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.

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.

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 and ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.