RESUMO
Asparagine-linked glycosylation is a common posttranslational modification of diverse secretory and membrane proteins in eukaryotes, where it is catalyzed by the multiprotein complex oligosaccharyltransferase. The functions of the protein subunits of oligoasccharyltransferase, apart from the catalytic Stt3p, are ill defined. Here we describe functional and structural investigations of the Ost3/6p components of the yeast enzyme. Genetic, biochemical and structural analyses of the lumenal domain of Ost6p revealed oxidoreductase activity mediated by a thioredoxin-like fold with a distinctive active-site loop that changed conformation with redox state. We found that mutation of the active-site cysteine residues of Ost6p and its paralogue Ost3p affected the glycosylation efficiency of a subset of glycosylation sites. Our results show that eukaryotic oligosaccharyltransferase is a multifunctional enzyme that acts at the crossroads of protein modification and protein folding.
Assuntos
Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Motivos de Aminoácidos , Domínio Catalítico , Glicosilação , Modelos Biológicos , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Compostos de Sulfidrila/metabolismoRESUMO
Despite their favorable pharmacokinetic properties, single-chain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 x 10(-9) to 1.5 x 10(-11) m. Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 x 10(-10) to <1.8 x 10(-12) m. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86 degrees C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains.
Assuntos
Anticorpos Monoclonais/química , Região Variável de Imunoglobulina/química , Engenharia de Proteínas/métodos , Animais , Regiões Determinantes de Complementaridade/química , Células Endoteliais/citologia , Escherichia coli/metabolismo , Humanos , Hibridomas/metabolismo , Cinética , Camundongos , Ligação Proteica , Coelhos , Fator de Necrose Tumoral alfa/química , Fator A de Crescimento do Endotélio Vascular/químicaRESUMO
Sulfotransferases are a versatile class of enzymes involved in numerous physiological processes. In mammals, adenosine 3'-phosphate-5'-phosphosulfate (PAPS) is the universal sulfuryl donor, and PAPS-dependent sulfurylation of small molecules, including hormones, sugars, and antibiotics, is a critical step in hepatic detoxification and extracellular signaling. In contrast, little is known about sulfotransferases in bacteria, which make use of sulfurylated molecules as mediators of cell-cell interactions and host-pathogen interactions. Bacterial arylsulfate sulfotransferases (also termed aryl sulfotransferases), in contrast to PAPS-dependent sulfotransferases, transfer sulfuryl groups exclusively among phenolic compounds in a PAPS-independent manner. Here, we report the crystal structure of the virulence factor arylsulfate sulfotransferase (ASST) from the prototypic, pyelonephritogenic Escherichia coli strain CFT073 at 2.0-A resolution, and 2 catalytic intermediates, at 2.1-A and 2.4-A resolution, with substrates bound in the active site. ASST is one of the largest periplasmic enzymes and its 3D structure differs fundamentally from all other structurally characterized sulfotransferases. Each 63.8-kDa subunit of the ASST homodimer comprises a 6-bladed beta-propeller domain and a C-terminal beta-sandwich domain. The active sites of the dimer are situated at the center of the channel formed by each beta-propeller and are defined by the side chains of His-252, His-356, Arg-374, and His-436. We show that ASST follows a ping-pong bi-bi reaction mechanism, in which the catalytic residue His-436 undergoes transient sulfurylation, a previously unreported covalent protein modification. The data provide a framework for understanding PAPS-independent sulfotransfer and a basis for drug design targeting this bacterial virulence factor.
Assuntos
Arilsulfotransferase/química , Arilsulfotransferase/metabolismo , Escherichia coli/enzimologia , Animais , Arilsulfotransferase/genética , Domínio Catalítico , Cristalografia , Dimerização , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Cinética , Mamíferos , Mutagênese Sítio-Dirigida , Fosfoadenosina Fosfossulfato/metabolismo , Estrutura Terciária de Proteína , Pielonefrite/microbiologia , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de Virulência/química , Fatores de Virulência/metabolismoRESUMO
Disulfide bond formation is a critical step in the folding of many secretory proteins. In bacteria, disulfide bonds are introduced by the periplasmic dithiol oxidase DsbA, which transfers its catalytic disulfide bond to folding polypeptides. Reduced DsbA is reoxidized by ubiquinone Q8, catalyzed by inner membrane quinone reductase DsbB. Here, we report the preparation of a kinetically stable ternary complex between wild-type DsbB, containing all essential cysteines, Q8 and DsbA covalently bound to DsbB. The crystal structure of this trapped DsbB reaction intermediate exhibits a charge-transfer interaction between Q8 and the Cys44 in the DsbB reaction center providing experimental evidence for the mechanism of de novo disulfide bond generation in DsbB.
Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Membrana Celular/metabolismo , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Dissulfetos/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Oxirredução , Estrutura Secundária de ProteínaRESUMO
GrpE is the nucleotide-exchange factor of the DnaK chaperone system. Escherichia coli cells with the classical temperature-sensitive grpE280 phenotype do not grow under heat-shock conditions and have been found to carry the G122D point mutation in GrpE. To date, the molecular mechanism of this defect has not been investigated in detail. Here, we examined the structural and functional properties of isolated GrpE(G122D) in vitro. Similar to wild-type GrpE, GrpE(G122D) is an elongated dimer in solution. Compared to wild-type GrpE, GrpE(G122D) catalyzed the ADP/ATP exchange in DnaK only marginally and did not compete with wild-type GrpE in interacting with DnaK. In the presence of ADP, GrpE(G122D) in contrast to wild-type GrpE, did not form a complex with DnaK detectable by size-exclusion chromatography with on-line static light-scattering and differential refractometry. Apparently, GrpE(G122D) in the presence of ADP binds to DnaK only with much lower affinity than wild-type GrpE. GrpE(G122D) could not substitute for wild-type GrpE in the refolding of denatured proteins by the DnaK/DnaJ/GrpE chaperone system. In the crystal structure of a (Delta1-33)GrpE(G122D).DnaK-ATPase complex, which as yet is the only available structure of a GrpE variant, Asp122 does not interact directly with neighboring residues of GrpE or DnaK. The far-UV circular dichroism spectra of mutant and wild-type GrpE proved slightly different. Possibly, a discrete change in conformation impairs the formation of the complex with DnaK and renders GrpE(G122D) virtually inactive as a nucleotide exchange factor. In view of the drastically reduced ADP/ATP-exchange activity of GrpE(G122D), the heat sensitivity of grpE280 cells might be explained by the ensuing slowing of the chaperone cycle and the increased sequestering of target proteins by high-affinity, ADP-liganded DnaK, both effects being incompatible with efficient chaperone action required for cell growth.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Mutação/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Cromatografia em Gel , Dicroísmo Circular , Dimerização , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glucosefosfato Desidrogenase/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Cinética , Luciferases/metabolismo , Fenótipo , Ligação Proteica , Desnaturação Proteica , Dobramento de ProteínaRESUMO
Sustained-release formulations of a single-chain anti-VEGF-A antibody fragment were investigated in vitro toward their potential use for intravitreal applications. The hydrophobic polyester hexylsubstituted poly(lactic acid) (hexPLA) was selected as the sustained-release excipient for its biodegradability and semi-solid aggregate state, allowing an easy and mild formulation procedure. The lyophilized antibody fragment ESBA903 was micronized and incorporated into the liquid polymer matrix by cryo-milling, forming homogeneous and injectable suspensions. The protein showed excellent compatibility with the hexPLA polymer and storage stability at 4°C for 10 weeks. Additionally, hexPLA shielded the incorporated active substance from the surrounding medium, resulting in a better stability of ESBA903 inside the polymer than after its release in the buffer solution. Formulations of ESBA903 with hexPLA having drug loadings between 1.25% and 5.0% and polymer molecular weights of 1500 g/mol, 2500 g/mol, 3500 g/mol and 5000 g/mol were investigated regarding their in vitro release. All formulations except with the highest molecular weight formed spherical depots in aqueous buffer solutions and released the antibody fragment for at least 6-14 weeks. The polymer viscosity derived from the molecular weight strongly influenced the release rate, while the drug loading had minor influence, allowing customization of the release profile and the daily drug release. Size exclusion chromatography and SDS-PAGE revealed that the antibody fragment structure was kept intact during incorporation and release from the liquid matrix. Furthermore, the released protein monomer maintained its high affinity to human VEGF-A, as measured by surface plasmon resonance analysis. Formulations of ESBA903 in hexPLA meet the basic needs to be used for intravitreal sustained-release applications in age-related macular degeneration treatment.
Assuntos
Excipientes/química , Ácido Láctico/química , Polímeros/química , Anticorpos de Cadeia Única/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Química Farmacêutica/métodos , Preparações de Ação Retardada , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Eletroforese em Gel de Poliacrilamida , Liofilização , Humanos , Interações Hidrofóbicas e Hidrofílicas , Injeções Intravítreas , Peso Molecular , Poliésteres/química , Anticorpos de Cadeia Única/imunologia , Ressonância de Plasmônio de Superfície/métodos , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/imunologia , ViscosidadeRESUMO
The familiar heat shock response in cells comprises the enhanced expression of molecular chaperones. In recent experiments with the Hsp70 system of Escherichia coli, the co-chaperone GrpE has been found to undergo a reversible thermal transition in the physiological temperature range. Here, we tested whether this thermal transition is of functional significance in the complete DnaK/DnaJ/GrpE chaperone system. We found that a mere increase in temperature resulted in a higher fraction of fluorescence-labeled peptides being sequestered by DnaK. This direct adaptation of the DnaK/DnaJ/GrpE chaperone system to heat shock conditions may serve to bridge the time lag of enhanced chaperone expression.
Assuntos
2-Naftilamina/análogos & derivados , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Chaperonas Moleculares/metabolismo , 2-Naftilamina/metabolismo , Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Corantes Fluorescentes/metabolismo , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico/química , Substâncias Macromoleculares , Chaperonas Moleculares/química , Peptídeos/metabolismoRESUMO
Disulfide bond formation in the Escherichia coli periplasm requires the transfer of electrons from substrate proteins to DsbA, which is recycled as an oxidant by the membrane protein DsbB. The highly virulent, uropathogenic E. coli strain CFT073 contains a second, homologous pair of proteins, DsbL and DsbI, which are encoded in a tri-cistronic operon together with a periplasmic, uropathogen-specific arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI form a functional redox pair, and that ASST is a substrate of DsbL/DsbI in vivo. DsbL is the most reactive oxidizing thioredoxin-like protein known to date. In contrast to DsbA, however, DsbL oxidizes reduced RNaseA with a much lower rate and prevents unspecific aggregation of reduced insulin. The 1.55 A resolution crystal structure of reduced DsbL provides insight into the reduced state of thioredoxin-like dithiol oxidases at high resolution, and reveals an unusual cluster of basic residues stabilizing the thiolate anion of the nucleophilic active-site cysteine. We propose that the DsbL/DsbI pair of uropathogenic E. coli was acquired as an additional, specific redox couple that guarantees biological activity of ASST.
Assuntos
Arilsulfotransferase/metabolismo , Dissulfetos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Oxirredutases/metabolismo , Periplasma/enzimologia , Sequência de Aminoácidos , Arilsulfotransferase/química , Arilsulfotransferase/genética , Sítios de Ligação , Cristalografia por Raios X , Dissulfetos/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Teste de Complementação Genética , Glutationa/metabolismo , Ligação de Hidrogênio , Insulina/química , Insulina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Conformação Proteica , Estrutura Terciária de Proteína , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismo , Alinhamento de SequênciaRESUMO
Proteins of the thioredoxin (Trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. All members of the Trx family with thiol-disulfide oxidoreductase activity contain the characteristic Cys-X-X-Cys motif in their active site. Here, using Poisson-Boltzmann-based protonation-state calculations based on 100-ns molecular dynamics simulations, we investigate the catalytic mechanism of DsbL, the most oxidizing Trx-like protein known to date. We observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. These results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. Intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. A consistent catalytic mechanism for DsbL, probably conferrable to other proteins of the same class, is presented. Our results suggest a functional role of hydration entropy of active-site groups.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Estrutura Terciária de Proteína , Prótons , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Sítios de Ligação , Catálise , Simulação por Computador , Proteínas de Escherichia coli/genética , Modelos Moleculares , Oxirredução , Isomerases de Dissulfetos de Proteínas/genética , Tiorredoxinas/genéticaRESUMO
Temperature directly controls functional properties of the DnaK/DnaJ/GrpE chaperone system. The rate of the high to low affinity conversion of DnaK shows a non-Arrhenius temperature dependence and above approximately 40 degrees C even decreases. In the same temperature range, the ADP/ATP exchange factor GrpE undergoes an extensive, fully reversible thermal transition (Grimshaw, J. P. A., Jelesarov, I., Schönfeld, H. J., and Christen, P. (2001) J. Biol. Chem. 276, 6098-6104). To show that this transition underlies the thermal regulation of the chaperone system, we introduced an intersubunit disulfide bond into the paired long helices of the GrpE dimer. The transition was absent in disulfide-linked GrpE R40C but was restored by reduction. With disulfide-stabilized GrpE, the rate of ADP/ATP exchange and conversion of DnaK from its ADP-liganded high affinity R state to the ATP-liganded low affinity T state continuously increased with increasing temperature. With reduced GrpE R40C, the conversion became slower at temperatures >40 degrees C, as observed with wild-type GrpE. Thus, the long helix pair in the GrpE dimer acts as a thermosensor that, by decreasing its ADP/ATP exchange activity, induces a shift of the DnaK.substrate complexes toward the high affinity R state and in this way adapts the DnaK/DnaJ/GrpE system to heat shock conditions.