Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site

OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram-negative bacteria. Here we present the crystal structure of OmpT, which shows a 10-stranded antiparallel beta-barrel that protrudes far from the lipid bilayer into the extracellular space. We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. The proteolytic site is located in a groove at the extracellular top of the vase-shaped beta-barrel. Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His-Asp dyad and an Asp-Asp couple that activate a putative nucleophilic water molecule. The active site is fully conserved within the omptin family. Therefore, the structure described here provides a sound basis for the design of drugs against omptin-mediated bacterial pathogenesis. Coordinates are in the Protein Data Bank (accession No. 1I78)     

Possible involvement of a novel STAM-associated molecule "AMSH" in intracellular signal transduction mediated by cytokines.

STAM containing an SH3 (Src homology 3) domain and an immunoreceptor tyrosine-based activation motif was previously revealed to be implicated in signaling pathways immediately downstream of Jak2 and Jak3 tyrosine kinases associated with cytokine receptors. We molecularly cloned a novel molecule interacting with the SH3 domain of STAM, which was named AMSH (associated molecule with the SH3 domain of STAM). AMSH contains a putative bipartite nuclear localization signal and a homologous region of a c-Jun activation domain-binding protein 1 (JAB1) subdomain in addition to a binding site for the SH3 domain of STAM. AMSH mutant deleted of the C-terminal half conferred dominant negative effects on signaling for DNA synthesis and c-myc induction mediated by interleukin 2 and granulocyte macrophage-colony-stimulating factor. These results suggest that AMSH plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3.STAM complex.     

Rat plasma alpha 1-inhibitor3: a member of the alpha-macroglobulin family

The overall mechanism of interaction with proteinases of alpha 1-inhibitor3, a plasma proteinase inhibitor so far specific to the rat, has been shown to be closely similar to that described for alpha-macroglobulins. This mechanism includes: (i) the cleavage of at least one susceptible peptidic bond which leads to structural changes in the molecule. (ii) The cleavage of a putative thiol ester bond in another site of the molecule which permits the covalent linkage of the enzyme. Moreover, fragmentation of alpha 1-inhibitor3 upon heating as observed for alpha-macroglobulin quarter subunits has been demonstrated. The question is raised of the presence of such a molecule in rat plasma in addition to two alpha-macroglobulin species, all of these proteinase inhibitors being antigenically unrelated.     

RecA protein-promoted homologous pairing between duplex molecules: functional role of duplex regions of gapped duplex DNA

RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped DNA.     

Phytochrome phosphorylation modulates light signaling by influencing the protein-protein interaction

Plant photoreceptor phytochromes are phosphoproteins, but the question as to the functional role of phytochrome phosphorylation has remained to be elucidated. We investigated the functional role of phytochrome phosphorylation in plant light signaling using a Pfr-specific phosphorylation site mutant, Ser598Ala of oat (Avena sativa) phytochrome A (phyA). The transgenic Arabidopsis thaliana (phyA-201 background) plants with this mutant phyA showed hypersensitivity to light, suggesting that phytochrome phosphorylation at Serine-598 (Ser598) in the hinge region is involved in an inhibitory mechanism. The phosphorylation at Ser598 prevented its interaction with putative signal transducers, Nucleoside Diphosphate Kinase-2 and Phytochrome-Interacting Factor-3. These results suggest that phosphorylation in the hinge region of phytochromes serves as a signal-modulating site through the protein-protein interaction between phytochrome and its putative signal transducer proteins.     

Nucleotide channel of RNA-dependent RNA polymerase used for intermolecular uridylylation of protein primer

Poliovirus VPg is a 22 amino acid residue peptide that serves as the protein primer for replication of the viral RNA genome. VPg is known to bind directly to the viral RNA-dependent RNA polymerase, 3D, for covalent uridylylation, yielding mono and di-uridylylated products, VPg-pU and VPg-pUpU, which are subsequently elongated. To model the docking of the VPg substrate to a putative VPg-binding site on the 3D polymerase molecule, we performed a variety of structure-based computations followed by experimental verification. First, potential VPg folded structures were identified, yielding a suite of predicted beta-hairpin structures. These putative VPg structures were then docked to the region of the polymerase implicated by genetic experiments to bind VPg, using grid-based and fragment-based methods. Residues in VPg predicted to affect binding were identified through molecular dynamics simulations, and their effects on the 3D-VPg interaction were tested computationally and biochemically. Experiments with mutant VPg and mutant polymerase molecules confirmed the predicted binding site for VPg on the back side of the polymerase molecule during the uridylylation reaction, opposite to that predicted to bind elongating RNA primers.     

Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis

The (R)-specific enoyl coenzyme A hydratase ((R)-hydratase) from Aeromonas caviae catalyzes the addition of a water molecule to trans-2-enoyl coenzyme A (CoA), with a chain-length of 4-6 carbons, to produce the corresponding (R)-3-hydroxyacyl-CoA. It forms a dimer of identical subunits with a molecular weight of about 14,000 and is involved in polyhydroxyalkanoate (PHA) biosynthesis. The crystal structure of the enzyme has been determined at 1.5-A resolution. The structure of the monomer consists of a five-stranded antiparallel beta-sheet and a central alpha-helix, folded into a so-called "hot dog" fold, with an overhanging segment. This overhang contains the conserved residues including the hydratase 2 motif residues. In dimeric form, two beta-sheets are associated to form an extended 10-stranded beta-sheet, and the overhangs obscure the putative active sites at the subunit interface. The active site is located deep within the substrate-binding tunnel, where Asp(31) and His(36) form a catalytic dyad. These residues are catalytically important as confirmed by site-directed mutagenesis and are possibly responsible for the activation of a water molecule and the protonation of a substrate molecule, respectively. Residues such as Leu(65) and Val(130) are situated at the bottom of the substrate-binding tunnel, defining the preference of the enzyme for the chain length of the substrate. These results provide target residues for protein engineering, which will enhance the significance of this enzyme in the production of novel PHA polymers. In addition, this study provides the first structural information of the (R)-hydratase family and may facilitate further functional studies for members of the family.     

The binding site on ICAM-1 for Plasmodium falciparum-infected erythrocytes overlaps, but is distinct from, the LFA-1-binding site.

The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of three putative endothelial receptors that mediate in vitro cytoadherence of P. falciparum-infected erythrocytes. Since cytoadherence to postcapillary venular endothelium is thought to be a major factor in the virulence of P. falciparum malaria, we have examined the interaction between ICAM-1 and the P. falciparum-infected cell, and have compared it with the interaction to the physiological counter receptor, the leukocyte integrin LFA-1. Our results demonstrate that the malaria-binding site resides in the first two domains of the ICAM-1 molecule and overlaps, but is distinct from, the LFA-1 site.     

GTP-dependent binding of ADP-ribosylation factor to coatomer in close proximity to the binding site for dilysine retrieval motifs and p23.

A site-directed photocross-linking approach was employed to determine components that act downstream of ADP-ribosylation factor (ARF). To this end, a photolabile phenylalanine analog was incorporated at various positions of the putative effector region of the ARF molecule. Depending on the position of incorporation, we find specific and GTP-dependent interactions of ARF with two subunits of the coatomer complex, beta-COP and gamma-COP, as well as an interaction with a cytosolic protein (approximately 185 kDa). In addition, we observe homodimer formation of ARF molecules at the Golgi membrane. These data suggest that the binding site of ARF to coatomer is at the interface of its beta- and gamma-subunits, and this is in close proximity to the second site of interaction of coatomer with the Golgi membrane, the binding site within gamma-COP for cytosolic dibasic/diphenylalanine motifs.     

Genomic organization of the region encoding guinea pig lipoprotein lipase; evidence for exon fusion and unconventional splicing

The coding sequence of guinea pig lipoprotein lipase (LPL) is organized into nine exons and spans a region of approximately 14 kb of the guinea pig genome. A non-conforming 5'-splice site is located on the first intron, which exhibits a 12-nucleotide perfect match with the 5'-end of the second exon. A previously described tryptic cleavage site is located on exon V, close to the 3' end of this exon. A similarity to vitellogenin resides on exons IV and V, and a putative active site is found on exon IV. A novel similarity to a fatty-acid-binding protein is noted on exon VI, adjacent to the postulated heparin-binding region. We suggest that free fatty acids (FFA) and heparin to some extent share the same site of interaction on the LPL molecule; and that a high local concentration of FFA can displace LPL from its site of action--the vascular endothelium--by competing for binding to heparan sulfate.     

Crystal structure of the archaeal holliday junction resolvase Hjc and implications for DNA recognition

BACKGROUND: Homologous recombination is a crucial mechanism in determining genetic diversity and repairing damaged chromosomes. Holliday junction is the universal DNA intermediate whose interaction with proteins is one of the major events in the recombinational process. Hjc is an archaeal endonuclease, which specifically resolves the junction DNA to produce two separate recombinant DNA duplexes. The atomic structure of Hjc should clarify the mechanisms of the specific recognition with Holliday junction and the catalytic reaction. RESULTS: The crystal structure of Hjc from the hyperthermophilic archaeon Pyrococcus furiosus has been determined at 2.0 A resolution. The active Hjc molecule forms a homodimer, where an extensive hydrophobic interface tightly assembles two subunits of a single compact domain. The folding of the Hjc subunit is clearly different from any other Holliday junction resolvases thus far known. Instead, it resembles those of type II restriction endonucleases, including the configurations of the active site residues, which constitute the canonical catalytic motifs. The dimeric Hjc molecule displays an extensive basic surface on one side, which contains many conserved amino acids, including those in the active site. CONCLUSIONS: The architectural similarity of Hjc to restriction endonucleases allowed us to construct a putative model of the complex with Holliday junction. This model accounts for how Hjc recognizes and resolves the junction DNA in a specific manner. Mutational and biochemical analyses highlight the importance of some loops and the amino terminal region in interaction with DNA.     

Arrestin binding to calmodulin: a direct interaction between two ubiquitous signaling proteins

Arrestins serve as multi-functional regulators of G-protein coupled receptors, interacting with hundreds of different receptor subtypes and a variety of other signaling proteins. Here we identify calmodulin as a novel arrestin interaction partner using three independent methods in vitro and in cells. Arrestin preferentially binds calcium-loaded calmodulin with a Kd value of approximately 7 microM, which is within range of endogenous calmodulin concentrations. The calmodulin binding site is localized on the concave side of the C-domain and a loop in the center of the arrestin molecule, significantly overlapping with receptor and microtubule-binding sites. Using purified proteins, we found that arrestins sequester calmodulin, preventing its binding to microtubules. Nanomolar affinity of arrestins for their cognate receptors makes calmodulin an ineffective competitor for arrestin binding at relatively high receptor concentrations. The arrestin-calmodulin interaction likely regulates the localization of both proteins and their availability for other interaction partners.     

Approaches to Nuclease Models: Synthesis and Conformational Studies of dTprA Conjugated with Histamine AT C8(A) and Ethylenediamine at C5′(T)

Abstract

Synthesis and spectroscopic characterization of a dinucleotide dT_prA 2 having imidazole containing side chain at C8 of adenine and aminoethylamino side chain at C5′ of nucleosugar is presented. Spectroscopic results suggest that such modifications do not drastically change the conformation of the new molecule relative to that of parent derivative. An anti glycosyl torsion would place the C8-conjugated histamine close to phosphate in right orientation for putative intramolecular interaction as in ribonuclease catalytic site.     

IFN-γ-induced BST2 mediates monocyte adhesion to human endothelial cells

BST2 is a type II transmembrane protein that had been initially identified as a surface molecule expressed on terminally differentiated B cells. Here, we characterize the expression of BST2 in human endothelial cells, HUVECs. IFN-γ, among various inflammatory stimuli, dramatically upregulates BST2 expression in HUVECs. We also address a novel putative role of BST2 in IFN-γ-stimulated HUVECs as an intercellular adhesion-related molecule. We show that purified extracellular domain of BST2 protein specifically and significantly decreased the adhesion of human monocytes to HUVECs, which suggests that IFN-γ-induced BST2 expression may be involved in monocyte migration from blood through the endothelium to the inflammation site. Furthermore, we show that the monocytic cell line U937 can directly adhere to BST2 extracellular domain-coated tissue culture wells. These results provide experimental evidence to support a novel role for BST2 in the interaction between human monocyte and IFN-γ-stimulated endothelium.     

Structural characterization of the N-terminal kinase-interacting domain of an Hsp90-cochaperone Cdc37 by CD and solution NMR spectroscopy

Cdc37 is a protein kinase-targeting molecular chaperone, which cooperates with Hsp90 to assist the folding, assembly and maturation of various signaling kinases. It consists of three distinct domains: the N-terminal, middle, and C-terminal domain. While the middle domain is an Hsp90-binding domain, the N-terminal domain is recognized as a kinase-interacting domain. The N-terminal domain contains a well-conserved Ser residue at position 13, and the phosphorylation at this site has been shown to be a prerequisite for the interaction between Cdc37 and signaling kinases. Although the phosphorylation of Ser13 might induce some conformational change in Cdc37 molecule, little is known about the structure of the N-terminal domain of Cdc37. We examined the structural and dynamic properties of several fragment proteins corresponding to the N-terminal region of Cdc37 by circular dichroism and solution NMR spectroscopy. We found that the N-terminal domain of Cdc37 exhibits highly dynamic structure, and it exists in the equilibrium between α-helical and more disordered structures. We also found that phosphorylation at Ser13 did not significantly change the overall structure of N-terminal fragment protein of Cdc37. The results suggested that more complicated mechanisms might be necessary to explain the phosphorylation-activated interaction of Cdc37 with various kinases.     

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

Two-dimensional crystals of yeast RNA polymerase I dimers were obtained upon interaction with positively charged lipid layers. A three-dimensional surface model of the enzyme was determined by analyzing tilted crystalline areas and by taking advantage of the non-crystallographic internal symmetry of the dimer to correct for the missing viewing directions. The structure shows, at approximately 3 nm resolution, an irregularly shaped molecule 11 nm x 11 nm x 15 nm in size characterized by a 3 nm wide and 10 nm long groove which constitutes a putative DNA binding site. The overall structure is similar to the Escherichia coli holo enzyme and the yeast RNA polymerase II delta 4/7 structures. The most remarkable structural feature is a finger-shaped stalk which partially occludes the entrance of the groove and forms a 2.5 nm wide channel. We discuss the possible location of the catalytic centre and of the carboxy-terminal region of the beta-like subunit in the channel. The interference of different DNA fragments with RNA polymerase dimerization and crystallization indicates the orientation of the template in the putative DNA binding groove.     

Biophysics of ceramide signaling: interaction with proteins and phase transition of membranes

Ceramides have been implied in intracellular signal transduction systems regulating cellular differentiation, activation, survival and apoptosis and thus appear capable of changing the life style of virtually any cell type. Ceramide belongs to the group of sphingosine-based lipid second messenger molecules that are critically involved in the regulation of diverse cellular responses to exogenous stimuli. The emerging picture suggests that coupling of ceramide to specific signaling cascades is both stimulus and cell-type specific and depends on the subcellular topology of its production. However, little is understood about the molecular mode of ceramide action. In particular, in lieu of a defined ceramide binding motif it is not clear how ceramide would directly interact with putative target signaling proteins. This article proposes two modes of ceramide action. First, a protruding alkyl chain of ceramide may interact with a hydrophobic cavity of a signaling protein providing a lipid anchor to attach proteins to membranes. Second, the generation of ceramide generally increases the volume of hydrocarbon chains within the lipid bilayer thereby enhancing its propensity of to form a hexagonal II phase (Hex II). Besides the generation of a hydrophobic interaction site for proteins local hexagonal phase II formation can also change the membrane fluidity and permeability, which may impinge on membrane fusion processes, solubilization of detergent-resistant signaling rafts, or membrane receptor internalization. Thus, ceramide production by sphingomyelinases (SMase) can play a pivotal signaling role through direct interaction with signaling proteins or through facilitating the formation and trafficking of signal transduction complexes.