NMR structure of a fungal virulence factor reveals structural homology with mammalian saposin B

The fungal protein CBP ( c alcium b inding p rotein) is a known virulence factor with an unknown virulence mechanism. The protein was identified based on its ability to bind calcium and its prevalence as Histoplasma capsulatum 's most abundant secreted protein. However, CBP has no sequence homology with other CBPs and contains no known calcium binding motifs. Here, the NMR structure of CBP reveals a highly intertwined homodimer and represents the first atomic level NMR model of any fungal virulence factor. Each CBP monomer is comprised of four α-helices that adopt the saposin fold, characteristic of a protein family that binds to membranes and lipids. This structural homology suggests that CBP functions as a lipid binding protein, potentially interacting with host glycolipids in the phagolysosome of host cells.     

Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens

The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX) family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.     

Equilibrium unfolding CD studies of bovine beta-lactoglobulin and its 14-52 fragment at acidic pH

Bovine beta-lactoglobulin represents an interesting example of context-dependent secondary structure induction. In fact, secondary structure predictions indicated that this beta-barrel protein has a surprisingly high alpha-helical preference, which was retained for short fragments. Cooperative transitions from the native beta-sheet to alpha-helical structures were additionally induced by organic solvents, in particular trifluoroethanol. As a result of this high alpha-helical preference, it has been proposed that non-native alpha-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better understanding of the processes that underlie conformational plasticity in this protein, CD measurements in the presence of increasing amounts of urea and in the presence of organic solvents were performed. Urea unfolding studies, performed at pH 2.1 and 37 degrees C, revealed an apparent two-state transition, and afforded no evidence of non native alpha-helical intermediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with higher molar concentration urea, lead to partial misfolding. A 29-mer peptide covering the region of strands a and b of the intact protein, characterized by the presence of 4/3 heptad repeats, was synthesized and studied by CD in the presence of different solvents. On the basis of the obtained results, a mechanism was proposed to explain the structural transition from the beta to alpha structure, provoked by organic solvents in the intact protein.     

Global shape and pH stability of ovorubin, an oligomeric protein from the eggs of Pomacea canaliculata

Ovorubin, a 300-kDa thermostable oligomer, is the major egg protein from the perivitellin fluid that surrounds the embryos of the apple snail Pomacea canaliculata. It plays essential roles in embryo development, including transport and protection of carotenoids, protease inhibition, photoprotection, storage, and nourishment. Here, we report ovorubin dimensions and global shape, and test the role of electrostatic interactions in conformational stability by analyzing the effects of pH, using small-angle X-ray scattering (SAXS), transmission electron microscopy, CD, and fluorescence and absorption spectroscopy. Analysis of SAXS data shows that ovorubin is an anisometric particle with a major axis of 130 A and a minor one varying between 63 and 76 A. The particle shape was not significantly affected by the absence of the cofactor astaxanthin. The 3D model presented here is the first for an invertebrate egg carotenoprotein. The quaternary structure is stable over a wide pH range (4.5-12.0). At a pH between 2.0 and 4.0, a reduction in the gyration radius and a loss of tertiary structure are observed, although astaxanthin binding is not affected and only minor alterations in secondary structure are observed. In vitro pepsin digestion indicates that ovorubin is resistant to this protease action. The high stability over a considerable pH range and against pepsin, together with the capacity to bear temperatures > 95 degrees C, reinforces the idea that ovorubin is tailored to withstand a wide variety of conditions in order to play its key role in embryo protection during development.     

Structural intermediates in the putative pathway from the cellular prion protein to the pathogenic form

The conversion of the alpha-helical, protease sensitive and noninfectious form of the prion protein (PrP(C)) into an insoluble, protease resistant, predominantly beta-sheeted and infectious form (PrP(Sc)) is the fundamental event in prion formation. In the present work, two soluble and stable intermediate structural states are newly identified for recombinant Syrian hamster PrP(90-231) (recPrP), a dimeric alpha-helical state and a tetra- or oligomeric, beta-sheet rich state. In 0.2% SDS at room temperature, recPrP is soluble and exhibits alpha-helical and random coil secondary structure as determined by circular dichroism. Reduction of the SDS concentration to 0.06% leads first to a small increase in alpha-helical content, whereas further dilution to 0.02% results in the aquisition of beta-sheet structure. The reversible transition curve is sigmoidal within a narrow range of SDS concentrations (0.04 to 0.02%). Size exclusion chromatography and chemical crosslinking revealed that the alpha-helical form is dimeric, while the beta-sheet rich form is tetra- or oligomeric. Both the alpha-helical and beta-sheet rich intermediates are soluble and stable. Thus, they should be accessible to further structural and mechanistic studies. At 0.01% SDS, the oligomeric intermediates aggregated into large, insoluble structures as observed by fluorescence correlation spectroscopy. Our results are discussed with respect to the mechanism of PrP(Sc) formation and the propagation of prions.     

Effect of p-nitrophenol metabolites on microbial cell electro-optical characteristics

The major cell wall-associated protein of the equine pathogen Streptococcus equi subsp. equi is a fibrinogen-binding protein (FgBP) which binds horse fibrinogen and equine IgG-Fc avidly through residues located in the N-terminal half and central regions of the molecule, respectively. The molecule is a major virulence factor for the organism and displays protective potential. In the present study, we use circular dichroism spectroscopy to investigate the secondary structure of the protein and show through the analysis of a panel of recombinant FgBP truncates that the C-terminal portion of FgBP contains an extensive alpha-helical coiled-coil structure that contributes to the thermal stability of the molecule.     

Sequence-specific 1H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX.

Factor IX is a blood clotting protein that contains three regions, including a gamma-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF-like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel beta-sheets, and preliminary distance geometry calculations indicate that the protein has two domains, separated by Trp28, with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the beta-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself.     

Unusual effect of salts on the homodimeric structure of NADH oxidase from Thermus thermophilus in acidic pH

The unusual salt-dependent behavior of the homodimeric flavoenzyme NADH oxidase from Thermus thermophilus in acidic pH has been studied using circular dichroism (CD) and sedimentation velocity. The native-like secondary and quaternary structures in acidic low ionic strength conditions were significantly perturbed by the addition of salts. The peptide region of the CD spectra showed a major salt-induced conformational change in the protein secondary structure. Sedimentation velocity experiments showed dissociation of the homodimeric structure of NADH oxidase in the presence of salt (>1 M). The new acidic conformation of the protein was stabilized by high ionic strength as indicated by a salt-induced increase in the melting temperature of the protein, and by a shift in the apparent pK(a) values of the conformational transition to a less acidic pH. Distortion of the dominant alpha-helical signal was expressed as the disappearance of the parallel polarized Moffitt exciton band at 208 nm without an accompanying loss of amplitude of n-->pi* electronic transitions at 222 nm. The unusual CD spectra correlated qualitatively with the theoretically calculated CD spectra of short alpha-helical structures and/or twisted beta-sheets. Differences between the experimentally obtained CD spectra and theoretical calculations (AGADIR) of the alpha-helical content of NADH oxidase indicate a role for non-local interactions in the protein conformation at high ionic strength and low pH. These findings indicate the importance of the homodimeric interface and electrostatic interactions for maintaining the structural integrity of this thermophilic protein.     

Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding

pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are beta-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of beta-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG(1)) into another (MG(2)). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway.     

Quaternary structure of the severe acute respiratory syndrome (SARS) coronavirus main protease

SARS (severe acute respiratory syndrome) has been one of the most severe viral infectious diseases last year and still remains as a highly risky public health problem around the world. Exploring the types of interactions responsible for structural stabilities of its component protein molecules constitutes one of the approaches to find a destabilization method for the virion particle. In this study, we performed a series of experiments to characterize the quaternary structure of the dimeric coronavirus main protease (M(pro), 3CL(pro)). By using the analytical ultracentrifuge, we demonstrated that the dimeric SARS coronavirus main protease exists as the major form in solution at protein concentration as low as 0.10 mg/mL at neutral pH. The enzyme started to dissociate at acidic and alkali pH values. Ionic strength has profound effect on the dimer stability indicating that the major force involved in the subunit association is ionic interactions. The effect of ionic strength on the protease molecule was reflected by the drastic change of electrostatic potential contour of the enzyme in the presence of NaCl. Analysis of the crystal structures indicated that the interfacial ionic interaction was attributed to the Arg-4...Glu-290 ion pair between the subunits. Detailed examination of the dimer-monomer equilibrium at different pH values reveals apparent pK(a) values of 8.0 +/- 0.2 and 5.0 +/- 0.1 for the Arg-4 and Glu-290, respectively. Mutation at these two positions reduces the association affinity between subunits, and the Glu-290 mutants had diminished enzyme activity. This information is useful in searching for substances that can intervene in the subunit association, which is attractive as a target to neutralize the virulence of SARS coronavirus.     

Energetics of outer membrane phospholipase A (OMPLA) dimerization

Outer membrane phospholipase A (OMPLA) is a widely conserved transmembrane enzyme found in Gram-negative bacteria, and it is implicated in the virulence of a number of pathogenic organisms. The regulation of the protein's phospholipase activity is not well understood despite the existence of a number of high resolution structures. Previous biochemical studies have demonstrated that dimerization of OMPLA is a prerequisite for its phospholipase activity, and it has been shown in vitro that this dimerization is dependent on calcium and substrate binding. Therefore, to fully understand the regulation of OMPLA, it is necessary to understand the stability of the protein dimer and the extent to which it is influenced by its effector molecules. We have used sedimentation equilibrium analytical ultracentrifugation to dissect the energetics of Escherichia coli OMPLA dimerization in detergent micelles. We find that calcium contributes relatively little stability to the dimer, while interactions with the substrate acyl chain are the predominant force in stabilizing the dimeric conformation of the enzyme. The resulting thermodynamic cycle suggests that interactions between effector molecules are additive. These energetic measurements not only provide insight into the activation of OMPLA, but they also represent the first quantitative investigation of the association energetics of a transmembrane beta-barrel. This thermodynamic study allows us to begin to address the differences between protein-protein interfaces in transmembrane proteins with a helical fold to those of a beta-barrel fold and to more fully understand the forces involved in membrane protein interactions.     

Calcium-induced folding and stabilization of the intrinsically disordered RTX domain of the CyaA toxin

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. Its C-terminal region, the receptor-binding domain (RD), contains ∼40 calcium-binding Repeat in ToXin (RTX) motifs, which are characteristic of many virulence factors of pathogenic bacteria. We previously showed that RD is intrinsically disordered in the absence of calcium and acquires its functional three-dimensional structure upon calcium binding. To gain further insight into the physicochemical properties of RD, we characterized its calcium-induced conformational and stability changes by combining spectroscopic approaches. We show that RD, in the absence of calcium, adopts premolten globule conformations, due in part to the strong internal electrostatic repulsions between the negative charges of the aspartate-rich polypeptide sequence. Accordingly, sodium is able to screen these electrostatic repulsions, allowing a partial compaction of the polypeptide, whereas calcium triggers a strong compaction as well as the acquisition of secondary and tertiary structures in a highly cooperative manner. The differential sensitivity of the calcium-loaded state to guanidinium- and urea-induced denaturations provides further evidence that electrostatic interactions play a critical role in the folding and stability of RD. These results provide new insights into the folding/function relationship of the RTX motifs.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

Binding specificity of mannose-specific carbohydrate-binding protein from the cell surface of Trypanosoma cruzi

The sugar binding specificity of the recently described mannose-specific carbohydrate-binding proteins (CBP) isolated to homogeneity from both the epimastigote and trypomastigote stages of the pathogenic protozoa Trypanosoma cruzi has been studied by quantitative hapten inhibition of the biotinylated CBPs to immobilized thyroglobulin using model oligosaccharides. The results clearly show a differential specificity toward high-mannose glycans between the CBPs from the two developmental stages. Thus, the isolated CBP from epimastigotes exhibited stronger affinity for higher mannose oligomers containing the Manalpha1-2Manalpha1-6Manalpha1-6 structure. Its affinity decreased, as did the number of mannose residues on the oligomer or removal of the terminal Manalpha1-2-linked mannose. By contrast the CBP isolated from the trypomastigote stage showed about 400-fold lower avidity than the epimastigote form, and contrary to it, it was slightly more specific toward Man5GlcNAc than Man9GlcNAc. Analysis of the interaction of epimastigote-Man-CBP with its ligands by UV difference spectroscopy indicates the existence of an extended binding site in that protein with a large enthalpic contribution to the binding. The thermodynamic parameters of binding were obtained by isothermal titration calorimetry and been found that the DeltaH values to be in good agreement with the van't Hoff values. The binding reactions are mainly enthalpically driven and exhibit enthalpy-enthropy compensation. In addition, analysis of the high-mannose glycans from different parts of the digestive tract of the reduviid insect vector of T. cruzi suggest a role of the CBP in the retention of the epimastigote stage in the anterior portion of the gut.     

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol.

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of alpha-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.     

Role of buried polar residues in helix bundle stability and lipid binding of apolipophorin III: destabilization by threonine 31

Apolipophorin III (apoLp-III) from Locusta migratoria is a model exchangeable apolipoprotein that plays a key role in neutral lipid transport. The protein is comprised of a bundle of five amphipathic alpha-helices, with most hydrophobic residues buried in the protein interior. The low stability of apoLp-III is thought to be crucial for lipid-induced helix bundle opening, to allow protein-lipid interactions. The presence of polar residues in the hydrophobic protein interior may facilitate this role. To test this, two buried polar residues, Thr-31 and Thr-144, were changed into alanine by site-directed mutagenesis. Secondary structure analysis and GdnHCl- and temperature-induced denaturation studies indicated an increase in alpha-helical content and protein stability for T31A apoLp-III compared to wild-type apoLp-III. In contrast, T144A had a decreased alpha-helical content and protein stability, while tryptophan fluorescence indicated increased exposure of the hydrophobic interior to buffer. Two mutant proteins that had lysine residues introduced in the hydrophobic core displayed a more pronounced decrease in secondary structure and protein stability. Lipid binding studies using phospholipid vesicles showed that T31A apoLp-III was able to transform phospholipid vesicles into discoidal particles but at a 3-fold reduced rate compared to wild-type apoLp-III. In contrast, the less stable apoLp-III mutants displayed an increased ability to transform phospholipid vesicles. These results demonstrate the inverse correlation between protein stability and the ability to transform phospholipid vesicles into discoidal protein-lipid complexes and that Thr-31 is a key determinant of the relatively low protein stability, thereby promoting apoLp-III to interact with lipid surfaces.     

Osmolyte trimethylamine N-oxide converts recombinant alpha-helical prion protein to its soluble beta-structured form at high temperature

The thermal unfolding of full-length human recombinant alpha-helical prion protein (alpha-PrP) in neutral pH is reversible, whereas, in the presence of the osmolyte N-trimethylamine oxide (TMAO), the protein acquires a beta-sheet structure at higher temperatures and the thermal unfolding of the protein is irreversible. Lysozyme, an amyloidogenic protein similar to prion protein, regains alpha-helical structure on cooling from its thermally unfolded form in buffer and in TMAO solutions. The thermal stability of alpha-PrP decreases, whereas that of lysozyme increases in TMAO solution. Light-scattering and turbidity values indicate that beta-sheet prion protein exists as soluble oligomers that increase thioflavin T fluorescence and bind to 1-anilino 8-naphthalene sulfonic acid (ANS). The oligomers are resistant to proteinase K digestion and during incubation for long periods they form linear amyloids>5 microm long. The comparable fluorescence polarization of the tryptophan groups and their accessibility to acrylamide in alpha-PrP and oligomers indicate that the unstructured N-terminal segments of the protein, which contain the tryptophan groups, do not associate among themselves during oligomerization. Partial unfolding of alpha-helical prion protein in TMAO solution leads to its structural conversion to misfolded beta-sheet form. The formation of the misfolded prion protein oligomers and their polymerization to amyloids in TMAO are unusual, since the osmolyte generally induces denatured protein to fold to a native-like state and protects proteins from thermal denaturation and aggregation.     

Detection and characterization of an acid-induced folding intermediate of endostatin

Endostatin, an important angiogenesis inhibitor, is very acid resistant. We are particularly interested in knowing that whether or not endostatin can form a folding intermediate during acid titration. 1H-NMR, CD spectrum, and ANS binding assay show that endostatin at pH 2.0 contains little tertiary structure, but retains substantial secondary structure with strong ANS binding, and Na2SO4 or TFE is found to strongly stabilize endostatin at pH 2.0. All these observations are consistent with the formation of a folding intermediate at pH 2.0. Kinetics studies show that sulfate anions significantly slow down the process for endostatin to reach its equilibrium state at pH 2.0. A regular increase in the amount of alpha-helix content of the intermediate of endostatin at pH 2.0 is found when the concentration of TFE is increased in the range of 0-40%, suggesting that endostatin has an intrinsic alpha-helical propensity.