Probing the origin of prion protein misfolding via reconstruction of ancestral proteins.

Prion diseases are fatal neurodegenerative diseases caused by pathogenic misfolding of the prion protein, PrP. They are transmissible between hosts, and sometimes between different species, as with transmission of bovine spongiform encephalopathy to humans. Although PrP is found in a wide range of vertebrates, prion diseases are seen only in certain mammals, suggesting that infectious misfolding was a recent evolutionary development. To explore when PrP acquired the ability to misfold infectiously, we reconstructed the sequences of ancestral versions of PrP from the last common primate, primate-rodent, artiodactyl, placental, bird, and amniote. Recombinant ancestral PrPs were then tested for their ability to form ß-sheet aggregates, either spontaneously or when seeded with infectious prion strains from human, cervid, or rodent species. The ability to aggregate developed after the oldest ancestor (last common amniote), and aggregation capabilities diverged along evolutionary pathways consistent with modern-day susceptibilities. Ancestral bird PrP could not be seeded with modern-day prions, just as modern-day birds are resistant to prion disease. Computational modeling of structures suggested that differences in helix 2 could account for the resistance of ancestral bird PrP to seeding. Interestingly, ancestral primate PrP could be converted by all prion seeds, including both human and cervid prions, raising the possibility that species descended from an ancestral primate have retained the susceptibility to conversion by cervid prions. More generally, the results suggest that susceptibility to prion disease emerged prior to ~100 million years ago, with placental mammals possibly being generally susceptible to disease.

Integration of light scattering with machine learning for label free cell detection.

Light scattering has been used for label-free cell detection. The angular light scattering patterns from the cells are unique to them based on the cell size, nucleus size, number of mitochondria, and cell surface roughness. The patterns collected from the cells can then be classified based on different image characteristics. We have also developed a machine learning (ML) method to classify these cell light scattering patterns. As a case study we have used this light scattering technique integrated with the machine learning to analyze staurosporine-treated SH-SY5Y neuroblastoma cells and compare them to non-treated control cells. Experimental results show that the ML technique can provide a classification accuracy (treated versus non-treated) of over 90%. The predicted percentage of the treated cells in a mixed solution is within 5% of the reference (ground-truth) value and the technique has the potential to be a viable method for real-time detection and diagnosis.

Unfolded and intermediate states of PrP play a key role in the mechanism of action of an antiprion chaperone.

Prion and prion-like diseases involve the propagation of misfolded protein conformers. Small-molecule pharmacological chaperones can inhibit propagated misfolding, but how they interact with disease-related proteins to prevent misfolding is often unclear. We investigated how pentosan polysulfate (PPS), a polyanion with antiprion activity in vitro and in vivo, interacts with mammalian prion protein (PrP) to alter its folding. Calorimetry showed that PPS binds two sites on natively folded PrP, but one PPS molecule can bind multiple PrP molecules. Force spectroscopy measurements of single PrP molecules showed PPS stabilizes not only the native fold of PrP but also many different partially folded intermediates that are not observed in the absence of PPS. PPS also bound tightly to unfolded segments of PrP, delaying refolding. These observations imply that PPS can act through multiple possible modes, inhibiting misfolding not only by stabilizing the native fold or sequestering natively folded PrP into aggregates, as proposed previously, but also by binding to partially or fully unfolded states that play key roles in mediating misfolding. These results underline the likely importance of unfolded states as critical intermediates on the prion conversion pathway.

Characterizing the inhibition of α-synuclein oligomerization by a pharmacological chaperone that prevents prion formation by the protein PrP.

Aggregation of the disordered protein α-synuclein into amyloid fibrils is a central feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease. Small, pre-fibrillar oligomers of misfolded α-synuclein are thought to be the key toxic entities, and α-synuclein misfolding can propagate in a prion-like way. We explored whether a compound with anti-prion activity that can bind to unfolded parts of the protein PrP, the cyclic tetrapyrrole Fe-TMPyP, was also active against α-synuclein aggregation. Observing the initial stages of aggregation via fluorescence cross-correlation spectroscopy, we found that Fe-TMPyP inhibited small oligomer formation in a dose-dependent manner. Fe-TMPyP also inhibited the formation of mature amyloid fibrils in vitro, as detected by thioflavin T fluorescence. Isothermal titration calorimetry indicated Fe-TMPyP bound to monomeric α-synuclein with a stoichiometry of 2, and two-dimensional heteronuclear single quantum coherence NMR spectra revealed significant interactions between Fe-TMPyP and the C-terminus of the protein. These results suggest commonalities among aggregation mechanisms for α-synuclein and the prion protein may exist that can be exploited as therapeutic targets.

Early stages of aggregation of engineered α-synuclein monomers and oligomers in solution.

α-Synuclein is a protein that aggregates as amyloid fibrils in the brains of patients with Parkinson's disease and dementia with Lewy bodies. Small oligomers of α-synuclein are neurotoxic and are thought to be closely associated with disease. Whereas α-synuclein fibrillization and fibril morphologies have been studied extensively with various methods, the earliest stages of aggregation and the properties of oligomeric intermediates are less well understood because few methods are able to detect and characterize early-stage aggregates. We used fluorescence spectroscopy to investigate the early stages of aggregation by studying pairwise interactions between α-synuclein monomers, as well as between engineered tandem oligomers of various sizes (dimers, tetramers, and octamers). The hydrodynamic radii of these engineered α-synuclein species were first determined by fluorescence correlation spectroscopy and dynamic light scattering. The rate of pairwise aggregation between different species was then monitored using dual-color fluorescence cross-correlation spectroscopy, measuring the extent of association between species labelled with different dyes at various time points during the early aggregation process. The aggregation rate and extent increased with tandem oligomer size. Self-association of the tandem oligomers was found to be the preferred pathway to form larger aggregates: interactions between oligomers occurred faster and to a greater extent than interactions between oligomers and monomers, indicating that the oligomers were not as efficient in seeding further aggregation by addition of monomers. These results suggest that oligomer-oligomer interactions may play an important role in driving aggregation during its early stages.

Structural characteristics and membrane interactions of tandem α-synuclein oligomers.

Pre-fibrillar oligomers of α-synuclein are thought to be pathogenic molecules leading to neurotoxicity associated with Parkinson's disease and other neurodegenerative disorders. However, small oligomers are difficult to isolate for study. To gain better insight into the properties of small α-synuclein oligomers, we investigated engineered oligomers of specific size (dimers, tetramers, and octamers) linked head-to-tail in tandem, comparing the behavior of the oligomers to monomeric α-synuclein. All oligomeric constructs remained largely disordered in solution, as determined from dynamic light scattering and size-exclusion chromatography. Electron microscopy revealed that each construct could aggregate to form fibrils similar to those formed by monomeric α-synuclein. The interactions with large unilamellar vesicles (LUVs) composed of negatively-charged lipids differed depending on size, with smaller oligomers forming more extensive helical structure as determined by CD spectroscopy. Monitoring the influx of a fluorescence bleaching agent into vesicles showed that larger oligomers were somewhat more effective at degrading vesicular integrity and inducing membrane permeabilization.

Partially native intermediates mediate misfolding of SOD1 in single-molecule folding trajectories.

Prion-like misfolding of superoxide dismutase 1 (SOD1) is associated with the disease ALS, but the mechanism of misfolding remains unclear, partly because misfolding is difficult to observe directly. Here we study the most misfolding-prone form of SOD1, reduced un-metallated monomers, using optical tweezers to measure unfolding and refolding of single molecules. We find that the folding is more complex than suspected, resolving numerous previously undetected intermediate states consistent with the formation of individual ß-strands in the native structure. We identify a stable core of the protein that unfolds last and refolds first, and directly observe several distinct misfolded states that branch off from the native folding pathways at specific points after the formation of the stable core. Partially folded intermediates thus play a crucial role mediating between native and non-native folding. These results suggest an explanation for SOD1's propensity for prion-like misfolding and point to possible targets for therapeutic intervention.

Expression, purification, crystallization and preliminary crystallographic analysis of the phosphoglycerate kinase from Acinetobacter baumannii.

Acinetobacter baumannii is a common multidrug-resistant clinical pathogen that is often found in hospitals. The A. baumannii phosphoglycerate kinase (AbPGK) is involved in the key energy-producing pathway of glycolysis and presents a potential target for antibiotic development. AbPGK has been expressed and purified; it was crystallized using lithium sulfate as the precipitant. The AbPGK crystals belonged to space group P222(1). They diffracted to a resolution of 2.5 Šusing synchrotron radiation at the Canadian Light Source.

The structures of Thermoplasma volcanium phosphoribosyl pyrophosphate synthetase bound to ribose-5-phosphate and ATP analogs.

Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs ß,γ-methylene ATP and ADP in two monoclinic crystal forms, P2(1). The ß,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP-Mg(2+) and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively.

Expression, purification and preliminary crystallographic analysis of Rv3002c, the regulatory subunit of acetolactate synthase (IlvH) from Mycobacterium tuberculosis.

Branched amino-acid biosynthesis is important to bacterial pathogens such as Mycobacterium tuberculosis (Mtb), a microorganism that presently causes more deaths in humans than any other prokaryotic pathogen (http://www.who.int/tb). In this study, the molecular cloning, expression, purification, crystallization and preliminary crystallographic analysis of recombinant IlvH, the small regulatory subunit of acetohydroxylic acid synthase (AHAS) in Mtb, are reported. AHAS carries out the first common reaction in the biosynthesis of valine, leucine and isoleucine. AHAS is an essential enzyme in Mtb and its inactivation leads to a lethal phenotype [Sassetti et al. (2001), Proc. Natl Acad. Sci. USA, 98, 12712-12717]. Thus, inhibitors of AHAS could potentially be developed into novel anti-Mtb therapies.

Expression, purification and preliminary crystallographic analysis of O-acetylhomoserine sulfhydrylase from Mycobacterium tuberculosis.

The gene product of the open reading frame Rv3340 from Mycobacterium tuberculosis is annotated as encoding a probable O-acetylhomoserine (OAH) sulfhydrylase (MetC), an enzyme that catalyzes the last step in the biosynthesis of methionine, which is an essential amino acid in bacteria and plants. Following overexpression in Escherichia coli, the M. tuberculosis MetC enzyme was purified and crystallized using the hanging-drop vapor-diffusion method. Native diffraction data were collected from crystals belonging to space group P2(1) and were processed to a resolution of 2.1 Å.

Crystal structure of the intermediate complex of the arginine repressor from Mycobacterium tuberculosis bound with its DNA operator reveals detailed mechanism of arginine repression.

The concentration of L-arginine in Mycobacterium tuberculosis (Mtb) and in many other bacteria is controlled by a transcriptional factor called the arginine repressor (ArgR). It regulates the transcription of the biosynthetic genes of the arginine operon by interacting with the approximately 16- to 20-bp ARG boxes in the promoter site of the operon. ArgRs in the arginine bound form are hexamers in which each protomer has two separately folded domains. The C-terminal domains form a hexameric core, whereas the N-terminal domains have the winged helix-turn-helix DNA-binding motif. Here, we present the crystal structure of the MtbArgR hexamer bound to three copies of the 16-bp DNA operator in the presence of trace amounts of L-arginine, determined to 2.15 A resolution. In contrast to our previously published structure of the ternary MtbArgR-DNA complex in the presence of 10 mM L-arginine, the DNA operators do not form a double ARG box in the structure reported here. The present structure not only retains the noncrystallographic 32 symmetry of the core (as in the earlier structure), but it also has the 3-fold axis for the whole complex. The core trimers are rotated relative to one another as in the other holo hexamers of MtbArgR, although the L-arginine ligands have only partial density and do not fully occupy the arginine-binding sites. Refinement of the occupancies and B-factors of ligands resulted in a value of approximately 4.4 arginine ligands per hexamer. This has allowed the dissociation constant of arginine from the arginine-binding site to be estimated. The present structure also has two protomer conformations, folded and extended. However, they are different from the conformations in the complex determined at an L-arginine concentration of 10 mM and do not form an interlocking arrangement. The new complex is less stable than the earlier described complex bound with nine arginine residues. Thus, the former can be considered as an intermediate in a pathway to the latter. On the basis of the structure of this intermediate complex, a more detailed mechanism of the arginine biosynthesis regulation in Mtb is proposed.

The molecular structure of ornithine acetyltransferase from Mycobacterium tuberculosis bound to ornithine, a competitive inhibitor.

Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to L-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 A resolutions, respectively. ORN is a competitive inhibitor of this enzyme against L-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O(gamma 1) of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN.

The structure of the arginine repressor from Mycobacterium tuberculosis bound with its DNA operator and Co-repressor, L-arginine.

The biosynthesis of arginine is an essential function for the metabolism of Mycobacterium tuberculosis (Mtb) and for the metabolism of many other microorganisms. The arginine repressor (ArgR) proteins control the transcription of genes encoding the arginine biosynthetic enzymes; they belong to repressors having one of the most intricate oligomerization patterns. Here, we present the crystal structure of the MtbArgR hexamer bound to three copies of the 20 base-pair DNA operator and to the co-repressor, L-arginine, determined to 3.3 A resolution. This is the first ternary structure of an intact hexameric ArgR in complex with its DNA operator. The structure reported here is very different from the suggested models of the ternary ArgR-DNA complexes; it has revealed the sophisticated symmetry of the complex and the presence of two remarkably different protomer conformations, folded and extended. Both features provide flexibility to DNA binding and are important for understanding the detailed function of ArgRs. Two of the 20 base-pair DNA operators align in a unified double-helical structure, suggesting the possible presence of a double ARG box in the promoter region of the Mtb arginine operon. Two pairs of protomers bind to the unified double ARG box so that the two folded protomers bind to the central half-sites of the double ARG box, whereas the two extended protomers bind to the remote half-sites. The protomers of the third pair bound to the single DNA operator also have a folded and an extended conformation. A probable mechanism for arginine repression is suggested on the basis of this structure.

Crystal structure of the arginine repressor protein in complex with the DNA operator from Mycobacterium tuberculosis.

The arginine repressor (ArgR) from Mycobacterium tuberculosis (Mtb) is a gene product encoded by the open reading frame Rv1657. It regulates the L-arginine concentration in cells by interacting with ARG boxes in the promoter regions of the arginine biosynthesis and catabolism operons. Here we present a 2.5-A structure of MtbArgR in complex with a 16-bp DNA operator in the absence of arginine. A biological trimer of the protein-DNA complex is formed via the crystallographic 3-fold symmetry axis. The N-terminal domain of MtbArgR has a winged helix-turn-helix motif that binds to the major groove of the DNA. This structure shows that, in the absence of arginine, the ArgR trimer can bind three ARG box half-sites. It also reveals the structure of the whole MtbArgR molecule itself containing both N-terminal and C-terminal domains.

Expression, purification and preliminary crystallographic analysis of N-acetylglucosamine-1-phosphate uridylyltransferase from Mycobacterium tuberculosis.

The gene product of open reading frame Rv1018c from Mycobacterium tuberculosis is annotated as encoding a probable N-acetylglucosamine 1-phosphate uridylyltransferase (MtbGlmU), an enzyme that catalyzes the biosynthesis of UDP-N-acetylglucosamine, a precursor common to lipopolysaccharide and peptidoglycan biosynthesis. Following overexpression in Escherichia coli, the enzyme was purified and crystallized using the hanging-drop vapour-diffusion method. Native diffraction data were collected from crystals belonging to space group R32 and processed to a resolution of 2.2 A.

Structure of the C-terminal domain of the arginine repressor protein from Mycobacterium tuberculosis.

The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the L-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of arginine repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 A resolution with bound arginine and at 2.15 A resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11 degrees upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.

Crystal structure of Mycobacterium tuberculosis Rv0760c at 1.50 A resolution, a structural homolog of Delta(5)-3-ketosteroid isomerase.

We have determined the X-ray crystal structure of the Mycobacterium tuberculosis (Mtb) gene product encoded by the open reading frame Rv0760c at 1.50 A resolution by single-wavelength anomalous dispersion (SAD) phasing of diffraction data from crystals of the selenomethionine-substituted protein. Refinement against diffraction data from the native protein resulted in R(work)=19.5% and R(free)=21.4%. The X-ray crystal structure shows that the homodimeric Rv0760c polypeptide has an alpha + beta conical barrel fold placing it among many structural neighbors of the nuclear transport factor 2 family (NTF2). This family is highly conserved in terms of structure; however the substrates and individual protein functions are diverse. The structures of native Rv0760c in several different crystal forms and Rv0760c bound to 17beta-estradiol 17-hemisuccinate (EH) have also been solved and analyzed.

The molecular structure of epoxide hydrolase B from Mycobacterium tuberculosis and its complex with a urea-based inhibitor.

Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC(50) approximately 19 nM) urea-based inhibitor at 2.1 and 2.4 A resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical alpha/beta hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the binding-site residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120 degrees about its C(alpha)-C(beta) bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the alpha/beta type.

Expression, purification, crystallization and preliminary X-ray analysis of Rv3117, a probable thiosulfate sulfurtransferase (CysA3) from Mycobacterium tuberculosis.

The gene product of open reading frame Rv3117 from Mycobacterium tuberculosis (Mtb) strain H37Rv is annotated as encoding a probable rhodanese-like thiosulfate sulfurtransferase (MtbCysA3). MtbCysA3 was expressed and purified and then crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.5 A. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 38.86, b = 91.43, c = 83.57 A, beta = 96.6 degrees . Preliminary diffraction data shows that two molecules are present in the asymmetric unit; this corresponds to a V(M) of 2.4 A(3) Da(-1).