Vitamin C inhibits the enzymatic activity of Streptococcus pneumoniae hyaluronate lyase

Enzyme activity measurement showed that L-ascorbic acid (vitamin C (Vc)) competitively inhibits the hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. The complex crystal structure of this enzyme with Vc was determined at 2.0 A resolution. One Vc molecule was found to bind to the active site of the enzyme. The Vc carboxyl group provides the negative charges that lead the molecule into the highly positively charged cleft of the enzyme. The Vc ring system forms hydrophobic interactions with the side chain of Trp-292, which is one of the aromatic patch residues of this enzyme responsible for the selection of the cleavage sites on the substrate chain. The binding of Vc inhibits the substrate binding at hyaluronan 1, 2, and 3 (HA1, HA2, and HA3) catalytic positions. The high concentration of Vc in human tissues probably provides a low level of natural resistance to the pneumococcal invasion. This is the first time that Vc the direct inhibition on the bacterial "spreading factor" was reported, and Vc is also the first chemical that has been shown experimentally to have an inhibitory effect on bacterial hyaluronate lyase. These studies also highlight the possible structural requirement for the design of a stronger inhibitor of bacterial hyaluronate lyase.     

Crystal structure at high resolution of ferric-pyochelin and its membrane receptor FptA from Pseudomonas aeruginosa

Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. We describe at 2.0 A resolution the crystal structure of the pyochelin outer membrane receptor FptA bound to the iron-pyochelin isolated from Pseudomonas aeruginosa. One pyochelin molecule bound to iron is found in the protein structure, providing the first three-dimensional structure at the atomic level of this siderophore. The pyochelin molecule provides a tetra-dentate coordination of iron, while the remaining bi-dentate coordination is ensured by another molecule not specifically recognized by the protein. The overall structure of the pyochelin receptor is typical of the TonB-dependent transporter superfamily, which uses the proton motive force from the cytoplasmic membrane through the TonB-ExbB-ExbD energy transducing complex to transport ferric ions across the bacterial outer membrane: a transmembrane 22 beta-stranded barrel occluded by a N-terminal domain that contains a mixed four-stranded beta-sheet. The N-terminal TonB box is disordered in two crystal forms, and loop L8 is found to point towards the iron-pyochelin complex, suggesting that the receptor is in a transport-competent conformation.     

The structure of melittin in the form I crystals and its implication for melittin's lytic and surface activities.

Melittin from bee venom is water-soluble, yet integrates into membranes and lyses cells. Each melittin chain consists of 26 amino acid residues and in aqueous salt solutions it exists as a tetramer. We have determined the molecular structure of the tetramer in two crystal forms grown from concentrated salt solutions. In both crystal forms the melittin polypeptide is a bent alpha-helical rod, with the "inner" surface largely consisting of hydrophobic sidechains and the "outer" surface consisting of hydrophilic side chains. Thus, the helix is strongly amphiphilic. In the tetramer, four such helices contribute their hydrophobic side chains to the center of the molecule. The packing of melittin tetramers is also very similar in the two crystal forms: they are packed in planar layers with the outsides forming hydrophilic surfaces and the insides (the centers of melittin tetramers) forming a hydrophobic surface. We suggest that the surface activity of melittin can be rationalized in terms of these surfaces. The lytic activity of melittin can also be interpreted in terms of the molecular structure observed in the crystals: the hydrophobic inner surface of a melittin helix may integrate into the apolar region of a bilayer with the helix axis approximately parallel to the plane of the bilayer, and with the hydrophilic surface exposed to the aqueous phase. This integration would be expected to disrupt the bilayer because of melittin helix would penetrate only a short distance into it. Additionally, the integration of melittin from one side of a bilayer would produce a surface area difference across the bilayer, perhaps leading to lysis. In this view, melittin is distinct from membrane proteins that penetrate evenly into both leaflets of a bilayer or exactly halfway through a bilayer, and hence we refer to melittin as a surface-active protein.     

Crystal structure and subunit dynamics of the abalone sperm lysin dimer: egg envelopes dissociate dimers, the monomer is the active species

Lysin is a 16-kD acrosomal protein used by abalone spermatozoa to create a hole in the egg vitelline envelope (VE) by a nonenzymatic mechanism. The crystal structure of the lysin monomer is known at 1.9 A resolution. The surface of the molecule reveals two tracks of basic residues running the length of one surface of the molecule and a patch of solvent-exposed hydrophobic residues on the opposite surface. Here we report that lysin dimerizes via interaction of the hydrophobic patches of monomers. Triton X-100 dissociates the dimer. The crystal structure of the dimer is described at 2.75 A resolution. Fluorescence energy transfer experiments show that the dimer has an approximate KD of 1 microM and that monomers exchange rapidly between dimers. Addition of isolated egg VE dissociates dimers, implicating monomers as the active species in the dissolution reaction. This work represents the first step in the elucidation of the mechanism by which lysin enables abalone spermatozoa to create a hole in the egg envelope during fertilization.     

Structure of human estrone sulfatase suggests functional roles of membrane association

Estrone sulfatase (ES; 562 amino acids), one of the key enzymes responsible for maintaining high levels of estrogens in breast tumor cells, is associated with the membrane of the endoplasmic reticulum (ER). The structure of ES, purified from the microsomal fraction of human placentas, has been determined at 2.60-A resolution by x-ray crystallography. This structure shows a domain consisting of two antiparallel alpha-helices that protrude from the roughly spherical molecule, thereby giving the molecule a "mushroom-like" shape. These highly hydrophobic helices, each about 40 A long, are capable of traversing the membrane, thus presumably anchoring the functional domain on the membrane surface facing the ER lumen. The location of the transmembrane domain is such that the opening to the active site, buried deep in a cavity of the "gill" of the "mushroom," rests near the membrane surface, thereby suggesting a role of the lipid bilayer in catalysis. This simple architecture could be a prototype utilized by the ER membrane in dictating the form and the function of ER-resident enzymes.     

Insights into redox partner interactions and substrate binding in nitrite reductase from Alcaligenes xylosoxidans: crystal structures of the Trp138His and His313Gln mutants

Dissimilatory nitrite reductase catalyses the reduction of nitrite to nitric oxide within the key biological process of denitrification. We present biochemical and structural results on two key mutants, one postulated to be important for the interaction with the partner protein and the other for substrate entry. Trp138, adjacent to one of the type-1 Cu ligands, is one of the residues surrounding a small depression speculated to be important in complex formation with the physiological redox partners, azurin I and II. Our data reveal that the Trp138His mutant is fully active using methyl viologen as an artificial electron donor, but there is a large decrease in activity using azurin I. These observations together with its crystal structure at a high resolution of 1.6 A confirm the importance of Trp138 in electron transfer and thus in productive interaction with azurin. A "hydrophobic pocket" on the protein surface has been identified as the channel through which nitrite may be guided to the catalytic type-2 Cu site. Glu133 and His313 at the opening of the pocket are conserved among most blue and green copper nitrite reductases (CuNiRs). The failure to soak the substrate into our high-resolution crystal form of native and mutant CuNiRs has been linked to the observation of an extraneous poly(ethylene glycol) (PEG) molecule interacting with His313. We present the crystal structure of His313Gln and the substrate-bound mutant at high resolutions of 1.65 and 1.72 A, respectively. The observation of the substrate-bound structure for the His313Gln mutant and inhibitory studies with PEG establishes the role of the hydrophobic pocket as the port of substrate entry.     

Crystal structure of trimeric carbohydrate recognition and neck domains of surfactant protein A

Surfactant protein A (SP-A), one of four proteins associated with pulmonary surfactant, binds with high affinity to alveolar phospholipid membranes, positioning the protein at the first line of defense against inhaled pathogens. SP-A exhibits both calcium-dependent carbohydrate binding, a characteristic of the collectin family, and specific interactions with lipid membrane components. The crystal structure of the trimeric carbohydrate recognition domain and neck domain of SP-A was solved to 2.1-A resolution with multiwavelength anomalous dispersion phasing from samarium. Two metal binding sites were identified, one in the highly conserved lectin site and the other 8.5 A away. The interdomain carbohydrate recognition domain-neck angle is significantly less in SP-A than in the homologous collectins, surfactant protein D, and mannose-binding protein. This conformational difference may endow the SP-A trimer with a more extensive hydrophobic surface capable of binding lipophilic membrane components. The appearance of this surface suggests a putative binding region for membrane-derived SP-A ligands such as phosphatidylcholine and lipid A, the endotoxic lipid component of bacterial lipopolysaccharide that mediates the potentially lethal effects of Gram-negative bacterial infection.     

The crystal structure of alpha-bungarotoxin at 2.5 A resolution: relation to solution structure and binding to acetylcholine receptor

We report collection of 2.5 A resolution X-ray diffraction data from newly grown crystals of the rare 'small unit cell' form of the long snake neurotoxin, alpha-bungarotoxin. The previous model of the molecule has been rebuilt, and refined using least-square methods to a crystallographic residual of 0.24 at 2.5 A resolution. alpha-Bungarotoxin's crystal structure is compared with the crystal structures of two other snake neurotoxins (cobratoxin and erabutoxin), and with its solution structure inferred from spectroscopic studies. Significant differences include less beta-sheet in bungarotoxin's crystal structure than in solution, or in the crystal structures of other neurotoxins, and an unusual orientation in the crystal of the invariant tryptophan. The functional, binding surface of bungarotoxin is described; it consists primarily of hydrophobic and hydrogen-bonding groups and only a few charged side-chains. The structure is compared with experimental binding parameters for neurotoxins.     

The outer membrane protein OmpW forms an eight-stranded beta-barrel with a hydrophobic channel

Escherichia coli OmpW belongs to a family of small outer membrane proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. To gain insight into the function of these proteins A we have determined the crystal structure of E. coli OmpW to 2.7-A resolution. The structure shows that OmpW forms an 8-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound n-dodecyl-N,N-dimethylamine-N-oxide detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of n-dodecyl-N,N-dimethylamine-N-oxide. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial outer membrane.     

Crystal structure of the trigonal form of bovine beta-lactoglobulin and of its complex with retinol at 2.5 A resolution

The structure of the trigonal crystal form of bovine beta-lactoglobulin has been determined by X-ray diffraction methods. An electron density map, calculated with phases obtained by the multiple isomorphous replacement method, served as a starting point for alternate cycles of model building and restrained least-squares refinement. The model of the molecule fitted to the initial Fourier map was the one built for the orthorhombic crystal form of beta-lactoglobulin, solved at 2.8 A resolution (1 A = 0.1 nm). The final R factor for 1456 atoms (1276 non-hydrogen protein atoms and 180 solvent atoms) is 0.22, including 5245 reflections from 6.0 to 2.5 A. The molecule shows significant differences in the two crystal forms mentioned, mainly due to different packing. In the trigonal form, the species crystallized does not appear to be dimeric, but a linear polymer with tight intermolecular contacts. A difference electron density map between the complex of beta-lactoglobulin with retinol and the native protein shows no significant peaks in the cavity which, in the similar retinol-binding protein, binds the chromophore. Instead, differences are found at a surface pocket, which is limited almost completely by hydrophobic residues.     

Intriguing conformation changes associated with the trans/cis isomerization of a prolyl residue in the active site of the DsbA C33A mutant

Escherichia coli DsbA belongs to the thioredoxin family and catalyzes the formation of disulfide bonds during the folding of proteins in the bacterial periplasm. It active site (C30-P31-H32-C33) consists of a disulfide bridge that is transferred to newly translocated proteins. The work reported here refers to the DsbA mutant termed C33A that retains, towards reduced unfolded thrombin inhibitor, an activity comparable with the wild-type enzyme. Besides, C33A is also able to form a stable covalent complex with DsbB, the membrane protein responsible for maintaining DsbA in its active form. We have determined the crystal structure of C33A at 2.0 angstroms resolution. Although the general architecture of wt DsbA is conserved, we observe the trans/cis isomerization of P31 in the active site and further conformational changes in the so-called "peptide binding groove" region. Interestingly, these modifications involve residues that are specific to DsbA but not to the thioredoxin family fold. The C33A crystal structure exhibits as well a hydrophobic ligand bound close to the active site of the enzyme. The structural analysis of C33A may actually explain the peculiar behavior of this mutant in regards with its interaction with DsbB and thus provides new insights for understanding the catalytic cycle of DsbA.     

Crystal structure of the bacterial protein export chaperone secB

SecB is a bacterial molecular chaperone involved in mediating translocation of newly synthesized polypeptides across the cytoplasmic membrane of bacteria. The crystal structure of SecB from Haemophilus influenzae shows that the molecule is a tetramer organized as a dimer of dimers. Two long channels run along the side of the molecule. These are bounded by flexible loops and lined with conserved hydrophobic amino acids, which define a suitable environment for binding non-native polypeptides. The structure also reveals an acidic region on the top surface of the molecule, several residues of which have been implicated in binding to SecA, its downstream target.     

Incorporation of surface tension into molecular dynamics simulation of an interface: a fluid phase lipid bilayer membrane.

In this paper we report on the molecular dynamics simulation of a fluid phase hydrated dimyristoylphosphatidylcholine bilayer. The initial configuration of the lipid was the x-ray crystal structure. A distinctive feature of this simulation is that, upon heating the system, the fluid phase emerged from parameters, initial conditions, and boundary conditions determined independently of the collective properties of the fluid phase. The initial conditions did not include chain disorder characteristic of the fluid phase. The partial charges on the lipids were determined by ab initio self-consistent field calculations and required no adjustment to produce a fluid phase. The boundary conditions were constant pressure and temperature. Thus the membrane was not explicitly required to assume an area/phospholipid molecule thought to be characteristic of the fluid phase, as is the case in constant volume simulations. Normal to the membrane plane, the pressure was 1 atmosphere, corresponding to the normal laboratory situation. Parallel to the membrane plane a negative pressure of -100 atmospheres was applied, derived from the measured surface tension of a monolayer at an air-water interface. The measured features of the computed membrane are generally in close agreement with experiment. Our results confirm the concept that, for appropriately matched temperature and surface pressure, a monolayer is a close approximation to one-half of a bilayer. Our results suggest that the surface area per phospholipid molecule for fluid phosphatidylcholine bilayer membranes is smaller than has generally been assumed in computational studies at constant volume. Our results confirm that the basis of the measured dipole potential is primarily water orientations and also suggest the presence of potential barriers for the movement of positive charges across the water-headgroup interfacial region of the phospholipid.     

Crystal Structure of the Complex of Concanavalin A and Hexapeptide

The X-ray structure analysis of a cross-linked crystal of concanavalin A soaked with a hexapeptide molecule as a probe molecule showed an electron density corresponding to full occupation in the binding pocket. The site lies on the surface of concanavalin A and is surrounded by three symmetry-related molecules. The crystal structure of the hexapeptide complex was refined at 1.93- resolution, to an R-factor of 19% (R _free factor of 25%), with an RMS deviation in bond distances of 0.01 . The model includes all 237 residues of concanavalin A, one manganese ion, one calcium ion, 95 water molecules, one glutaraldehyde molecule, one isopropanol molecule, and one hexapeptide molecule. This X-ray structure analysis also provides an approach to mapping the binding surface of crystalline protein with a probe molecule that is dissolved in the mixture of organic solvent with water or in neat organic solvent but is hardly dissolved in aqueous solution.     

From minichaperone to GroEL 1: information on GroEL-polypeptide interactions from crystal packing of minichaperones

We are reconstructing the mechanism of action of GroEL by a reductionist approach of isolating its minimal fragment that has residual activity (the "minichaperone" core) and then identifying how additional elements of structure confer further activity and function. We report here the 2.0 A resolution crystal structure of the minichaperone GroEL(193-345). The structure provides further clues on the nature of GroEL-polypeptide substrate interactions, because two molecules in the asymmetric unit interact by the binding of one molecule in the active site of its partner, thus mimicking a chaperone-polypeptide substrate complex. The results may explain some experimental observations, including the preference of GroEL for net positive charges (mediated by Glu238 and Glu257) and the key role of Tyr203 in mediating polypeptide binding. The larger binding site identified by these studies forms a continuous surface near the opening of the central cavity of GroEL that can accommodate a wide range of non-native protein conformations that differ in size and in structural and chemical properties.     

Structural basis of phospholipase A2 inhibition for the synthesis of prostaglandins by the plant alkaloid aristolochic acid from a 1.7 A crystal structure

This is the first structural observation of a plant product showing high affinity for phospholipase A(2) and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A(2) and a plant alkaloid aristolochic acid has been determined and refined to 1.7 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, beta-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp(31). Trp(31) is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp(31) is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp(31) in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His(48) and Asp(49).     

Crystal structure of the matrix protein VP40 from Ebola virus

Ebola virus maturation occurs at the plasma membrane of infected cells and involves the clustering of the viral matrix protein VP40 at the assembly site as well as its interaction with the lipid bilayer. Here we report the X-ray crystal structure of VP40 from Ebola virus at 2.0 A resolution. The crystal structure reveals that Ebola virus VP40 is topologically distinct from all other known viral matrix proteins, consisting of two domains with unique folds, connected by a flexible linker. The C-terminal domain, which is absolutely required for membrane binding, contains large hydrophobic patches that may be involved in the interaction with lipid bilayers. Likewise, a highly basic region is shared between the two domains. The crystal structure reveals how the molecule may be able to switch from a monomeric conformation to a hexameric form, as observed in vitro. Its implications for the assembly process are discussed.     

Crystal structure of Neisserial surface protein A (NspA), a conserved outer membrane protein with vaccine potential

The neisserial surface protein A (NspA) from Neisseria meningitidis is a promising vaccine candidate because it is highly conserved among meningococcal strains and induces bactericidal antibodies. NspA is a homolog of the Opa proteins, which mediate adhesion to host cells. Here, we present the crystal structure of NspA, determined to 2.55-A resolution. NspA forms an eight-stranded antiparallel beta-barrel. The four loops at the extracellular side of the NspA molecule form a long cleft, which contains mainly hydrophobic residues and harbors a detergent molecule, suggesting that the protein might function in the binding of hydrophobic ligands, such as lipids. In addition, the structure provides a starting point for structure-based vaccine design.     

Crystal structure of JlpA, a surface-exposed lipoprotein adhesin of Campylobacter jejuni

The Campylobacter jejuni JlpA protein is a surface-exposed lipoprotein that was discovered as an adhesin promoting interaction with host epithelium cells, an early critical step in the pathogenesis of C. jejuni disease. Increasing evidence ascertained that JlpA is antigenic, indicating a role of JlpA in immune response during the infectious process. Here, we report the crystal structure of JlpA at 2.7? resolution, revealing a catcher's mitt shaped unclosed half β-barrel. Although the apparent architecture of JlpA is somewhat reminiscent of other bacterial lipoproteins such as LolB, the topology of JlpA is unique among the bacterial surface proteins reported to date and therefore JlpA represents a novel bacterial cell surface lipoprotein. The concave face of the structure results in an unusually large hydrophobic basin with a localized acidic pocket, suggesting a possibility that JlpA may accommodate multiple ligands. Therefore, the structure provides framework for determining the molecular function of JlpA and new strategies for the rational design of small molecule inhibitors efficiently targeting JlpA.     

The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.