Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori.

Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH(3) and CO(2), neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.

RNA Editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting.

Terminal uridyltransferases (TUTases) execute 3' RNA uridylation across protists, fungi, metazoan and plant species. Uridylation plays a particularly prominent role in RNA processing pathways of kinetoplastid protists typified by the causative agent of African sleeping sickness, Trypanosoma brucei In mitochondria of this pathogen, most mRNAs are internally modified by U-insertion/deletion editing while guide RNAs and rRNAs are U-tailed. The founding member of TUTase family, RNA editing TUTase 1 (RET1), functions as a subunit of the 3' processome in uridylation of gRNA precursors and mature guide RNAs. Along with KPAP1 poly(A) polymerase, RET1 also participates in mRNA translational activation. RET1 is divergent from human TUTases and is essential for parasite viability in the mammalian host and the insect vector. Given its robust in vitro activity, RET1 represents an attractive target for trypanocide development. Here, we report high-resolution crystal structures of the RET1 catalytic core alone and in complex with UTP analogs. These structures reveal a tight docking of the conserved nucleotidyl transferase bi-domain module with a RET1-specific C2H2 zinc finger and RNA recognition (RRM) domains. Furthermore, we define RET1 region required for incorporation into the 3' processome, determinants for RNA binding, subunit oligomerization and processive UTP incorporation, and predict druggable pockets.

Structure of an Inward Proton-Transporting Anabaena Sensory Rhodopsin Mutant: Mechanistic Insights.

Microbial rhodopsins are light-activated, seven-α-helical, retinylidene transmembrane proteins that have been identified in thousands of organisms across archaea, bacteria, fungi, and algae. Although they share a high degree of sequence identity and thus similarity in structure, many unique functions have been discovered and characterized among them. Some function as outward proton pumps, some as inward chloride pumps, whereas others function as light sensors or ion channels. Unique among the microbial rhodopsins characterized thus far, Anabaena sensory rhodopsin (ASR) is a photochromic sensor that interacts with a soluble 14-kDa cytoplasmic transducer that is encoded on the same operon. The sensor itself stably interconverts between all-trans-15-anti and 13-cis-15-syn retinal forms depending on the wavelength of illumination, although only the former participates in a photocycle with a signaling M intermediate. A mutation in the cytoplasmic half-channel of the protein, replacing Asp217 with Glu (D217E), results in the creation of a light-driven, single-photon, inward proton transporter. We present the 2.3 Å structure of dark-adapted D217E ASR, which reveals significant changes in the water network surrounding Glu217, as well as a shift in the carbon backbone near retinal-binding Lys210, illustrating a possible pathway leading to the protonation of Glu217 in the cytoplasmic half-channel, located 15 Å from the Schiff base. Crystallographic evidence for the protonation of nearby Glu36 is also discussed, which was described previously by Fourier transform infrared spectroscopy analysis. Finally, two histidine residues near the extracellular surface and their possible role in proton uptake are discussed.

Challenging the state of the art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10.

For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins.

Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein.

Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.

Crystal structure of a conformation-dependent rabbit IgG Fab specific for amyloid prefibrillar oligomers.

BACKGROUND: Although rabbit antibodies are widely used in research, no structures of rabbit antigen-binding fragments (Fab) have been reported. M204 is a rabbit monoclonal antibody that recognizes a generic epitope that is common to prefibrillar amyloid oligomers formed from many different amyloidogenic sequences. Amyloid oligomers are widely suspected to be a primary causative agent of pathogenesis in several age-related neurodegenerative diseases, such as Alzheimer's disease. The detailed structure of these amyloid oligomers is not known nor is the mechanism for the recognition of the generic epitope by conformation-dependent monoclonal antibodies. METHOD: As a first approach to understanding the mechanism of conformation-dependent antibody recognition, we have crystallized the Fab of M204. RESULTS: We have determined the structure of the Fab of M204 at 1.54Å resolution. The crystal structure reveals details of the M204 antigen combining site and features unique to rabbit Fabs such as an interdomain disulfide bond on its light chain. GENERAL SIGNIFICANCE: Based on the structural features of the antigen-combining site of the M204, we rule out a "steric zipper" formation, as found in numerous amyloid fibril structures, as a mechanism of antibody-antigen recognition. The details of the first rabbit immunoglobulin Fab structure might also be useful for exploiting the potential of rabbit monoclonal antibodies for the development of humanized rabbit antibodies as therapeutic agents.

Structure of a C-terminal AHNAK peptide in a 1:2:2 complex with S100A10 and an acetylated N-terminal peptide of annexin A2.

AHNAK, a large 629 kDa protein, has been implicated in membrane repair, and the annexin A2-S100A10 heterotetramer [(p11)(2)(AnxA2)(2))] has high affinity for several regions of its 1002-amino-acid C-terminal domain. (p11)(2)(AnxA2)(2) is often localized near the plasma membrane, and this C2-symmetric platform is proposed to be involved in the bridging of membrane vesicles and trafficking of proteins to the plasma membrane. All three proteins co-localize at the intracellular face of the plasma membrane in a Ca(2+)-dependent manner. The binding of AHNAK to (p11)(2)(AnxA2)(2) has been studied previously, and a minimal binding motif has been mapped to a 20-amino-acid peptide corresponding to residues 5654-5673 of the AHNAK C-terminal domain. Here, the 2.5 Å resolution crystal structure of this 20-amino-acid peptide of AHNAK bound to the AnxA2-S100A10 heterotetramer (1:2:2 symmetry) is presented, which confirms the asymmetric arrangement first described by Rezvanpour and coworkers and explains why the binding motif has high affinity for (p11)(2)(AnxA2)(2). Binding of AHNAK to the surface of (p11)(2)(AnxA2)(2) is governed by several hydrophobic interactions between side chains of AHNAK and pockets on S100A10. The pockets are large enough to accommodate a variety of hydrophobic side chains, allowing the consensus sequence to be more general. Additionally, the various hydrogen bonds formed between the AHNAK peptide and (p11)(2)(AnxA2)(2) most often involve backbone atoms of AHNAK; as a result, the side chains, particularly those that point away from S100A10/AnxA2 towards the solvent, are largely interchangeable. While the structure-based consensus sequence allows interactions with various stretches of the AHNAK C-terminal domain, comparison with other S100 structures reveals that the sequence has been optimized for binding to S100A10. This model adds new insight to the understanding of the specific interactions that occur in this membrane-repair scaffold.

Cross-protomer interaction with the photoactive site in oligomeric proteorhodopsin complexes.

Proteorhodopsins (PRs), members of the microbial rhodopsin superfamily of seven-transmembrane-helix proteins that use retinal chromophores, comprise the largest subfamily of rhodopsins, yet very little structural information is available. PRs are ubiquitous throughout the biosphere and their genes have been sequenced in numerous species of bacteria. They have been shown to exhibit ion-pumping activity like their archaeal homolog bacteriorhodopsin (BR). Here, the first crystal structure of a proteorhodopsin, that of a blue-light-absorbing proteorhodopsin (BPR) isolated from the Mediterranean Sea at a depth of 12 m (Med12BPR), is reported. Six molecules of Med12BPR form a doughnut-shaped C6 hexameric ring, unlike BR, which forms a trimer. Furthermore, the structures of two mutants of a related BPR isolated from the Pacific Ocean near Hawaii at a depth of 75 m (HOT75BPR), which show a C5 pentameric arrangement, are reported. In all three structures the retinal polyene chain is shifted towards helix C when compared with other microbial rhodopsins, and the putative proton-release group in BPR differs significantly from those of BR and xanthorhodopsin (XR). The most striking feature of proteorhodopsin is the position of the conserved active-site histidine (His75, also found in XR), which forms a hydrogen bond to the proton acceptor from the same molecule (Asp97) and also to Trp34 of a neighboring protomer. Trp34 may function by stabilizing His75 in a conformation that favors a deprotonated Asp97 in the dark state, and suggests cooperative behavior between protomers when the protein is in an oligomeric form. Mutation-induced alterations in proton transfers in the BPR photocycle in Escherichia coli cells provide evidence for a similar cross-protomer interaction of BPR in living cells and a functional role of the inter-protomer Trp34-His75 interaction in ion transport. Finally, Wat402, a key molecule responsible for proton translocation between the Schiff base and the proton acceptor in BR, appears to be absent in PR, suggesting that the ion-transfer mechanism may differ between PR and BR.

Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue.

To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol(-1) (15.1 kJ mol(-1)). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the ß-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation.

Withaferin A binds covalently to the N-terminal domain of annexin A2.

Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

N-terminal acetylation of annexin A2 is required for S100A10 binding.

Annexin A2 (AnxA2), a Ca2+-regulated phospholipid binding protein involved in membrane-cytoskeleton contacts and membrane transport, exists in two physical states, as a monomer or in a heterotetrameric complex mediated by S100A10. Formation of the AnxA2-S100A10 complex is of crucial regulatory importance because only the complex is firmly anchored in the plasma membrane, where it functions in the plasma membrane targeting/recruitment of certain ion channels and receptors. The S100A10 binding motif is located in the first 12 residues of the AnxA2 N-terminal domain, but conflicting reports exist as to the importance of N-terminal AnxA2 acetylation with regard to S100A10 binding. We show here that AnxA2 is subject to N-terminal modification when expressed heterologously in Escherichia coli. Met1 is removed and Ser2 is acetylated, yielding the same modification as the authentic mammalian protein. Bacterially expressed and N-terminally acetylated AnxA2 binds S100A10 with an affinity comparable to AnxA2 from porcine tissue and is capable of forming the AnxA2-S100A10 heterotetramer. Complex formation is competitively inhibited by acetylated but not by non-acetylated peptides covering the N-terminal AnxA2 sequence. These results demonstrate that N-terminal acetylation of AnxA2 is required for S100A10 binding and that this common eukaryotic modification is also obtained upon expression in bacteria.

Structural basis of human LRG1 recognition by Magacizumab, a humanized monoclonal antibody with therapeutic potential.

The formation of new dysfunctional blood vessels is a crucial stage in the development of various conditions such as macular degeneration, diabetes, cardiovascular disease, neurological disease and inflammatory disorders, as well as during tumor growth, eventually contributing to metastasis. An important factor involved in pathogenic angiogenesis is leucine-rich α-2-glycoprotein 1 (LRG1), the antibody blockade of which has been shown to lead to a reduction in both choroidal neovascularization and tumor growth in mouse models. In this work, the structural interactions between the LRG1 epitope and the Fab fragment of Magacizumab, a humanized function-blocking IgG4 against LRG1, are analysed, determining its specific binding mode and the key residues involved in LRG1 recognition. Based on these structural findings, a series of mutations are suggested that could be introduced into Magacizumab to increase its affinity for LRG1, as well as a model of the entire Fab-LRG1 complex that could enlighten new strategies to enhance affinity, consequently leading towards an even more efficient therapeutic.

Structure prediction of honey bee vitellogenin: a multi-domain protein important for insect immunity.

Vitellogenin (Vg) has been implicated as a central protein in the immunity of egg-laying animals. Studies on a diverse set of species suggest that Vg supports health and longevity through binding to pathogens. Specific studies of honey bees (Apis mellifera) further indicate that the vitellogenin (vg) gene undergoes selection driven by local pathogen pressures. Determining the complete 3D structure of full-length Vg (flVg) protein will provide insights regarding the structure-function relationships underlying allelic variation. Honey bee Vg has been described in terms of function, and two subdomains have been structurally described, while information about the other domains is lacking. Here, we present a structure prediction, restrained by experimental data, of flVg from honey bees. To achieve this, we performed homology modeling and used AlphaFold before using a negative-stain electron microscopy map to restrict, orient, and validate our 3D model. Our approach identified a highly conserved Ca2+ -ion-binding site in a von Willebrand factor domain that might be central to Vg function. Thereafter, we used rigid-body fitting to predict the relative position of high-resolution domains in a flVg model. This mapping represents the first experimentally validated full-length protein model of a Vg protein and is thus relevant for understanding Vg in numerous species. Our results are also specifically relevant to honey bee health, which is a topic of global concern due to rapidly declining pollinator numbers.

Crystallographic structure of xanthorhodopsin, the light-driven proton pump with a dual chromophore.

Homologous to bacteriorhodopsin and even more to proteorhodopsin, xanthorhodopsin is a light-driven proton pump that, in addition to retinal, contains a noncovalently bound carotenoid with a function of a light-harvesting antenna. We determined the structure of this eubacterial membrane protein-carotenoid complex by X-ray diffraction, to 1.9-A resolution. Although it contains 7 transmembrane helices like bacteriorhodopsin and archaerhodopsin, the structure of xanthorhodopsin is considerably different from the 2 archaeal proteins. The crystallographic model for this rhodopsin introduces structural motifs for proton transfer during the reaction cycle, particularly for proton release, that are dramatically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine-aspartate complex for regulating the pK(a) of the primary proton acceptor not present in archaeal pumps but apparently conserved in eubacterial pumps. In addition to aiding elucidation of a more general proton transfer mechanism for light-driven energy transducers, the structure defines also the geometry of the carotenoid and the retinal. The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as approximately 45%, and the 46 degrees angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer.

Cryo-EM structure of Helicobacter pylori urease with an inhibitor in the active site at 2.0 Å resolution.

Infection of the human stomach by Helicobacter pylori remains a worldwide problem and greatly contributes to peptic ulcer disease and gastric cancer. Without active intervention approximately 50% of the world population will continue to be infected with this gastric pathogen. Current eradication, called triple therapy, entails a proton-pump inhibitor and two broadband antibiotics, however resistance to either clarithromycin or metronidazole is greater than 25% and rising. Therefore, there is an urgent need for a targeted, high-specificity eradication drug. Gastric infection by H. pylori depends on the expression of a nickel-dependent urease in the cytoplasm of the bacteria. Here, we report the 2.0 Å resolution structure of the 1.1 MDa urease in complex with an inhibitor by cryo-electron microscopy and compare it to a ß-mercaptoethanol-inhibited structure at 2.5 Å resolution. The structural information is of sufficient detail to aid in the development of inhibitors with high specificity and affinity.

Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha.

Oleylethanolamide (OEA) is a naturally occurring lipid that regulates satiety and body weight. Although structurally related to the endogenous cannabinoid anandamide, OEA does not bind to cannabinoid receptors and its molecular targets have not been defined. Here we show that OEA binds with high affinity to the peroxisome-proliferator-activated receptor-alpha (PPAR-alpha), a nuclear receptor that regulates several aspects of lipid metabolism. Administration of OEA produces satiety and reduces body weight gain in wild-type mice, but not in mice deficient in PPAR-alpha. Two distinct PPAR-alpha agonists have similar effects that are also contingent on PPAR-alpha expression, whereas potent and selective agonists for PPAR-gamma and PPAR-beta/delta are ineffective. In the small intestine of wild-type but not PPAR-alpha-null mice, OEA regulates the expression of several PPAR-alpha target genes: it initiates the transcription of proteins involved in lipid metabolism and represses inducible nitric oxide synthase, an enzyme that may contribute to feeding stimulation. Our results, which show that OEA induces satiety by activating PPAR-alpha, identify an unexpected role for this nuclear receptor in regulating behaviour, and raise possibilities for the treatment of eating disorders.

UTP-bound and Apo structures of a minimal RNA uridylyltransferase.

3'-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase beta. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 Angstroms crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 Angstroms crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.

Apo and calcium-bound crystal structures of Alpha-11 giardin, an unusual annexin from Giardia lamblia.

Alpha-11 giardin is a member of the multi-gene alpha-giardin family in the intestinal protozoan, Giardia lamblia. This gene family shares an ancestry with the annexin super family, whose common characteristic is calcium-dependent binding to membranes that contain acidic phospholipids. Several alpha giardins are highly expressed during parasite-induced diarrhea in humans. Despite being a member of a large family of proteins, little is known about the function and cellular localization of alpha-11 giardin, although giardins are often associated with the cytoskeleton. It has been shown that Giardia exhibits high levels of alpha-11 giardin mRNA transcript throughout its life cycle; however, constitutive over-expression of this protein is lethal to the parasite. Determining the three-dimensional structure of an alpha-giardin is essential to identifying functional domains shared in the alpha-giardin family. Here we report the crystal structures of the apo and Ca(2+)-bound forms of alpha-11 giardin, the first alpha giardin to be characterized structurally. Crystals of apo and Ca(2+)-bound alpha-11 giardin diffracted to 1.1 A and 2.93 A, respectively. The crystal structure of selenium-substituted apo alpha-11 giardin reveals a planar array of four tandem repeats of predominantly alpha-helical domains, reminiscent of previously determined annexin structures, making this the highest-resolution structure of an annexin to date. The apo alpha-11 giardin structure also reveals a hydrophobic core formed between repeats I/IV and II/III, a region typically hydrophilic in other annexins. Surprisingly, the Ca(2+)-bound structure contains only a single calcium ion, located in the DE loop of repeat I and coordinated differently from the two types of calcium sites observed in previous annexin structures. The apo and Ca(2+)-bound alpha-11 giardin structures assume overall similar conformations; however, Ca(2+)-bound alpha-11 giardin crystallized in a lower-symmetry space group with four molecules in the asymmetric unit. Vesicle-binding studies suggest that alpha-11 giardin, unlike most other annexins, does not bind to vesicles composed of acidic phospholipids in a calcium-dependent manner.

Crystal structure of the Anabaena sensory rhodopsin transducer.

We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured beta-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal alpha-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the alpha-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K(d) of 8 microM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible alpha-face to mediate transduction of the light signal are discussed.

URB602 inhibits monoacylglycerol lipase and selectively blocks 2-arachidonoylglycerol degradation in intact brain slices.

The N-aryl carbamate URB602 (biphenyl-3-ylcarbamic acid cyclohexyl ester) is an inhibitor of monoacylglycerol lipase (MGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Here, we investigated the mechanism by which URB602 inhibits purified recombinant rat MGL by using a combination of biochemical and structure-activity relationship (SAR) approaches. We found that URB602 weakly inhibits recombinant MGL (IC(50) = 223 +/- 63 microM) through a rapid and noncompetitive mechanism. Dialysis experiments and SAR analyses suggest that URB602 acts through a partially reversible mechanism rather than by irreversible carbamoylation of MGL. Finally, URB602 (100 microM) elevates 2-AG levels in hippocampal slice cultures without affecting levels of other endocannabinoid-related substances. Thus, URB602 may provide a useful tool by which to investigate the physiological roles of 2-AG and explore the potential interest of MGL as a therapeutic target.