phosphosensor’, leads to a constitutively active arrestin that is able to terminate phototransduction by binding to non-phosphorylated, light-activated rhodopsin . Crystal structure of a R175E mutant arrestin at 2.7 Å resolution reveals significant differences compared to the basal state reported in full-length arrestin structures. Most striking differences are disruption of hydrogen bond network in the polar core , and three-element interaction (between β-strand I, α-helix I, and the C-tail), including disordering of several residues in the receptor-binding finger loop and the C-terminus (residues 361–404). Additionally, R175E structure shows a 7.5° rotation of the amino and carboxy-terminal domains relative to each other. Comparison of the crystal structures of basal arrestin and R175E mutant provides insights into the mechanism of arrestin activation, where the latter likely represents an intermediate activation state prior to formation of the high-affinity complex with the G protein-coupled receptor

Structural evidence for the role of polar core residue Arg175 in arrestin activation

Binding mechanism of arrestin requires photoactivation and phosphorylation of the receptor protein rhodopsin, where the receptor bound phosphate groups cause displacement of the long C-tail ‘activating’ arrestin. Mutation of arginine 175 to glutamic acid (R175E), a central residue in the polar core and previously predicted as the ‘phosphosensor’ leads to a pre-active arrestin that is able to terminate phototransduction by binding to non-phosphorylated, light-activated rhodopsin. Here, we report the first crystal structure of a R175E mutant arrestin at 2.7 Å resolution that reveals significant differences compared to the basal state reported in full-length arrestin structures. These differences comprise disruption of hydrogen bond network in the polar core, and three-element interaction including disordering of several residues in the receptor-binding finger loop and the C-terminus (residues 361–404). Additionally, R175E structure shows a 7.5° rotation of the amino and carboxy-terminal domains relative to each other. Consistent to the biochemical data, our structure suggests an important role of R29 in the initial activation step of C-tail release. Comparison of the crystal structures of basal arrestin and R175E mutant provide insights into the mechanism of arrestin activation, where binding of the receptor likely induces structural changes mimicked as in R175E

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

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

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

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 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

Small-angle X-ray scattering study of the kinetics of light-dark transition in a LOV protein - Fig 3

<p>(A) and (B) Small-angle scattering data of PpSB1-LOV-R66I in the light- and dark-states. Distance distribution function <i>P</i>(<i>r</i>) is shown as inset in (A).</p