Structural evidence for visual arrestin priming via complexation of phosphoinositols.

Visual arrestin (Arr1) terminates rhodopsin signaling by blocking its interaction with transducin. To do this, Arr1 translocates from the inner to the outer segment of photoreceptors upon light stimulation. Mounting evidence indicates that inositol phosphates (InsPs) affect Arr1 activity, but the Arr1-InsP molecular interaction remains poorly defined. We report the structure of bovine Arr1 in a ligand-free state featuring a near-complete model of the previously unresolved C-tail, which plays a crucial role in regulating Arr1 activity. InsPs bind to the N-domain basic patch thus displacing the C-tail, suggesting that they prime Arr1 for interaction with rhodopsin and help direct Arr1 translocation. These structures exhibit intact polar cores, suggesting that C-tail removal by InsP binding is insufficient to activate Arr1. These results show how Arr1 activity can be controlled by endogenous InsPs in molecular detail.

The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants.

Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.

ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores.

Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

A Novel Polar Core and Weakly Fixed C-Tail in Squid Arrestin Provide New Insight into Interaction with Rhodopsin.

Photoreceptors of the squid Loligo pealei contain a G-protein-coupled receptor (GPCR) signaling system that activates phospholipase C in response to light. Analogous to the mammalian visual system, signaling of the photoactivated GPCR rhodopsin is terminated by binding of squid arrestin (sArr). sArr forms a light-dependent, high-affinity complex with squid rhodopsin, which does not require prior receptor phosphorylation for interaction. This is at odds with classical mammalian GPCR desensitization where an agonist-bound phosphorylated receptor is needed to break stabilizing constraints within arrestins, the so-called "three-element interaction" and "polar core" network, before a stable receptor-arrestin complex can be established. Biophysical and mass spectrometric analysis of the squid rhodopsin-arrestin complex indicates that in contrast to mammalian arrestins, the sArr C-tail is not involved in a stable three-element interaction. We determined the crystal structure of C-terminally truncated sArr that adopts a basal conformation common to arrestins and is stabilized by a series of weak but novel polar core interactions. Unlike mammalian arrestin-1, deletion of the sArr C-tail does not influence kinetic properties of complex formation of sArr with the receptor. Hydrogen-deuterium exchange studies revealed the footprint of the light-activated rhodopsin on sArr. Furthermore, double electron-electron resonance spectroscopy experiments provide evidence that receptor-bound sArr adopts a conformation different from the one known for arrestin-1 and molecular dynamics simulations reveal the residues that account for the weak three-element interaction. Insights gleaned from studying this system add to our general understanding of GPCR-arrestin interaction.

Crystallogenesis of Membrane Proteins Mediated by Polymer-Bounded Lipid Nanodiscs.

For some membrane proteins, detergent-mediated solubilization compromises protein stability and functionality, often impairing biophysical and structural analyses. Hence, membrane-protein structure determination is a continuing bottleneck in the field of protein crystallography. Here, as an alternative to approaches mediated by conventional detergents, we report the crystallogenesis of a recombinantly produced membrane protein that never left a lipid bilayer environment. We used styrene-maleic acid (SMA) copolymers to solubilize lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity and size-exclusion chromatography, and transferred proteins into the lipidic cubic phase (LCP) for in meso crystallization. The 2.0-Å structure of an α-helical seven-transmembrane microbial rhodopsin thus obtained is of high quality and virtually identical to the 2.2-Å structure obtained from traditional detergent-based purification and subsequent LCP crystallization.

Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography.

The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11 259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means of in situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited.

Development and Characterization of Potent Cyclic Acyldepsipeptide Analogues with Increased Antimicrobial Activity.

The problem of antibiotic resistance has prompted the search for new antibiotics with novel mechanisms of action. Analogues of the A54556 cyclic acyldepsipeptides (ADEPs) represent an attractive class of antimicrobial agents that act through dysregulation of caseinolytic protease (ClpP). Previous studies have shown that ADEPs are active against Gram-positive bacteria (e.g., MRSA, VRE, PRSP (penicillin-resistant Streptococcus pneumoniae)); however, there are currently few studies examining Gram-negative bacteria. In this study, the synthesis and biological evaluation of 14 novel ADEPs against a variety of pathogenic Gram-negative and Gram-positive organisms is outlined. Optimization of the macrocyclic core residues and N-acyl side chain culminated in the development of 26, which shows potent activity against the Gram-negative species Neisseria meningitidis and Neisseria gonorrheae and improved activity against the Gram-positive organisms Staphylococcus aureus and Enterococcus faecalis in comparison with known analogues. In addition, the co-crystal structure of an ADEP-ClpP complex derived from N. meningitidis was solved.

The C-terminal peptide plays a role in the formation of an intermediate form during the transition between xanthine dehydrogenase and xanthine oxidase.

UNLABELLED: Mammalian xanthine oxidoreductase can exist in both dehydrogenase and oxidase forms. Conversion between the two is implicated in such diverse processes as lactation, anti-bacterial activity, reperfusion injury and a growing number of diseases. We have constructed a variant of the rat liver enzyme that lacks the carboxy-terminal amino acids 1316-1331; it appears to assume an intermediate form, exhibiting a mixture of dehydrogenase and oxidase activities. The purified variant protein retained ~ 50-70% of oxidase activity even after prolonged dithiothreitol treatment, supporting a previous prediction that the C-terminal region plays a role in the dehydrogenase to oxidase conversion. In the crystal structure of the protein variant, most of the enzyme stays in an oxidase conformation. After 15 min of incubation with a high concentration of NADH, however, the corresponding X-ray structures showed a dehydrogenase-type conformation. On the other hand, disulfide formation between Cys535 and Cys992, which can clearly be seen in the electron density map of the crystal structure of the variant after removal of dithiothreitol, goes in parallel with the complete conversion to oxidase, resulting in structural changes identical to those observed upon proteolytic cleavage of the linker peptide. These results indicate that the dehydrogenase-oxidase transformation occurs rather readily and the insertion of the C-terminal peptide into the active site cavity of its subunit stabilizes the dehydrogenase form. We propose that the intermediate form can be generated (e.g. in endothelial cells) upon interaction of the C-terminal peptide portion of the enzyme with other proteins or the cell membrane. DATABASE: Coordinate sets and structure factors for the four crystal structures reported in the present study have been deposited in the Protein Data Bank under the identification numbers 4YRW, 4YTZ, 4YSW, and 4YTY.

Protein conformational gating of enzymatic activity in xanthine oxidoreductase.

In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E(sq/hq)) is ~170 mV, a striking difference. The former greatly prefers NAD(+) as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD(+)), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 Å resolution crystal structures for XDH, XO, the NAD(+)- and NADH-complexed XDH, E(sq/hq) were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E(sq/hq) difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD(+) binding at XDH. Instead, the positive charge of the NAD(+) ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD(+) molecule all contribute to altering E(sq/hq) upon NAD(+) binding to XDH.

Activators of cylindrical proteases as antimicrobials: identification and development of small molecule activators of ClpP protease.

ClpP is a cylindrical serine protease whose ability to degrade proteins is regulated by the unfoldase ATP-dependent chaperones. ClpP on its own can only degrade small peptides. Here, we used ClpP as a target in a high-throughput screen for compounds, which activate the protease and allow it to degrade larger proteins, hence, abolishing the specificity arising from the ATP-dependent chaperones. Our screen resulted in five distinct compounds, which we designate as Activators of Self-Compartmentalizing Proteases 1 to 5 (ACP1 to 5). The compounds are found to stabilize the ClpP double-ring structure. The ACP1 chemical structure was considered to have drug-like characteristics and was further optimized to give analogs with bactericidal activity. Hence, the ACPs represent classes of compounds that can activate ClpP and that can be developed as potential novel antibiotics.

Crystal structures of urate bound form of xanthine oxidoreductase: substrate orientation and structure of the key reaction intermediate.

Two contradictory models have been proposed for the binding mode of the substrate xanthine to and its activation mechanism by xanthine oxidoreductase. In an effort to distinguish between the two models, we determined the crystal structures of the urate complexes of the demolybdo-form of the D428A mutant of rat xanthine oxidoreductase at 1.7 Å and of the reduced bovine milk enzyme at 2.1 Å, the latter representing a reaction intermediate. The results clearly indicate the catalytically relevant binding mode of the substrate xanthine.

Mechanism of inhibition of xanthine oxidoreductase by allopurinol: crystal structure of reduced bovine milk xanthine oxidoreductase bound with oxipurinol.

Inhibitors of xanthine oxidoreductase block conversion of xanthine to uric acid and are therefore potentially useful for treatment of hyperuricemia or gout. We determined the crystal structure of reduced bovine milk xanthine oxidoreductase complexed with oxipurinol at 2.0 A resolution. Clear electron density was observed between the N2 nitrogen of oxipurinol and the molybdenum atom of the molybdopterin cofactor, indicating that oxipurinol coordinated directly to molybdenum. Oxipurinol forms hydrogen bonds with glutamate 802, arginine 880, and glutamate 1261, which have previously been shown to be essential for the enzyme reaction. We discuss possible differences in the hypouricemic effect of inhibitors, including allopurinol and newly developed inhibitors, based on their mode of binding in the crystal structures.

Mammalian xanthine oxidoreductase - mechanism of transition from xanthine dehydrogenase to xanthine oxidase.

Reactive oxygen species are generated by various biological systems, including NADPH oxidases, xanthine oxidoreductase, and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide. Recent X-ray crystallographic and site-directed mutagenesis studies have revealed a highly sophisticated mechanism of conversion from XDH to XO, suggesting that the conversion is not a simple artefact, but rather has a function in mammalian organisms. Furthermore, this transition seems to involve a thermodynamic equilibrium between XDH and XO; disulfide bond formation or proteolysis can then lock the enzyme in the XO form. In this review, we focus on recent advances in our understanding of the mechanism of conversion from XDH to XO.

Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: identification of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant.

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys(535) and Cys(992) residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4'-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4'-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD(+) binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4'-dithiodipyridine, indicating that Cys(1316) and Cys(1324) are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.

Y-700 [1-[3-Cyano-4-(2,2-dimethylpropoxy)phenyl]-1H-pyrazole-4-carboxylic acid]: a potent xanthine oxidoreductase inhibitor with hepatic excretion.

Y-700 (1-[3-Cyano-4-(2,2-dimethylpropoxy)phenyl]-1H-pyrazole-4-carboxylic acid) is a newly synthesized inhibitor of xanthine oxidoreductase (XOR). Steady-state kinetics with the bovine milk enzyme indicated a mixed type inhibition with K(i) and K(i) ' values of 0.6 and 3.2 nM, respectively. Titration experiments showed that Y-700 bound tightly both to the active sulfo-form and to the inactive desulfo-form of the enzyme with K(d) values of 0.9 and 2.8 nM, respectively. X-ray crystallographic analysis of the enzyme-inhibitor complex revealed that Y-700 closely interacts with the channel leading to the molybdenum-pterin active site but does not directly coordinate to the molybdenum ion. In oxonate-treated rats, orally administered Y-700 (1-10 mg/kg) dose dependently lowered plasma urate levels. At a dose of 10 mg/kg, the hypouricemic action of Y-700 was more potent and of longer duration than that of 4-hydroxypyrazolo(3,4-d)pyrimidine, whereas its action was approximately equivalent to that of 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid, a nonpurine inhibitor of XOR. In normal rats, orally administered Y-700 (0.3-3 mg/kg) dose dependently reduced the urinary excretion of urate and allantoin, accompanied by an increase in the excretion of hypoxanthine and xanthine. Y-700 (1 mg/kg) was absorbed rapidly by the oral route with high bioavailability (84.1%). Y-700 was hardly excreted via the kidneys but was mainly cleared via the liver. These results suggest that Y-700 will be a promising candidate for the treatment of hyperuricemia and other diseases in which XOR may be involved.

The crystal structure of xanthine oxidoreductase during catalysis: implications for reaction mechanism and enzyme inhibition.

Molybdenum is widely distributed in biology and is usually found as a mononuclear metal center in the active sites of many enzymes catalyzing oxygen atom transfer. The molybdenum hydroxylases are distinct from other biological systems catalyzing hydroxylation reactions in that the oxygen atom incorporated into the product is derived from water rather than molecular oxygen. Here, we present the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon-oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable broad charge-transfer band at approximately 640 nm. The crystal structure of the complex indicates that the catalytically labile Mo-OH oxygen has formed a bond with a carbon atom of the substrate. In addition, the MoS group of the oxidized enzyme has become protonated to afford Mo-SH on reduction of the molybdenum center. In contrast to previous assignments, we find this last ligand at an equatorial position in the square-pyramidal metal coordination sphere, not the apical position. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide.

Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase.

In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO) either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated that atoms in the highly charged active-site loop (Gln-423-Lys-433) around the FAD cofactor underwent large dislocations during the conversion, blocking the approach of the NAD+ substrate to FAD in the XO form as well as changing the electrostatic environment around FAD. Here we identify a unique cluster of amino acids that plays a dual role by forming the core of a relay system for the XDH/XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and site-directed mutagenesis analysis experiments showed that Phe-549, Arg-335, Trp-336, and Arg-427 sit at the center of a relay system that transmits modifications of the linker peptide by cysteine oxidation or proteolytic cleavage to the active-site loop (Gln-423-Lys-433). The tight interactions of these residues are crucial in the stabilization of the XDH conformation and for keeping the solvent channel closed. Both oxidative and proteolytic generation of XO effectively leads to the removal of Phe-549 from the cluster causing a reorientation of the bulky side chain of Trp-336, which then in turn forces a dislocation of Arg-427, an amino acid located in the active-site loop. The conformational change also opens the gate for the solvent channel, making it easier for oxygen to reach the reduced FAD in XO.

An extremely potent inhibitor of xanthine oxidoreductase. Crystal structure of the enzyme-inhibitor complex and mechanism of inhibition.

TEI-6720 (2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid) is an extremely potent inhibitor of xanthine oxidoreductase. Steady state kinetics measurements exhibit mixed type inhibition with K(i) and K(i)' values of 1.2 +/- 0.05 x 10(-10) m and 9 +/- 0.05 x 10(-10) m, respectively. Fluorescence-monitored titration experiments showed that TEI-6720 bound very tightly to both the active and the inactive desulfo-form of the enzyme. The dissociation constant determined for the desulfo-form was 2 +/- 0.03 x 10(-9) m; for the active form, the corresponding number was too low to allow accurate measurements. The crystal structure of the active sulfo-form of milk xanthine dehydrogenase complexed with TEI-6720 and determined at 2.8-A resolution revealed the inhibitor molecule bound in a long, narrow channel leading to the molybdenum-pterin active site of the enzyme. It filled up most of the channel and the immediate environment of the cofactor, very effectively inhibiting the activity of the enzyme through the prevention of substrate binding. Although the inhibitor did not directly coordinate to the molybdenum ion, numerous hydrogen bonds as well as hydrophobic interactions with the protein matrix were observed, most of which are also used in substrate recognition.