RESUMO
Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.
Assuntos
Domínio Catalítico , Endopeptidases , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Especificidade por Substrato , Substituição de Aminoácidos , Potyvirus/enzimologia , Potyvirus/genética , Potyvirus/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cinética , Sequência de Aminoácidos , Mutação , Proteólise , Expressão GênicaRESUMO
The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 Å) crystal structure of a truncated nsP2pro (residues 463-785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.
Assuntos
Domínio Catalítico , Cisteína Proteases , Vírus da Encefalite Equina Venezuelana , Animais , Humanos , Cristalografia por Raios X , Cisteína Proteases/química , Cisteína Proteases/genética , Vírus da Encefalite Equina Venezuelana/enzimologia , Cavalos , Simulação de Dinâmica MolecularRESUMO
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3'- and 5'-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3'-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke-Blackburn-Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand-protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors.
RESUMO
N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 µM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 µM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.
Assuntos
Proteínas de Bactérias/metabolismo , Cadaverina/metabolismo , Desferroxamina/metabolismo , Oxigenases de Função Mista/biossíntese , Sideróforos/biossíntese , Streptomyces/enzimologia , Biocatálise , Domínio Catalítico , Dinitrocresóis/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/metabolismo , Holoenzimas/metabolismo , Hidroxilação , Cinética , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , NADP/metabolismo , Ornitina/metabolismo , OxirreduçãoRESUMO
The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1', and P2' variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1' amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses.
Assuntos
Vírus da Encefalite Equina Venezuelana/enzimologia , Proteases Virais/química , Domínio Catalítico , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Especificidade por Substrato , Proteases Virais/genética , Proteases Virais/metabolismoRESUMO
Yersinia pestis, the causative agent of bubonic plague, is one of the most lethal pathogens in recorded human history. Today, the concern is the possible misuse of Y. pestis as an agent in bioweapons and bioterrorism. Current therapies for the treatment of plague include the use of a small number of antibiotics, but clinical cases of antibiotic resistance have been reported in some areas of the world. Therefore, the discovery of new drugs is required to combat potential Y. pestis infection. Here, the crystal structure of the Y. pestis UDP-glucose pyrophosphorylase (UGP), a metabolic enzyme implicated in the survival of Y. pestis in mouse macrophages, is described at 2.17â Å resolution. The structure provides a foundation that may enable the rational design of inhibitors and open new avenues for the development of antiplague therapeutics.
Assuntos
UTP-Glucose-1-Fosfato Uridililtransferase/química , Yersinia pestis/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Peste/tratamento farmacológico , Conformação ProteicaRESUMO
Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3' end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.
Assuntos
Inibidores Enzimáticos/química , Ligantes , Diester Fosfórico Hidrolases/química , Conformação Proteica , Sequência de Bases , Domínio Catalítico/genética , Cristalografia , Reparo do DNA/genética , Histidina/análogos & derivados , Histidina/química , Histidina/isolamento & purificação , Humanos , Modelos Moleculares , Diester Fosfórico Hidrolases/genética , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Members of the ribonuclease (RNase) III family regulate gene expression by processing dsRNAs. It was previously shown that Escherichia coli (Ec) RNase III recognizes dsRNA with little sequence specificity and the cleavage products are mainly 11 nucleotides (nt) long. It was also shown that the mutation of a glutamate (EcE38) to an alanine promotes generation of siRNA-like products typically 22 nt long. To fully characterize substrate specificity and product size of RNase IIIs, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) enzymes and delineated their products by next-generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Furthermore, we identified a second glutamate (EcE65/AaE64), which, when mutated to alanine, also enhances the production of siRNA-like products. Based on these findings, we created a bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.
Assuntos
Proteínas Mutantes/genética , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Alanina/genética , Sequência de Aminoácidos/genética , Dimerização , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Ácido Glutâmico/genética , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Mutação , Ribonuclease III/química , Ribonuclease III/isolamento & purificação , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
Chemical library screening approaches that focus exclusively on catalytic events may overlook unique effects of protein-protein interactions that can be exploited for development of specific inhibitors. Phosphotyrosyl (pTyr) residues embedded in peptide motifs comprise minimal recognition elements that determine the substrate specificity of protein tyrosine phosphatases (PTPases). We incorporated aminooxy-containing amino acid residues into a 7-residue epidermal growth factor receptor (EGFR) derived phosphotyrosine-containing peptide and subjected the peptides to solution-phase oxime diversification by reacting with aldehyde-bearing druglike functionalities. The pTyr residue remained unmodified. The resulting derivatized peptide library was printed in microarrays on nitrocellulose-coated glass surfaces for assessment of PTPase catalytic activity or on gold monolayers for analysis of kinetic interactions by surface plasmon resonance (SPR). Focusing on amino acid positions and chemical features, we first analyzed dephosphorylation of the peptide pTyr residues within the microarrayed library by the human dual-specificity phosphatases (DUSP) DUSP14 and DUSP22, as well as by PTPases from poxviruses (VH1) and Yersinia pestis (YopH). In order to identify the highest affinity oxime motifs, the binding interactions of the most active derivatized phosphopeptides were examined by SPR using noncatalytic PTPase mutants. On the basis of high-affinity oxime fragments identified by the two-step catalytic and SPR-based microarray screens, low-molecular-weight nonphosphate-containing peptides were designed to inhibit PTP catalysis at low micromolar concentrations.
Assuntos
Biblioteca de Peptídeos , Fosfopeptídeos/química , Análise Serial de Proteínas/métodos , Proteínas Tirosina Fosfatases/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Sequência de Aminoácidos , Catálise , Colódio/química , Fosfatases de Especificidade Dupla/química , Receptores ErbB/química , Humanos , Cinética , Fosfatases da Proteína Quinase Ativada por Mitógeno/química , Estrutura Molecular , Fosfotirosina/química , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Propriedades de SuperfícieRESUMO
A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.
RESUMO
Here, new crystal structures are presented of the isolated membrane-proximal D1 and distal D2 domains of protein tyrosine phosphatase epsilon (PTPâ), a protein tyrosine phosphatase that has been shown to play a positive role in the survival of human breast cancer cells. A triple mutant of the PTPâ D2 domain (A455N/V457Y/E597D) was also constructed to reconstitute the residues of the PTPâ D1 catalytic domain that are important for phosphatase activity, resulting in only a slight increase in the phosphatase activity compared with the native D2 protein. The structures reported here are of sufficient resolution for structure-based drug design, and a microarray-based assay for high-throughput screening to identify small-molecule inhibitors of the PTPâ D1 domain is also described.
Assuntos
Desenho de Fármacos , Análise Serial de Proteínas/métodos , Domínios Proteicos/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/química , Cristalografia por Raios X/métodos , Humanos , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Bibliotecas de Moléculas PequenasRESUMO
The production of high-quality crystals is the main bottleneck in determining the structures of proteins using X-ray crystallography. In addition to being recognized as a very effective solubility-enhancing fusion partner, Escherichia coli maltose-binding protein (MBP) has also been successfully employed as a `fixed-arm' crystallization chaperone in more than 100 cases. Here, it is reported that designed ankyrin-repeat proteins (DARPins) that bind with high affinity to MBP can promote the crystallization of an MBP fusion protein when the fusion protein alone fails to produce diffraction-quality crystals. As a proof of principle, three different co-crystal structures of MBP fused to the catalytic domain of human dual-specificity phosphatase 1 in complex with DARPins are reported.
Assuntos
Fosfatase 1 de Especificidade Dupla/química , Proteínas Ligantes de Maltose/química , Chaperonas Moleculares/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38-alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.
Assuntos
Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fosfotreonina/química , Fosfotirosina/química , Conformação Proteica , Especificidade por Substrato , Sulfatos/químicaRESUMO
Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His6- or a dual His6-MBP tagged fusion protein by Gateway® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His6 tag or a His6-MBP tag can be made on the basis of this solubility test.
Assuntos
Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Vetores Genéticos/química , Histidina/genética , Proteínas Ligantes de Maltose/genética , Oligopeptídeos/genética , Proteínas Recombinantes de Fusão/genética , Animais , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Endopeptidases/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Histidina/metabolismo , Humanos , Corpos de Inclusão/química , Proteínas Ligantes de Maltose/metabolismo , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , SolubilidadeRESUMO
Although affinity tags are highly effective tools for the expression and purification of recombinant proteins, they generally need to be removed prior to structural and functional studies. This chapter describes a simple method for overproducing a soluble form of a stable variant of tobacco etch virus (TEV) protease in Escherichia coli and a protocol for purifying it to homogeneity so that it can be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. Further, we cleave a model substrate protein (MBP-NusG) in vitro using the purified TEV protease to illustrate a protease cleavage protocol that can be employed for simple pilot experiments and large-scale protein preparations.
Assuntos
Cromatografia de Afinidade/métodos , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Escherichia coli/genética , Potyvirus/enzimologia , Clonagem Molecular/métodos , Endopeptidases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
E2 enzymes in ubiquitin-like conjugation pathways are important, highly challenging pharmacological targets, and despite significant efforts, few noncovalent modulators have been discovered. Small-molecule microarray (SMM)-based screening was employed to identify an inhibitor of the "undruggable" small ubiquitin-like modifier (SUMO) E2 enzyme Ubc9. The inhibitor, a degradation product from a commercial screening collection, was chemically synthesized and evaluated in biochemical, mechanistic, and structure-activity relationship studies. Binding to Ubc9 was confirmed through the use of ligand-detected nuclear magnetic resonance, and inhibition of sumoylation in a reconstituted enzymatic cascade was found to occur with an IC50 of 75 µM. This work establishes the utility of the SMM approach for identifying inhibitors of E2 enzymes, targets with few known small-molecule modulators.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Cromatografia Líquida , Descoberta de Drogas/métodos , Humanos , Espectrometria de Massas , Estrutura Molecular , Transdução de Sinais , Relação Estrutura-Atividade , Sumoilação , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme. An X-ray crystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at a site distal to its catalytic cysteine. These fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9-5.8â mm. Mechanistic and biophysical analyses, coupled with molecular dynamics simulations, point toward ligand-induced rigidification of Ubc9 as a mechanism of inhibition.
Assuntos
Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação Alostérica , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Especificidade por Substrato , Sumoilação , Ressonância de Plasmônio de Superfície , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification.
Assuntos
Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Virais/metabolismo , Proteases Virais 3C , Escherichia coli/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Rhinovirus/metabolismo , Nicotiana/virologiaRESUMO
Although chaperone-assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose-binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter-domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP-mediated structural distortions are very rare.
Assuntos
Cristalização/métodos , Proteínas Ligantes de Maltose/química , Peptídeos/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Animais , Clonagem Molecular/métodos , Cristalografia/métodos , Entropia , Humanos , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/isolamento & purificação , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Mutação , Peptídeos/genética , Peptídeos/isolamento & purificação , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
A common problem encountered during the production of recombinant proteins, particularly in bacteria, is their tendency to accumulate in an insoluble and inactive form (i.e., as inclusion bodies). Although sometimes it is possible to convert the aggregated material into native, biologically active protein, this is a time-consuming, costly, and uncertain undertaking. Consequently, a general means of circumventing the formation of inclusion bodies is highly desirable. During the 1990s, it was serendipitously discovered that certain highly soluble proteins have the ability to enhance the solubility of their fusion partners, thereby preventing them from forming insoluble aggregates. In the ensuing years, Escherichia coli maltose-binding protein (MBP) has emerged as one of the most effective solubility enhancers. Moreover, once rendered soluble by fusion to MBP, many proteins are able to fold into their biologically active conformations. This brief review article focuses on our current understanding of what makes MBP such an effective solubility enhancer and how it facilitates the proper folding of its fusion partners.