RESUMO
As part of ongoing efforts to design improved nerve agent antidotes, two X-ray crystal structures of Torpedo californica acetylcholinesterase (TcAChE) bound to the bis-pyridinium oxime, Ortho-7, or its experimental bis-imidazolium analogue, 2BIM-7, were determined. Bis-oximes contain two oxime groups connected by a hydrophobic linker. One oxime group of Ortho-7 binds at the entrance to the active-site gorge near Trp279, and the second binds at the bottom near Trp84 and Phe330. In the Ortho-7-TcAChE complex the oxime at the bottom of the gorge was directed towards the nucleophilic Ser200. In contrast, the oxime group of 2BIM-7 was rotated away from Ser200 and the oxime at the entrance induced a significant conformational change in the peripheral anionic site (PAS) residue Trp279. The conformational change alters the surface of the PAS and positions the imidazolium oxime of 2BIM-7 further from Ser200. The relatively weaker binding and poorer reactivation of VX-inhibited, tabun-inhibited or sarin-inhibited human acetylcholinesterase by 2BIM-7 compared with Ortho-7 may in part be owing to the unproductively bound states caught in crystallo. Overall, the reactivation efficiency of 2BIM-7 was comparable to that of 2-pyridine aldoxime methyl chloride (2-PAM), but unlike 2-PAM the bis-imidazolium oxime lacks a fixed charge, which may affect its membrane permeability.
Assuntos
Acetilcolinesterase/química , Imidazóis/química , Oximas/química , Animais , Ânions , Sítios de Ligação , Cristalografia por Raios X , Cinética , Modelos Moleculares , TorpedoRESUMO
While the general catalytic mechanism of the widespread serine hydrolase superfamily has been documented extensively, much less is known about its varied modes of functional modulation within biological systems. Under oxidizing conditions, inhibition of Saccharomyces cerevisiae S-formylglutathione hydrolase (SFGH, homologous to human esterase D) activity is attributable to a cysteine (Cys-60) adjacent to its catalytic triad and approximately 8.0 Šaway from the Oγ of the nucleophilic serine. Cys-60 is oxidized to a sulfenic acid in the structure of the Paraoxon-inhibited W197I variant (PDB 3C6B). The structural snap-shot captured an unstable reversibly oxidized state, but it remained unclear as to whether the oxidation occurred before, during, or after the reaction with the organophosphate inhibitor. To determine if the oxidation of Cys-60 was functionally linked to ester hydrolysis, we used kinetic analysis and site-directed mutagenesis in combination with X-ray crystallography. The essential nature of Cys-60 for oxidation is demonstrated by the C60S variant, which is not inhibited by peroxide in the presence or absence of substrate. In the presence of substrate, the rate of inhibition of the WT SFGH by peroxide increases 14-fold, suggesting uncompetitive behavior linking oxidation to ester hydrolysis. Here we found one variant, H160I, which is activated by peroxide. This variant is activated at comparable rates in the presence or absence of substrate, indicating that the conserved His-160 is involved in the inhibitory mechanism linking ester hydrolysis to the oxidation of Cys-60. Copper chloride inhibition experiments show that at least two metal ions bind and inhibit both WT and H160I. A structure of the Paraoxon-inhibited W197I variant soaked with CuCl(2) shows density for one metal ion per monomer at the N-terminus, and density around the Cys-60 sulfur consistent with a sulfinic acid, Cys-SO(2). A Dali structural similarity search uncovered two other enzymes (Bacillus subtilis RsbQ, 1WOM and Clostridium acetobutylicum Lipase-esterase, 3E0X) that contain a similar Cys adjacent to a catalytic triad. We speculate that the regulatory motif uncovered is conserved in some D-type esterases and discuss its structural similarities in the active site of human protective protein (HPP; also known as Cathepsin A).
Assuntos
Peróxidos/metabolismo , Saccharomyces cerevisiae/enzimologia , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Histidina/química , Histidina/genética , Histidina/metabolismo , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Tioléster Hidrolases/genéticaRESUMO
RTA1-33/44-198 is a catalytically inactive, single-domain derivative of the ricin toxin A-chain (RTA) engineered to serve as a stable protein scaffold for presentation of native immunogenic epitopes (Olson et al., Protein Eng Des Sel 2004;17:391-397). To improve the stability and solubility of RTA1-33/44-198 further, we have undertaken the design challenge of introducing a disulfide (SS) bond. Nine pairs of residues were selected for placement of the SS-bond based on molecular dynamics simulation studies of the modeled single-domain chain. Disulfide formation at either of two positions (R48C/T77C or V49C/E99C) involving a specific surface loop (44-55) increased the protein melting temperature by ~5°C compared with RTA1-33/44-198 and by ~13°C compared with RTA. Prolonged stability studies of the R48C/T77C variant (> 60 days at 37°C, pH 7.4) confirmed a > 40% reduction in self-aggregation compared with RTA1-33/44-198 lacking the SS-bond. The R48C/T77C variant retained affinity for anti-RTA antibodies capable of neutralizing ricin toxin, including a monoclonal that recognizes a human B-cell epitope. Introduction of either R48C/T77C or V49C/E99C promoted crystallization of RTA1-33/44-198, and the X-ray structures of the variants were solved to 2.3 A or 2.1 A resolution, respectively. The structures confirm formation of an intramolecular SS-bond, and reveal a single-domain fold that is significantly reduced in volume compared with RTA. Loop 44 to 55 is partly disordered as predicted by simulations, and is positioned to form self-self interactions between symmetry-related molecules. We discuss the importance of RTA loop 34 to 55 as a nucleus for unfolding and aggregation, and draw conclusions for ongoing structure-based minimalist design of RTA-based immunogens.
Assuntos
Dissulfetos/química , Epitopos/química , Ricina/química , Animais , Western Blotting , Cristalização , Cristalografia por Raios X , Dissulfetos/metabolismo , Epitopos/genética , Epitopos/metabolismo , Humanos , Imunoglobulina G/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Ricina/genética , Ricina/metabolismo , Temperatura de TransiçãoRESUMO
RiVax is a recombinant protein that is currently under clinical development as part of a human vaccine to protect against ricin poisoning. RiVax includes ricin A-chain (RTA) residues 1-267 with two intentional amino-acid substitutions, V76M and Y80A, aimed at reducing toxicity. Here, the crystal structure of RiVax was solved to 2.1â Å resolution and it was shown that it is superposable with that of the ricin toxin A-chain from Ricinus communis with a root-mean-square deviation of 0.6â Å over 258 C(α) atoms. The RiVax structure is also compared with the recently determined structure of another potential ricin-vaccine immunogen, RTA 1-33/44-198 R48C/T77C. Finally, the locations and solvent-exposure of two toxin-neutralizing B-cell epitopes were examined and it was found that these epitopes are within or near regions predicted to be involved in catalysis. The results demonstrate the composition of the RiVax clinical material and will guide ongoing protein-engineering strategies to develop improved immunogens.
Assuntos
Vacinas/química , Cristalografia por Raios X , Epitopos de Linfócito B/química , Humanos , Proteínas Recombinantes/químicaRESUMO
Macromolecules of the cholinergic basal lamina are essential elements of the complex signaling processes governing development, function, and repair of the vertebrate neuromuscular junction. One special form of acetylcholinesterase (AChE) is anchored within BL through a collagen tail (ColQ) that binds heparan sulfate proteoglycans, such as perlecan, and the post-synaptic muscle specific kinase MuSK. New experimental approaches are probing the spatio-temporal dynamics of ColQ-AChE over days or weeks in vivo, thereby unraveling its interactions with other BL components, as well as pre-and post-synaptic elements. Concurrent advances in understanding of the biological effects of specific ColQ-AChE mutations prefigure improved diagnostics and clinical approaches for some congenital myasthenic syndromes.
Assuntos
Acetilcolinesterase/metabolismo , Membrana Basal/enzimologia , Junção Neuromuscular/enzimologia , Animais , Colágeno/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Proteínas Musculares/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/metabolismo , VertebradosRESUMO
Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 A resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme "acyl pocket". The W197I substitution enhances ySFGH reactivity with paraoxon by >1000-fold ( k i (W197I) = 16 +/- 2 mM (-1) h (-1)), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 A); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic acid may play a role in the regulation of a "D-type" esterase. The structure of the W197I variant is the first reported cysteine sulfenic acid in a serine esterase. We constructed five Cys60/W197I variants and show that introducing a positive charge near the oxyanion hole, W197I/C60R or W197I/C60K, results in a further enhancement of the rates of phosphorylation with paraoxon ( k i = 42 or 80 mM (-1) h (-1), respectively) but does not affect the dephosphorylation of the enzyme. We also characterized three histidine substitutions near the oxyanion hole, G57H, L58H, and M162H, which significantly decrease esterase activity.
Assuntos
Inibidores da Colinesterase/química , Farmacorresistência Fúngica/fisiologia , Paraoxon/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Tioléster Hidrolases/química , Substituição de Aminoácidos , Sítios de Ligação , Carboxilesterase/química , Carboxilesterase/genética , Carboxilesterase/metabolismo , Inibidores da Colinesterase/metabolismo , Cristalografia por Raios X , Humanos , Hidrólise , Mutação de Sentido Incorreto , Paraoxon/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
BACKGROUND: Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions. RESULTS: We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a DeltaH of -13 +/- 2 kJ/mol and a DeltaS of -0.04 +/- 0.01 kJ/(K*mol). The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site. CONCLUSION: We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ricina/química , Ricina/metabolismo , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Espectrometria de FluorescênciaRESUMO
Ricin is an A-B ribosome inactivating protein (RIP) toxin composed of an A-chain subunit (RTA) that contains a catalytic N-glycosidase and a B-chain (RTB) lectin domain that binds cell surface glycans. Ricin exploits retrograde transport to enter into the Golgi and the endoplasmic reticulum, and then dislocates into the cytoplasm where it can reach its substrate, the rRNA. A subset of isolated antibodies (Abs) raised against the RTA subunit protect against ricin intoxication, and RTA-based vaccine immunogens have been shown to provide long-lasting protective immunity against the holotoxin. Anti-RTA Abs are unlikely to cross a membrane and reach the cytoplasm to inhibit the enzymatic activity of the A-chain. Moreover, there is not a strict correlation between the apparent binding affinity (Ka) of anti-RTA Abs and their ability to successfully neutralize ricin toxicity. Some anti-RTA antibodies are toxin-neutralizing, whereas others are not. We hypothesize that neutralizing anti-RTA Abs may interfere selectively with conformational change(s) or partial unfolding required for toxin internalization. To test this hypothesis, we measured the melting temperatures (Tm) of neutralizing single-domain Ab (sdAb)-antigen (Ag) complexes relative to the Tm of the free antigen (Tm-shift = Tmcomplex - TmAg), and observed increases in the Tmcomplex of 9-20 degrees. In contrast, non-neutralizing sdAb-Ag complexes shifted the TmComplex by only 6-7 degrees. A strong linear correlation (r2 = 0.992) was observed between the magnitude of the Tm-shift and the viability of living cells treated with the sdAb and ricin holotoxin. The Tm-shift of the sdAb-Ag complex provided a quantitative biophysical parameter that could be used to predict and rank-order the toxin-neutralizing activities of Abs. We determined the first structure of an sdAb-RTA1-33/44-198 complex, and examined other sdAb-RTA complexes. We found that neutralizing sdAb bound to regions involved in the early stages of unfolding. These Abs likely interfere with steps preceding or following endocytosis that require conformational changes. This method may have utility for the characterization or rapid screening of other Ab that act to prevent conformational changes or unfolding as part of their mechanism of action.
Assuntos
Anticorpos Neutralizantes/química , Complexo Antígeno-Anticorpo/química , Dicroísmo Circular/métodos , Ricina/química , Anticorpos de Cadeia Única/química , Temperatura de Transição , Animais , Humanos , Conformação Proteica , Desnaturação Proteica , Estabilidade ProteicaRESUMO
The crystal structure of acetylcholinesterase from Torpedo californica complexed with the uncharged inhibitor, PEG-SH-350 (containing mainly heptameric polyethylene glycol with a terminal thiol group) is determined at 2.3 A resolution. This is an untypical acetylcholinesterase inhibitor, since it lacks the cationic moiety typical of the substrate (acetylcholine). In the crystal structure, the elongated ligand extends along the whole of the deep and narrow active-site gorge, with the terminal thiol group bound near the bottom, close to the catalytic site. Unexpectedly, the cation-binding site (formed by the faces of aromatic side-chains) is occupied by CH(2) groups of the inhibitor, which are engaged in C-H...pi interactions that structurally mimic the cation-pi interactions made by the choline moiety of acetylcholine. In addition, the PEG-SH molecule makes numerous other weak but specific interactions of the C-H...O and C-H...pi types.
Assuntos
Acetilcolinesterase/química , Inibidores da Colinesterase/química , Polietilenoglicóis/química , Compostos de Sulfidrila/química , Animais , Sítios de Ligação , Cátions , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , TorpedoRESUMO
A microfluidic assay was developed for screening botulinum neurotoxin serotype A (BoNT-A) by using a fluorescent resonance energy transfer (FRET) assay. Molded silicone microdevices with integral valves, pumps, and reagent reservoirs were designed and fabricated. Electrical and pneumatic control hardware were constructed, and software was written to automate the assay protocol and data acquisition. Detection was accomplished by fluorescence microscopy. The system was validated with a peptide inhibitor, running 2 parallel assays, as a feasibility demonstration. The small footprint of each bioreactor cell (0.5 cm2) and scalable fluidic architecture enabled many parallel assays on a single chip. The chip is programmable to run a dilution series in each lane, generating concentration-response data for multiple inhibitors. The assay results showed good agreement with the corresponding experiments done at a macroscale level. Although the system has been developed for BoNT-A screening, a wide variety of assays can be performed on the microfluidic chip with little or no modification.
Assuntos
Bioensaio/métodos , Toxinas Botulínicas Tipo A/análise , Microfluídica/instrumentação , Bioensaio/instrumentação , Estudos de Viabilidade , Microquímica/instrumentação , MiniaturizaçãoRESUMO
Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide-linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N-acetylgalactosamine-binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267-amino-acid A chain consisting of residues 1-33/44-198 or 1-198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier-transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule-like state at 45 degrees C, rRTA 1-33/44-198 and 1-198 are significantly more thermostable, remaining completely folded at temperatures up to 53 degrees C and 51 degrees C, respectively. Deleting both an exposed loop region (amino acids 34-43) and the C-terminal domain (199-267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three-state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C-terminal domain. Removing the C-terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens.
Assuntos
Substâncias para a Guerra Química/química , Ricina/química , Ricina/genética , Vacinas/química , Animais , Humanos , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ricina/imunologia , Deleção de Sequência/genética , TemperaturaRESUMO
Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is "native-like," and inactivation can be reversed by exposure to reduced glutathione. The allicin-modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t(1/2) approximately 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine-N-oxide, glycerol, and sucrose, and the divalent cations, Ca(2+), Mg(2+), and Mn(2+) can prevent this transition of the native-like state for >24 h at room temperature. Trimethylamine-N-oxide and Mg(2+) can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39 degrees C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native-like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.
Assuntos
Acetilcolinesterase/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Perileno/análogos & derivados , Ácidos Sulfínicos/farmacologia , Torpedo/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Algoritmos , Naftalenossulfonato de Anilina/farmacologia , Animais , Antracenos , Varredura Diferencial de Calorimetria , Catálise/efeitos dos fármacos , Catálise/efeitos da radiação , Dicroísmo Circular , Reagentes de Ligações Cruzadas/química , Cisteína/química , Dissulfetos , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Glutationa/farmacologia , Glicerol/farmacologia , Temperatura Alta , Hidrólise , Cinética , Magnésio/farmacologia , Metilaminas/química , Metilaminas/farmacologia , Modelos Teóricos , Perileno/química , Conformação Proteica , Desnaturação Proteica , Espectrometria de Fluorescência , Reagentes de Sulfidrila/farmacologia , Termodinâmica , TripsinaRESUMO
Previous attempts to produce a vaccine for ricin toxin have been hampered by safety concerns arising from residual toxicity and the undesirable aggregation or precipitation caused by exposure of hydrophobic surfaces on the ricin A-chain (RTA) in the absence of its natural B-chain partner. We undertook a structure-based solution to this problem by reversing evolutionary selection on the 'ribosome inactivating protein' fold of RTA to arrive at a non-functional, compacted single-domain scaffold (sequence RTA1-198) for presentation of a specific protective epitope (RTA loop 95-110). An optimized protein based upon our modeling design (RTA1-33/44-198) showed greater resistance to thermal denaturation, less precipitation under physiological conditions and a reduction in toxic activity of at least three orders of magnitude compared with RTA. Most importantly, RTA1-198 or RTA1-33/44-198 protected 100% of vaccinated animals against supra-lethal challenge with aerosolized ricin. We conclude that comparative protein analysis and engineering yielded a superior vaccine by exploiting a component of the toxin that is inherently more stable than is the parent RTA molecule.
Assuntos
Ricina/imunologia , Vacinas/imunologia , Dicroísmo Circular , Luz , Dobramento de Proteína , Espalhamento de RadiaçãoRESUMO
The X-ray crystal structure of the reaction product of acetylcholinesterase (AChE) with the inhibitor diisopropylphosphorofluoridate (DFP) showed significant structural displacement in a loop segment of residues 287-290. To understand this conformational selection, a Monte Carlo (MC) simulation study was performed of the energy landscape for the loop segment. A computational strategy was applied by using a combined simulated annealing and room temperature Metropolis sampling approach with solvent polarization modeled by a generalized Born (GB) approximation. Results from thermal annealing reveal a landscape topology of broader basin opening and greater distribution of energies for the displaced loop conformation, while the ensemble average of conformations at 298 K favored a shift in populations toward the native by a free-energy difference in good agreement with the estimated experimental value. Residue motions along a reaction profile of loop conformational reorganization are proposed where Arg-289 is critical in determining electrostatic effects of solvent interaction versus Coulombic charging.
Assuntos
Acetilcolinesterase/química , Transferência de Energia , Isoflurofato/metabolismo , Conformação Proteica , Torpedo/metabolismo , Acetilcolinesterase/metabolismo , Acilação , Animais , Simulação por Computador , Cristalografia por Raios X , Modelos Moleculares , Método de Monte Carlo , TermodinâmicaRESUMO
We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine Oγ. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its k cat/K m for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1 (hCE1). We discuss the use of pNBE as a surrogate scaffold for the mammalian esterases, and the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.
RESUMO
Vitetta and colleagues identified and characterized a putative vascular leak peptide (VLP) consensus sequence in recombinant ricin toxin A-chain (RTA) that contributed to dose-limiting human toxicity when RTA was administered intravenously in large quantities during chemotherapy. We disrupted this potentially toxic site within the more stable RTA1-33/44-198 vaccine immunogen and determined the impact of these mutations on protein stability, structure and protective immunogenicity using an experimental intranasal ricin challenge model in BALB/c mice to determine if the mutations were compatible. Single amino acid substitutions at the positions corresponding with RTA D75 (to A, or N) and V76 (to I, or M) had minor effects on the apparent protein melting temperature of RTA1-33/44-198 but all four variants retained greater apparent stability than the parent RTA. Moreover, each VLP(-) variant tested provided protection comparable with that of RTA1-33/44-198 against supralethal intranasal ricin challenge as judged by animal survival and several biomarkers. To understand better how VLP substitutions and mutations near the VLP site impact epitope structure, we introduced a previously described thermal stabilizing disulfide bond (R48C/T77C) along with the D75N or V76I substitutions in RTA1-33/44-198. The D75N mutation was compatible with the adjacent stabilizing R48C/T77C disulfide bond and the T(m) was unaffected, whereas the V76I mutation was less compatible with the adjacent disulfide bond involving C77. A crystal structure of the RTA1-33/44-198 R48C/T77C/D75N variant showed that the structural integrity of the immunogen was largely conserved and that a stable immunogen could be produced from E. coli. We conclude that it is feasible to disrupt the VLP site in RTA1-33/44-198 with little or no impact on apparent protein stability or protective efficacy in mice and such variants can be stabilized further by introduction of a disulfide bond.
Assuntos
Substâncias para a Guerra Química/toxicidade , Peptídeos/administração & dosagem , Ricina/genética , Ricina/toxicidade , Vacinas Sintéticas/administração & dosagem , Administração Intranasal , Animais , Glicemia/análise , Líquido da Lavagem Broncoalveolar/química , Feminino , Injeções Intramusculares , Dose Letal Mediana , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Peptídeos/genética , Ricina/administração & dosagem , Ricina/químicaRESUMO
Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2), produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIPâ¢SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to proteinâ¢polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other proteinâ¢polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.
Assuntos
Polinucleotídeos/farmacologia , Proteínas Inativadoras de Ribossomos/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Camundongos , Estrutura Molecular , Polinucleotídeos/química , Proteínas Inativadoras de Ribossomos/química , Ricina/química , Toxina Shiga II/químicaRESUMO
Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism that could afflict large, unprotected populations if the toxin were employed in an act of bioterrorism. Current post-exposure therapy is limited to symptomatic treatment or passive immunization that is effective for treating infant botulism at a cost of US $45,300 per treatment regimen. Antibodies can neutralize the extracellular but not the intracellular BoNTA. Moreover, antibody production, storage, and administration in a mass casualty scenario pose logistical challenges. Alternatively, small-molecule inhibitors of BoNTA endopeptidase (BoNTAe) are sought to antagonize the extracellular or intracellular toxin. While several such molecules reportedly demonstrated efficacy in protecting cells against BoNTA, there is scant information to show that small molecules can significantly protect mammals against BoNTA. Herein we report the development of effective small-molecules BoNTAe inhibitors with promising in vivo pharmacokinetics. One such molecule has an in vivo half-life of 6.5 hours and is devoid of obvious sign of toxicity. Pre-treatment with this molecule at 2 mg/kg protected 100% and 70% of treated mice against BoNTA at 5 times of its median-lethal dose during the periods of 2 and 4 half-lives of the inhibitor, respectively. In contrast, 40% and 0% of untreated mice survived during the respective periods. Similar levels of protection were also observed with two other small molecules. These results demonstrate that small molecules can significantly protect mice against BoNTA and support the pursuit of small-molecule antagonists as a cost-effective alternative or as an adjunct to passive immunity for treating botulism.
Assuntos
Toxinas Botulínicas Tipo A/antagonistas & inibidores , Inibidores de Proteases/farmacocinética , Animais , Bioterrorismo , Custos e Análise de Custo , Camundongos , Neurotoxinas , Inibidores de Proteases/economia , Inibidores de Proteases/uso terapêutico , Substâncias Protetoras , Taxa de SobrevidaRESUMO
Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism. Current treatment for post exposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAe) are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously we reported a serotype-selective, small-molecule BoNTAe inhibitor with a K(i) (app) value of 3.8+/-0.8 microM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC(50) value of 100 microM. However, it was difficult to further improve the lead from micromolar to even high nanomolar potency due to the unusually large enzyme-substrate interface of BoNTAe. The enzyme-substrate interface area of 4,840 A(2) for BoNTAe is about four times larger than the typical protein-protein interface area of 750-1,500 A(2). Inhibitors must carry several functional groups to block the unusually large interface of BoNTAe, and syntheses of such inhibitors are therefore time-consuming and expensive. Herein we report the development of a serotype-selective, small-molecule, and competitive inhibitor of BoNTAe with a K(i) value of 760+/-170 nM using synthesis-based computer-aided molecular design (SBCAMD). This new approach accounts the practicality and efficiency of inhibitor synthesis in addition to binding affinity and selectivity. We also report a three-dimensional model of BoNTAe in complex with the new inhibitor and the dynamics of the complex predicted by multiple molecular dynamics simulations, and discuss further structural optimization to achieve better in vivo efficacy in neutralizing BoNTA than those of our early micromolar leads. This work provides new insight into structural modification of known small-molecule BoNTAe inhibitors. It also demonstrates that SBCAMD is capable of improving potency of an inhibitor lead by nearly one order of magnitude, even for BoNTAe as one of the most challenging protein targets. The results are insightful for developing effective small-molecule inhibitors of protein targets with large active sites.
Assuntos
Toxinas Botulínicas Tipo A/química , Endopeptidases/química , Botulismo/terapia , Biologia Computacional/métodos , Simulação por Computador , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Cinética , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Software , Relação Estrutura-AtividadeRESUMO
Optimization of a serotype-selective, small-molecule inhibitor of botulinum neurotoxin serotype A (BoNTA) endopeptidase is a formidable challenge because the enzyme-substrate interface is unusually large and the endopeptidase itself is a large, zinc-binding protein with a complex fold that is difficult to simulate computationally. We conducted multiple molecular dynamics simulations of the endopeptidase in complex with a previously described inhibitor (K(i) (app) of 7+/-2.4 microM) using the cationic dummy atom approach. Based on our computational results, we hypothesized that introducing a hydroxyl group to the inhibitor could improve its potency. Synthesis and testing of the hydroxyl-containing analog as a BoNTA endopeptidase inhibitor showed a twofold improvement in inhibitory potency (K(i) (app) of 3.8+/-0.8 microM) with a relatively small increase in molecular weight (16 Da). The results offer an improved template for further optimization of BoNTA endopeptidase inhibitors and demonstrate the effectiveness of the cationic dummy atom approach in the design and optimization of zinc protease inhibitors.