Crystal structures of the signal transducing protein GlnK from Thermus thermophilus HB8

The Thermus thermophilus HB8 genome encodes a signal transducing PII protein, GlnK. The crystal structures of GlnK have been determined in two different space groups, P2(1)2(1)2(1) and P3(1)21. The PII protein has the T-loop, which is essential for interactions with receptor proteins. In both crystal forms, three GlnK molecules form a trimer in the asymmetric unit. In one P2(1)2(1)2(1) crystal form, the three T-loops in the trimer are disordered, while in another P2(1)2(1)2(1) crystal form, the T-loop from one molecule in the trimer is ordered. In the P3(1)21 crystal, one T-loop is ordered while the other two T-loops are disordered. The conformations of the ordered T-loops significantly differ between the two crystal forms; one makes the alpha-helix in the middle of the T-loop, while the other has an extension of the beta-hairpin. Two different conformations are captured by the crystal contacts. The observation of multiple T-loop conformations suggests that the T-loop could potentially exhibit "polysterism," which would be important for interactions with receptor proteins. The crystal structures of the nucleotide-bound forms, GlnK.ATP and GlnK.ADP, have also been determined. ATP/ADP binding within a cleft at the interface of two adjacent T. thermophilus GlnK monomers might affect the conformation of the T-loop.     

Loop-driven conformational transition between the alternative and collapsed form of prethrombin-2: targeted molecular dynamics study

Two distinct crystal structures of prethrombin-2, the alternative and collapsed forms, are elucidated by X-ray crystallogrphy. We analyzed the conformational transition from the alternative to the collapsed form employing targeted molecular dynamics (TMD) simulation. Despite small RMSD difference in the two X-ray crystal structures, some hydrophobic residues (W60d, W148, W215, and F227) show a significant difference between the two conformations. TMD simulation shows that the four hydrophobic residues undergo concerted movement from dimer to trimer transition via tetramer state in the conformational change from the alternative to the collapsed form. We reveal that the concerted movement of the four hydrophobic residues is controlled by movement of specific loop regions behind. In this paper, we propose a sequential scenario for the conformational transition from the alternative form to the collapsed form, which is partially supported by the mutant W148A simulation.     

The extracellular hemoglobin of the earthworm, Lumbricus terrestris. Oxygenation properties of isolated chains, trimer, and a reassociated product

The extracellular hemoglobin of the earthworm Lumbricus terrestris has a two-tiered hexagonal structure that can be dissociated into 1/12 subunits. The Hb contains four major kinds of oxygen-binding chains, a, b, c, and d, of which a-c form a disulfide-linked trimer. Additional non-heme chains are necessary for the assembly of the intact 3800-kDa molecule of approximately 200 subunits. Oxygen equilibria have been measured for chains c and d, the abc trimer, the partially reassembled product of addition of chain d to the trimer, and the intact molecule. The results show that oxygenation of the trimer but not the isolated c or d subunits is modulated by both pH and Ca2+ ions. Cooperativity of oxygen binding by the trimer is low (Hill coefficient approximately 1.3). However, addition of chain d results in a substantial decrease in oxygen affinity and a large increase in cooperativity so that the oxygen equilibrium becomes indistinguishable from that of the intact native molecule at pH 6.8. Light-scattering data show that the smallest observed trimeric abc unit is the dimer (abc)2 at pH 6.8. Analysis of the major sedimentation velocity boundary of the product of the abc unit and chain d in the CO form in the absence of calcium surprisingly can be accounted for entirely in terms of a nondissociating dimer, (abc)2, and chain d. The data for the CO form in the presence of calcium are best fitted in terms of (abc)2.d. Although both subunits c and d also form dimers, oxygen binding by subunit c, but not d, is highly cooperative. These observations, taken together, suggest that the two dimers (abc)2 and d2 are likely to be the major participants in forming the primary functional unit, (abcd)2, which at pH 7.4 is partially dissociated when in the CO form. Subunit d is clearly necessary for the formation of a cooperative unit. The hypothesis that (abcd)2 is a primary functional unit is consistent with a stoichiometry of 2 (abcd)2 units per 1/12 subunit or 24 such units in each molecule of Hb which would contain, in all, 192 heme-containing chains.     

Structural basis for a high affinity inhibitor bound to protein kinase MK2

The Ser/Thr protein kinase MAPKAP kinase 2 (MK2) plays a crucial role in inflammation. We determined the structure of the kinase domain of MK2 in complex with a low molecular mass inhibitor in two different crystal forms, obtained from soaking and co-crystallization. To our knowledge, these are the first structures of MK2 showing the binding mode of an inhibitor with high binding affinity (IC50 8.5 nM). The two crystal forms revealed conformational flexibility in the binding site and extend the experimental basis for rational drug design. Crystal form-1 contained one MK2 molecule per asymmetric unit. Form-2 contained 12 molecules, which arrange into two different types of MK2 trimers. One of them may serve as a model for an intermediate state during substrate phosphorylation, as each MK2 monomer places its activation segment into the substrate peptide binding groove of the trimer neighbor.     

The structure of a functional unit from the wall of a gastropod hemocyanin offers a possible mechanism for cooperativity

Structure-function relationships in a molluscan hemocyanin have been investigated by determining the crystal structure of the Rapana thomasiana (gastropod) hemocyanin functional unit RtH2e in deoxygenated form at 3.38 A resolution. This is the first X-ray structure of an unit from the wall of the molluscan hemocyanin cylinder. The crystal structure of RtH2e demonstrates molecular self-assembly of six identical molecules forming a regular hexameric cylinder. This suggests how the functional units are ordered in the wall of the native molluscan hemocyanins. The molecular arrangement is stabilized by specific protomer-to-protomer interactions, which are probably typical for the functional units building the wall of the cylinders. A molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins is proposed on the basis of the molecular interactions between the protomers. In particular, the deoxygenated RtH2e structure reveals a tunnel leading from two opposite sides of the molecule to the active site. The tunnel represents a possible entrance pathway for dioxygen molecules. No such tunnels have been observed in the crystal structure of the oxy-Odg, a functional unit from the Octopus dofleini (cephalopod) hemocyanin in oxygenated form.     

Structures of the two 3D domain-swapped RNase A trimers

When concentrated in mildly acidic solutions, bovine pancreatic ribonuclease (RNase A) forms long-lived oligomers including two types of dimer, two types of trimer, and higher oligomers. In previous crystallographic work, we found that the major dimeric component forms by a swapping of the C-terminal beta-strands between the monomers, and that the minor dimeric component forms by swapping the N-terminal alpha-helices of the monomers. On the basis of these structures, we proposed that a linear RNase A trimer can form from a central molecule that simultaneously swaps its N-terminal helix with a second RNase A molecule and its C-terminal strand with a third molecule. Studies by dissociation are consistent with this model for the major trimeric component: the major trimer dissociates into both the major and the minor dimers, as well as monomers. In contrast, the minor trimer component dissociates into the monomer and the major dimer. This suggests that the minor trimer is cyclic, formed from three monomers that swap their C-terminal beta-strands into identical molecules. These conclusions are supported by cross-linking of lysyl residues, showing that the major trimer swaps its N-terminal helix, and the minor trimer does not. We verified by X-ray crystallography the proposed cyclic structure for the minor trimer, with swapping of the C-terminal beta-strands. This study thus expands the variety of domain-swapped oligomers by revealing the first example of a protein that can form both a linear and a cyclic domain-swapped oligomer. These structures permit interpretation of the enzymatic activities of the RNase A oligomers on double-stranded RNA.     

Crystal structure of phosphatidylglycerophosphatase (PGPase), a putative membrane-bound lipid phosphatase, reveals a novel binuclear metal binding site and two "proton wires"

Phosphatidylglycerophosphatase (PGPase), an enzyme involved in lipid metabolism, catalyzes formation of phosphatidylglycerol from phosphatidylglycerophosphate. Phosphatidylglycerol is a multifunctional phospholipid, found in the biological membranes of many organisms. Here, we report the crystal structure of Listeria monocytogenes PGPase at 1.8 A resolution. PGPase, an all-helical molecule, forms a homotetramer. Each protomer contains an independent active site with two metal ions, Ca(2+) and Mg(2+), forming a hetero-binuclear center located in a hydrophilic cavity near the surface of the molecule. The binuclear center, conserved ligands, metal-bound water molecules, and an Asp-His dyad form the active site. The catalytic mechanism of this enzyme is likely to proceed via binuclear metal activated nucleophilic water. The binuclear metal-binding active-site environment of this structure should provide insights into substrate binding and metal-dependent catalysis. A long channel with inter-linked linear water chains, termed "proton wires," is observed at the tetramer interface. Comparison of similar water chain structures in photosynthetic reaction centers (RCs), Cytochrome f, gramicidin, and bacteriorhodopsin, suggests that PGPase may conduct protons via proton wires.     

Crystal structures of heptakis(2,6-di-O-ethyl)cyclomaltoheptaose

Heptakis(2,6-di-O-ethyl)-beta-cyclodextrin (DE-beta-CD) was crystallized in two forms from hexane and 95% aqueous methanol, respectively: A form I crystal with the space group P2(1)2(1)2(1) and a form II crystal with the space group P3(1). In both crystals, DE-beta-CD molecules are in a round shape with intramolecular O-3-H...O-2 hydrogen bonds. In the form I crystal, the DE-beta-CD molecules are arranged along the twofold screw axis to form a helically extended polymeric chain by including the 6-O-ethyl groups of the adjacent molecule. One hexane molecule with twofold disorder is located in the intermolecular channel along the a-axis. In contrast, the DE-beta-CD molecules in the form II crystal form a helical arrangement along the threefold screw axis. One methanol and one water molecule are included on the O-6 side of the molecular cavity. The water molecule links the methanol molecule and two ethoxy groups of the adjacent DE-beta-CD molecule with hydrogen bonds. The result suggests the important role of solvent in the formation of helical arrangement of DE-beta-CD molecules.     

Interactions between metal ions and carbohydrates: the coordination behavior of neutral erythritol to transition metal ions

The single crystals of coordinated complexes of neutral erythritol (C4H10O4) with various transition metal ions were synthesized and studied using FT-IR and single crystal X-ray diffraction analysis. Two CuCl2-erythritol complexes (denoted as CuE(I) and CuE(II)) were obtained. In CuE(I), Cu2+ coordinates with two chloride ions and four OH groups from two erythritol molecules. Two copper centers are linked by one erythritol molecule to form a zigzag chain. For CuE(II), each Cu2+ coordinates with two OH groups from an erythritol molecule and two chloride ions. The crystal of CuE(II) contains complexed and free erythritol, the dimers of [Cu2Cl4(C4H10O4)] further form a [Cu2Cl4(C4H10O4)]infinity chain via secondary Cu...Cl bonds, both the dimer unit of [Cu2Cl4.(C4H10O4)] and non-coordinated C4H10O4 unit exist side by side in the crystal. MnCl2-erythritol complex whose structure is similar to CuE(I) is also acquired. The OH groups of erythritol act as ligand to coordinate to metal ions on one hand, one the other hand, OH groups form hydrogen bonds network that link chain and layer together to build three-dimensional structures.     

Synthesis and characteristics of an aspartame analogue, L-asparaginyl L-3-phenyllactic acid methyl ester

Aspartame (L-aspartyl L-phenylalanine methyl ester) isan artificial sweetener as shown in Fig.1 (A) [1]. Studieson its structure and function showed that its N-terminalL-aspartyl residue could only be replaced by aminomalonyl[2] or L-asparaginyl [3] residue. When its peptide bondwas replaced by an ester bond [Fig. 1(B)] or the hydrogenof amide in the peptide bond replaced by a methyl group[Fig. 1(C)], its sweetness was lost [4]. According to thecrystal structure of aspartame, between the …     

Assembly and packing of clathrin into coats

We present a model for the packing of clathrin molecules into the characteristic hexagons and pentagons covering coated pits and vesicles. The assembly unit is a symmetrical trimer with three extended legs. Polymerization of these units occurs in seconds under suitable conditions, giving empty polyhedral cages resembling the structures around coated vesicles. Images of small, negatively stained fragments of cages, assembled directly on electron microscope grids, reveal details of the structure, which correlate well with the predicted features of the model. There is one clathrin trimer at each polyhedral vertex, and each leg of the trimer extends along two neighboring polyhedral edges. Quasi-equivalent packing in pentagons and hexagons in polyhedra of different sizes requires a variable joint at the vertex of the molecule and a hinge in each leg. The construction of clathrin coats is remarkable for the extended fibrous contacts that each molecule makes with many others. Such contacts may confer mechanical strength combined with flexibility needed when a vesicle is pinched off from the membrane.     

A theoretical and experimental study of calcium,iron, zinc,cadmium, and sodium ions absorption by aspartame

Aspartame (L-Aspartyl-L-phenylalanine methyl ester) is a sweet dipeptide used in some foods and beverages. Experimental studies show that aspartame causes osteoporosis and some illnesses, which are similar to those of copper and calcium deficiency. This raises the issue that aspartame in food may interact with cations and excrete them from the body. This study aimed to study aspartame interaction with calcium, zinc, iron, sodium, and cadmium ions via molecular dynamics simulation (MD) and spectroscopy. Following a 480-ns molecular dynamics simulation, it became clear that the aspartame is able to sequester Fe²⁺, Ca²⁺, Cd²⁺, and Zn²⁺ ions for a long time. Complexation led to increasing UV–Vis absorption spectra and emission spectra of the complexes. This study suggests a potential risk of cationic absorption of aspartame. This study suggests that purification of cadmium-polluted water by aspartame needs a more general risk assessment.     

A kinetic model for the molecular basis of the contractile activity of Acanthamoeba myosins IA and IB

Acanthamoeba myosins IA and IB are single-headed, monomeric molecules consisting of one heavy chain and one light chain. Both have high actin-activated Mg2+-ATPase activity, when the heavy chain is phosphorylated, but neither seems to be able to form the bipolar filaments that are generally thought to be required for actomyosin-dependent contractility. In this paper, we show that, at fixed F-actin concentration, the actin-activated Mg2+-ATPase activities of myosins IA and IB increase about 5-fold in specific activity in a cooperative manner as the myosin concentration is increased. The myosin concentration range over which this cooperative change occurs depends on the actin concentration. More myosin I is required for the cooperative increase in activity at high concentrations of F-actin. The cooperative increase in specific activity at limiting actin concentrations is caused by a decrease in the KATPase for F-actin. The high and low KATPase states of the myosin have about the same Vmax at infinite actin concentration. Both myosins are completely bound to the F-actin long before the Vmax values are reached. Therefore, much of the actin activation must be the result of interactions between F-actin and actomyosin. These kinetic data can be explained by a model in which the cooperative shift of myosin I from the high KATPase to the low KATPase state results from the cross-linking of actin filaments by myosin I. Cross-linking might occur either through two actin-binding sites on a single molecule or by dimers or oligomers of myosin I induced to form by the interaction of myosin I monomers with the actin filaments. The ability of Acanthamoeba myosins IA and IB to cross-link actin filaments is demonstrated in the accompanying paper (Fujisaki, H., Albanesi, J.P., and Korn, E.D. (1985) J. Biol. Chem. 260, 11183-11189).     

The structure of the saccharide-binding site of concanavalin A.

A complex of concanavalin A with methyl alpha-D-mannopyranoside has been crystallized in space group P212121 with a = 123.9 A, b = 129.1 A and c = 67.5 A. X-ray diffraction intensities to 2.9 A resolution have been collected on a Xentronics/Nicolet area detector. The structure has been solved by molecular replacement where the starting model was based on refined coordinates of an I222 crystal of saccharide-free concanavalin A. The structure of the saccharide complex was refined by restrained least-squares methods to an R-factor value of 0.19. In this crystal form, the asymmetric unit contains four protein subunits, to each of which a molecule of mannoside is bound in a shallow crevice near the surface of the protein. The methyl alpha-D-mannopyranoside molecule is bound in the C1 chair conformation 8.7 A from the calcium-binding site and 12.8 A from the transition metal-binding site. A network of seven hydrogen bonds connects oxygen atoms O-3, O-4, O-5 and O-6 of the mannoside to residues Asn14, Leu99, Tyr100, Asp208 and Arg228. O-2 and O-1 of the mannoside extend into the solvent. O-2 is hydrogen-bonded through a water molecule to an adjacent asymmetric unit. O-1 is not involved in any hydrogen bond and there is no fixed position for its methyl substituent.     

Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain

We have studied the structure of bovine heart mitochondrial NADH:ubiquinone (Q) oxidoreductase (EC 1.6.99.3) by image analysis of electron micrographs. A three-dimensional reconstruction was calculated from a tilt-series of a two-dimensional crystal of the molecule. Our interpretation of the position of the molecule in the unit cell of the crystal is supported by additional (low-resolution) analysis of images of single molecules. The three-dimensional reconstruction was calculated with the aid of an iterative real-space reconstruction algorithm. The various projections used as input to the algorithm were obtained by averaging the images of the tilted crystal through a Fourier-space peak-filtering procedure. The reconstructed unit cell measures 15.2 X 15.2 nm in the plane of the two-dimensional crystal and has a height of 10-11 nm. The unit cell contains one molecule consisting of four large subunits. At the present resolution of about 1.3 nm in the untilted projection, these four monomers are seen as two dimers related by a two-fold axis. Two views of the single particles have been recognized; they are the top and side view of the building block of the crystal. After computer image alignment and correspondence analysis, clusters of similar particles have been averaged. In the averages an uneven stain distribution is seen around the molecules, which may result from preferential staining of hydrophilic parts of the molecule. The molecular mass of the whole molecule was determined from scanning transmission electron microscopy measurements as (1.6 +/- 0.2) X 10(6) daltons.     

Naturally occurring isolates of Neisseria gonorrhoea, which display anomalous serovar properties, express PIA/PIB hybrid porins, deletions in PIB or novel PIA molecules

The por gene of Neisseria gonorrhoeae encodes the Protein I porin responsible for serovar specificity. In this study the por genes have been sequenced from clinical isolates which exhibited anomalous serovar reactivity. One group of `intermediate' strains differed significantly from both Protein IA and IB strains, were more closely related to IA but appeared to represent a distinct class of Protein I. Another strain was closely related to Protein IB of serovar IB-6 but contained a deletion of six amino acids in surface exposed loop 6 which removed epitopes recognised by IB specific monoclonal antibodies. The third group of strains, which reacted with both IA and IB specific monoclonal antibodies, expressed hybrid Protein I molecules containing both IA and IB epitopes. These strains appeared to originate from a double crossover between Proteins IA and IB with the amino and carboxy terminal residues homologous to IB while the surface exposed loop 6 demonstrated close homology to IA. This is the first demonstration of naturally occurring gonococci expressing a hybrid Protein IA/IB.     

Models of delta-hemolysin membrane channels and crystal structures

Molecular modeling and energy calculations have been used to study how delta-hemolysin and melittin helices may aggregate on membrane surfaces and insert through membranes to form channels. In these models adjacent antiparallel amphipathic helices form planar "raft" structures, in which one surface is hydrophobic and the other hydrophilic. Models of delta-hemolysin crystal structure were developed using these "rafts." These models are based on the unit cell constants and the crystal symmetry obtained from the preliminary crystal data. Energy calculations favor channel models of delta-hemolysin with six or eight monomers per channel.     

Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes

Acne is a common skin disorder of the pilosebaceous unit. In addition to genetic, hormonal and environmental factors, abnormal colonization by Propionibacterium acnes has been implicated in the occurrence of acne via the induction of inflammatory mediators. To gain more insight into the role that sebocytes play in the innate immune response of the skin, particularly in acne, we compared the antimicrobial peptide and proinflammatory cytokine/chemokine expression at mRNA and protein levels, as well as the viability and differentiation of SZ95 sebocytes in response to co-culture with representative isolates of P. acnes type IA and type IB as well as Escherichia coli-derived lipopolysaccharide (LPS). We found that, in vitro, P. acnes type IA and IB isolates and LPS induced human beta-defensin-2 and proinflammatory cytokine/chemokine expression, and influenced sebocyte viability and differentiation. Our results provide evidence that sebocytes are capable of producing proinflammatory cytokines/chemokines and antimicrobial peptides, which may have a role in acne pathogenesis. Furthermore, since P. acnes types IA and IB differentially affect both the differentiation and viability of sebocytes, our data demonstrate that different strains of P. acnes vary in their capacity to stimulate an inflammatory response within the pilosebaceous follicle.     

Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites

BACKGROUND: Holo-(acyl carrier protein) synthase (AcpS), a member of the phosphopantetheinyl transferase superfamily, plays a crucial role in the functional activation of acyl carrier protein (ACP) in the fatty acid biosynthesis pathway. AcpS catalyzes the attachment of the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to the sidechain of a conserved serine residue on apo-ACP. RESULTS: We describe here the first crystal structure of a type II ACP from Bacillus subtilis in complex with its activator AcpS at 2.3 A. We also have determined the structures of AcpS alone (at 1.8 A) and AcpS in complex with CoA (at 1.5 A). These structures reveal that AcpS exists as a trimer. A catalytic center is located at each of the solvent-exposed interfaces between AcpS molecules. Site-directed mutagenesis studies confirm the importance of trimer formation in AcpS activity. CONCLUSIONS: The active site in AcpS is only formed when two AcpS molecules dimerize. The addition of a third molecule allows for the formation of two additional active sites and also permits a large hydrophobic surface from each molecule of AcpS to be buried in the trimer. The mutations Ile5-->Arg, Gln113-->Glu and Gln113-->Arg show that AcpS is inactive when unable to form a trimer. The co-crystal structures of AcpS-CoA and AcpS-ACP allow us to propose a catalytic mechanism for this class of 4'-phosphopantetheinyl transferases.