副溶血性弧菌耐热性直接溶血素(TDH)的研究进展

副溶血性弧菌(Vibrio parahaemolyticus)是海产品中一种常见的食源性致病菌,常导致水产养殖动物患病或者引起食物中毒。耐热性直接溶血素(thermotolerant direct hemolysin,TDH)是副溶血性弧菌最为重要的致病因子之一。本文围绕tdh基因在弧菌属中的广泛分布与传播、tdh基因的多样性及其表达调控、TDH的蛋白结构及其生物活性进行了综述,并对未来TDH的研究方向进行了展望。旨在进一步了解由副溶血性弧菌感染所引起的病症,为预防副溶血性弧菌的感染和临床治疗提供理论支撑。     

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts—using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023 Å^− 1, corresponding to spatially and statistically averaged repeat distance values of about 275–330 Å. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D₂O in dim laboratory light or transpired with D₂O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230 Å, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.     

Oligomerization propensity and flexibility of yeast frataxin studied by X-ray crystallography and small-angle X-ray scattering

Frataxin is a mitochondrial protein with a central role in iron homeostasis. Defects in frataxin function lead to Friedreich's ataxia, a progressive neurodegenerative disease with childhood onset. The function of frataxin has been shown to be closely associated with its ability to form oligomeric species; however, the factors controlling oligomerization and the types of oligomers present in solution are a matter of debate. Using small-angle X-ray scattering, we found that Co²⁺, glycerol, and a single amino acid substitution at the N-terminus, Y73A, facilitate oligomerization of yeast frataxin, resulting in a dynamic equilibrium between monomers, dimers, trimers, hexamers, and higher-order oligomers. Using X-ray crystallography, we found that Co²⁺ binds inside the channel at the 3-fold axis of the trimer, which suggests that the metal has an oligomer-stabilizing role. The results reveal the types of oligomers present in solution and support our earlier suggestions that the trimer is the main building block of yeast frataxin oligomers. They also indicate that different mechanisms may control oligomer stability and oligomerization in vivo.     

Integration of small-angle X-ray scattering data into structural modeling of proteins and their assemblies

A major challenge in structural biology is to determine the configuration of domains and proteins in multidomain proteins and assemblies, respectively. All available data should be considered to maximize the accuracy and precision of these models. Small-angle X-ray scattering (SAXS) efficiently provides low-resolution experimental data about the shapes of proteins and their assemblies. Thus, we integrated SAXS profiles into our software for modeling proteins and their assemblies by satisfaction of spatial restraints. Specifically, we modeled the quaternary structures of multidomain proteins with structurally defined rigid domains as well as quaternary structures of binary complexes of structurally defined rigid proteins. In addition to SAXS profiles and the component structures, we used stereochemical restraints and an atomic distance-dependent statistical potential. The scoring function is optimized by a biased Monte Carlo protocol, including quasi-Newton and simulated annealing schemes. The final prediction corresponds to the best scoring solution in the largest cluster of many independently calculated solutions. To quantify how well the quaternary structures are determined based on their SAXS profiles, we used a benchmark of 12 simulated examples as well as an experimental SAXS profile of the homotetramer d-xylose isomerase. Optimization of the SAXS-dependent scoring function generally results in accurate models if sufficiently precise approximations for the constituent rigid bodies are available; otherwise, the best scoring models can have significant errors. Thus, SAXS profiles can play a useful role in the structural characterization of proteins and assemblies if they are combined with additional data and used judiciously. Our integration of a SAXS profile into modeling by satisfaction of spatial restraints will facilitate further integration of different kinds of data for structure determination of proteins and their assemblies.     

Vibrio harveyi hemolysin induces ultrastructural changes and apoptosis in flounder (Paralichthys olivaceus) cells

Vibrio harveyi hemolysin (VHH) is considered a major pathogenic virulence factor to fish. However, the VHH active-site mutant has lost all hemolytic and phospholipase activities as well as pathogenicity. In this study, the effect of VHH on erythrocytes and a gill cell line from flounder was elucidated. Erythrocyte membranes formed thin tubular protrusions immediately after exposure to VHH, and membrane corrugations were evident after extended incubation. In contrast, the mutant VHH did not induce any gross morphological changes. With VHH-treated FG-9307 cells, a cell line derived from flounder gill, destruction of organelles and formation of features resembling apoptotic bodies were observed. Immunogold staining showed that a large amount of VHH was deposited on the membranes and membrane debris of erythrocytes and FG-9307 cells after treatment with VHH. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in VHH-treated FG-9307 cells using DAPI staining. Moreover, cell cycle analysis showed that VHH increased the proportion of cells in G1 phase. In addition, VHH significantly increased the percentage of apoptosis, the number of TUNEL positive apoptotic cells, and caspase-3 activity in FG-9307 cells when compared with the untreated controls. These data suggested that VHH killed the cells through apoptosis via the caspase activation pathway.     

Probing dimerization and structural flexibility of mammalian lipoxygenases by small-angle X-ray scattering

Human lipoxygenases (LOXs) and their metabolites have a great impact on human homeostasis and are of interest for targeted drug design. This goal requires detailed knowledge of their structures and an understanding of structure-function relationship. At the moment, there are two complete crystal structures for mammalian LOX [rabbit 12/15LOX (r-12/15LOX) and human 5LOX (h-5LOX)] and a fragment of human 12LOX. The low-resolution structures in solution for various LOX isoforms have brought about controversial results. Here we explored the behavior of r-12/15LOX in aqueous solution under different conditions (salt and pH) by small-angle X-ray scattering (SAXS) and compared it with human platelet-type 12S-LOX (hp-12LOX) and h-5LOX. Thermodynamic calculations concerning the stability of molecular assemblies, thermal motion analysis [TLSMD (translation, libration, and screw rotation motion detection based on crystallographic temperature factor B_j)], and results of SAXS analyses brought about the following conclusions: (i) in contrast to its crystal structure, r-12/15LOX functions as a monomer that dominates in solution; (ii) it dimerizes at higher protein concentrations in the presence of salt and with increasing degree of motional freedom of the N-terminal PLAT domain, as suggested by the Y98,614 → R double mutant; (iii) in aqueous solutions, hp-12LOX is stable as a dimer, in contrast to h-5LOX and r-12/15LOX, which are monomeric; and (iv) all three mammalian isozymes show a high level of flexibility not only for the PLAT domain but also for other subdomains of the catalytic part in TLS (translation, libration, and screw rotation) analysis and hp-12LOX in SAXS.     

X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB

The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB₃₂₁) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB₃₂₁ consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB₃₂₁ in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB₃₂₁ binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB₃₂₁. These results call into question whether receptor dimerization is the basic underlying event in InlB₃₂₁-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB₃₂₁ bind and activate the Met receptor.     

Solution structure of human and bovine beta(2)-glycoprotein I revealed by small-angle X-ray scattering

beta(2)-Glycoprotein I (beta(2)GPI) is a highly glycosylated phospholipid-binding plasma protein comprised of four complement control protein (CCP) domains and a distinct fifth domain. The structural organisation of human and bovine beta(2)GPI in aqueous solution was studied by small-angle X-ray scattering (SAXS). Low-resolution models that match the SAXS experimental data best were independently constructed by three different ab initio 3D-reconstruction algorithms. Similar elongated S-shaped models with distinct side-arms, which were correlated to the position of the carbohydrate chains, were restored from all three algorithms. Due to an additional glycosylation site located on the CCP2 domain of bovine beta(2)GPI a small change in the characteristic SAXS parameters was observed, which coincided with results obtained from SDS-PAGE. In comparison to the human analogue the corresponding restored low-resolution models displayed a similar S-shape with less bending in the middle part. As the experimental SAXS curves fit poorly to the simulated scattering curves calculated from the crystallographic coordinates of human beta(2)GPI, the crystal structure was modified. First, additional carbohydrate residues missing from the crystal structure were modelled. Second, on the basis of the low-resolution models, the J-shaped crystal structure was rotated between CCP3 and CCP2 assuming the greatest interdomain flexibility between these domains. An S-shaped model with a tilt angle of approximately 60 degrees between CCP3 and CCP2 yielded the best fit to the experimental SAXS data. Since there is evidence that beta(2)GPI can adopt different conformations, which reveal distinct differences in autoantibody recognition, our data clearly point to a reorientation of the flexible domains, which may be an essential feature for binding of autoantibodies.     

The signaling pathway in histidine kinase and the response regulator complex revealed by X-ray crystallography and solution scattering

The structure of a histidine kinase (ThkA) complexed with a response regulator (TrrA) in the two-component regulatory system from hyperthermophile Thermotoga maritima was determined by a combination of X-ray crystallography at a resolution of 4.2 A and small-angle X-ray scattering (SAXS). The boundary of the three component domains (PAS-sensor, dimerization and catalytic domains) of ThkA and the bound TrrA molecule were unambiguously assigned in the electron density map at 4.2 A resolution. ThkA forms a dimer with crystallographic 2-fold symmetry and two monomeric TrrAs bind to the ThkA dimer. SAXS experiments also confirmed this association state in solution and specific binding between ThkA and TrrA (Kd=8.2x10(-11) M(-2)). The association interface between ThkA and TrrA contains the phosphotransfer His residue in the ThkA, indicative of an efficient receipt of the phosphoryl group. One Per-Arnt-Sim (PAS) domain does not interact with the other PAS domain, but with the catalytic domain of the same polypeptide chain and with one TrrA molecule. Observed inter-domain and inter-molecular interactions reveal a definite pathway of signal transduction in the kinase/regulator complex. In addition, we propose a responsible role of TrrA for the feedback regulation of sensing and/or kinase activities of ThkA.     

Structural characterization of the active and inactive states of Src kinase in solution by small-angle X-ray scattering

Src kinase plays an important role in several signaling and regulation mechanisms in vivo. Enzymatic activity is tightly regulated through the phosphorylation and dephosphorylation of tyrosine 527, which is placed at the C-terminal tail. Here, we have addressed domain rearrangements involved in the regulation mechanism of Src kinase in solution using small-angle X-ray scattering. In the phosphorylated wild-type form of Src kinase corresponding to the inactive state of the protein, a single conformation compatible with a closed crystallographic structure was found in solution. In the Y527F point mutant representing the active state, analysis of scattering data reveals an equilibrium between two differently populated conformations differing in the radius of gyration by 5 Å. The major species (85% of the total population) presents a closed conformation indistinguishable from the crystallographic structure of the inactive state. The minor species (15% of the total population) is an open conformation similar to the crystallographic structure in the active state. The latter structure has the SH3, SH2, and SH2-catalytic domain linker assembled as a pseudo-two-domain protein. The regulation model emerging from this study, including at least three different conformational states, allows the tight regulation of the enzyme without compromising fast response in the presence of natural targets.     

Electron microscopy of the separated outer tegument of the sparganum and its antigenicity

The author reported previously on separation of the outer tegument of the spargana (plerocercoids of Spirometra mansoni) using high concentration of urea solution. To determine which layer of the tegument is separated by this method, an electron microscopic analysis has been processed in this study. It was confirmed that the basement layer of the tegument is separated from the parenchyme of the sparganum. In addition, the antigenicity of the separated outer tegument against the human sparganosis patient sera was evaluated. Numerous antigenic proteins, including 16 and 55 kDa proteins, were noticed in the separated tegument; however, there were no diagnostic 31/36 kDa molecules in this tegument. The molecules reactive with the patient sera in the tegument are to be characterized in future studies.     

Multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly

Lumazine synthases have been observed in the form of pentamers, dimers of pentamers, icosahedral capsids consisting of 60 subunits and larger capsids with unknown molecular structure. Here we describe the analysis of the assembly of native and mutant forms of lumazine synthases from Bacillus subtilis and Aquifex aeolicus at various pH values and in the presence of different buffers using small angle X-ray scattering and electron microscopy. Both wild-type lumazine synthases are able to form capsids with a diameter of roughly 160 A and larger capsids with diameters of around 300 A. The relative abundance of smaller and larger capsids is strongly dependent on buffer and pH. Both forms can co-exist and are in some cases accompanied by other incomplete or deformed capsids. Several mutants of the B. subtilis lumazine synthase, in which residues in or close to the active site were replaced, as well as an insertion mutant of A. aeolicus lumazine synthase form partially or exclusively larger capsids with a diameter of about 300 A. The mutations also reduce or inhibit enzymatic activity, suggesting that the catalytic function of the enzyme is tightly correlated with its quaternary structure. The data show that multiple assembly forms are a general feature of lumazine synthases.     

Towards a molecular description of intermediate filament structure and assembly

Intermediate filaments (IFs) represent one of the prominent cytoskeletal elements of metazoan cells. Their constituent proteins are coded by a multigene family, whose members are expressed in complex patterns that are controlled by developmental programs of differentiation. Hence, IF proteins found in epidermis differ significantly from those in muscle or neuronal tissues. Due to their fibrous nature, which stems from a fairly conserved central alpha-helical coiled-coil rod domain, IF proteins have long resisted crystallization and thus determination of their atomic structure. Since they represent the primary structural elements that determine the shape of the nucleus and the cell more generally, a major challenge is to arrive at a more rational understanding of how their nanomechanical properties effect the stability and plasticity of cells and tissues. Here, we review recent structural results of the coiled-coil dimer, assembly intermediates and growing filaments that have been obtained by a hybrid methods approach involving a rigorous combination of X-ray crystallography, small angle X-ray scattering, cryo-electron tomography, computational analysis and molecular modeling.     

A new side opening on prolyl oligopeptidase revealed by electron microscopy

Prolyl oligopeptidase (POP) has gained importance as a target for the treatment of neuropsychiatric diseases and cognitive disturbances. Therefore, a variety of strategies are currently used to identify POP inhibitors. Here we performed electron microscopy (EM) studies of human POP. Our data reveal for the first time the presence of a new side opening in POP that was not observed in any of the crystallographic structures described to date. Finally, molecular dynamics, the relevant normal modes that contribute to the fluctuation of the catalytic triad residues and the algorithm CAVERN also support the existence of a new large side opening on POP.     

Structural characterisation of the insecticidal toxin XptA1, reveals a 1.15 MDa tetramer with a cage-like structure

A recently identified class of proteins conferring insecticidal activity to several bacteria within the Enterobacteriaceae family have potential for control of commercially important insect pests. Here, we report the first purification, biophysical characterisation and 3-D structural analysis of one of the toxin components, XptA1, from Xenorhabdus nematophila PMFI296 to a resolution of 23 A. Membrane binding studies indicate that the three-component toxin system has a different mode of action from that of proteins from Bacillus thuringiensis (Bt). Biophysical characterisation of XptA1 suggests a mechanism of action of XptA1 whereby it first binds to the cell membrane forming a structure with a central cavity and forms a complex with its partners XptB1 and XptC1 producing the full insecticidal toxin. The structure of XptA1 is shown by a combination of electron microscopy, ultracentrifugation and circular dichroism spectroscopy to be a 1.15 MDa tetramer with a cage-like structure. Each of the four symmetry-related subunits has three well-defined domains and a longitudinal twist with one end narrower than the other. One third of the residues of XptA1 are alpha-helical and it is suggested the subunits associate partly via an alpha-helical coiled-coil interaction. XptA1 itself shows the same secondary structure at neutral pH and in an alkaline environment up to pH10.5. This pH tolerance indicates that the folded XptA1 can pass through the midgut of Lepidopteran insects susceptible to the insecticidal toxin complex. This implies therefore that its folded structure is important for its biological activity.     

Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by fourier transform infrared spectroscopy and scanning electron microscopy

Rhizobium meliloti produced a copolymer of short chain length polyhydroxyalkanoate (scl-PHA) on sucrose and rice bran oil as carbon substrates. Recombinant Escherichia coli (JC7623ABC1J4), bearing PHA synthesis genes, was used to synthesize short chain length-co-medium chain length PHA (scl-co-mcl-PHA) on glucose and decanoic acid. Fourier transform infrared spectroscopy (FTIR) spectra of the PHAs indicated strong characteristic bands at 1282, 1723, and 2934 cm⁻¹ for scl-PHA and at 2933 and 2976 cm⁻¹ for scl-co-mcl-PHA polymer. Differentiation of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate-P(HB-co-HV) copolymer was obseverd using FTIR, with absorption bands at 1723 and 1281 for PHB, and at 1738, 1134, 1215 cm⁻¹ for HV-copolymer. The copolymers were analyzed by GC and ¹H NMR spectroscopy. Films of polymer blends of PHA produced by R. meliloti and recombinant E. coli were prepared using glycerol, polyethylene glycol, polyvinyl acetate, individually (1:1 ratio), to modify the mechanical properties of the films and these films were evaluated by FTIR and scanning electron microscopy.     

Small-angle X-ray scattering reveals the solution structure of a bacteriophytochrome in the catalytically active Pr state

Phytochromes are light-sensing macromolecules that are part of a two component phosphorelay system controlling gene expression. Photoconversion between the Pr and Pfr forms facilitates autophosphorylation of a histidine in the dimerization domain (DHp). We report the low-resolution structure of a bacteriophytochrome (Bph) in the catalytic (CA) Pr form in solution determined by small-angle X-ray scattering (SAXS). Ab initio modeling reveals, for the first time, the domain organization in a typical bacteriophytochrome, comprising an chromophore binding and phytochrome (PHY) N terminal domain followed by a C terminal histidine kinase domain. Homologous high-resolution structures of the light-sensing chromophore binding domain (CBD) and the cytoplasmic part of a histidine kinase sensor allows us to model 75% of the structure with the remainder comprising the phytochrome domain which has no 3D representative in the structural database. The SAXS data reveal a dimeric Y shaped macromolecule and the relative positions of the chromophores (biliverdin), autophosphorylating histidine residues and the ATP molecules in the kinase domain. SAXS data were collected from a sample in the autophosphorylating Pr form and reveal alternate conformational states for the kinase domain that can be modeled in an open (no-catalytic) and closed (catalytic) state. This model suggests how light-induced signal transduction can stimulate autophosphorylation followed by phosphotransfer to a response regulator (RR) in the two-component system.     

The ultrastructure of Chlorobaculum tepidum revealed by cryo-electron tomography

Chlorobaculum (Cba) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. As other anoxygenic green photosynthetic bacteria, Cba tepidum synthesizes bacteriochlorophylls for the assembly of a large light-harvesting antenna structure, the chlorosome. Chlorosomes are sac-like structures that are connected to the reaction centers in the cytoplasmic membrane through the BChl α-containing Fenna–Matthews–Olson protein. Most components of the photosynthetic machinery are known on a biophysical level, however, the structural integration of light harvesting with charge separation is still not fully understood. Despite over two decades of research, gaps in our understanding of cellular architecture exist. Here we present an in-depth analysis of the cellular architecture of the thermophilic photosynthetic green sulfur bacterium of Cba tepidum by cryo-electron tomography. We examined whole hydrated cells grown under different electron donor conditions. Our results reveal the distribution of chlorosomes in 3D in an unperturbed cell, connecting elements between chlorosomes and the cytoplasmic membrane and the distribution of reaction centers in the cytoplasmic membrane.     

Photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina

Fourier transform infrared spectroscopy (FTIR) difference spectroscopy in combination with deuterium exchange experiments has been used to study the photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina. Comparison of (P740(+)-P740) and (P700(+)-P700) FTIR difference spectra show that P700 and P740 share many structural similarities. However, there are several distinct differences also: 1), The (P740(+)-P740) FTIR difference spectrum is significantly altered upon proton exchange, considerably more so than the (P700(+)-P700) FTIR difference spectrum. The P740 binding pocket is therefore more accessible than the P700 binding pocket. 2), Broad, "dimer" absorption bands are observed for both P700(+) and P740(+). These bands differ significantly in substructure, however, suggesting differences in the electronic organization of P700(+) and P740(+). 3), Bands are observed at 2727(-) and 2715(-) cm(-1) in the (P740(+)-P740) FTIR difference spectrum, but are absent in the (P700(+)-P700) FTIR difference spectrum. These bands are due to formyl CH modes of chlorophyll d. Therefore, P740 consists of two chlorophyll d molecules. Deuterium-induced modification of the (P740(+)-P740) FTIR difference spectrum indicates that only the highest frequency 13(3) ester carbonyl mode of P740 downshifts, indicating that this ester mode is weakly H-bonded. In contrast, the highest frequency ester carbonyl mode of P700 is free from H-bonding. Deuterium-induced changes in (P740(+)-P740) FTIR difference spectrum could also indicate that one of the chlorophyll d 3(1) carbonyls of P740 is hydrogen bonded.