RESUMO
The integration of diverse chemical tools like small-molecule inhibitors, activity-based probes (ABPs), and proteolysis targeting chimeras (PROTACs) advances clinical drug discovery and facilitates the exploration of various biological facets of targeted proteins. Here, we report the development of such a chemical toolbox for the human Parkinson disease protein 7 (PARK7/DJ-1) implicated in Parkinson's disease and cancers. By combining structure-guided design, miniaturized library synthesis, and high-throughput screening, we identified two potent compounds, JYQ-164 and JYQ-173, inhibiting PARK7 in vitro and in cells by covalently and selectively targeting its critical residue, Cys106. Leveraging JYQ-173, we further developed a cell-permeable Bodipy probe, JYQ-196, for covalent labeling of PARK7 in living cells and a first-in-class PARK7 degrader JYQ-194 that selectively induces its proteasomal degradation in human cells. Our study provides a valuable toolbox to enhance the understanding of PARK7 biology in cellular contexts and opens new opportunities for therapeutic interventions.
Assuntos
Proteína Desglicase DJ-1 , Proteólise , Compostos de Boro/farmacologia , Compostos de Boro/química , Compostos de Boro/síntese química , Proteína Desglicase DJ-1/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-AtividadeRESUMO
Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.
Assuntos
Cisteína Proteases , Processamento de Proteína Pós-Traducional , Ubiquitinação , Ubiquitina , CisteínaRESUMO
Proteotoxic stress causes profound endoplasmic reticulum (ER) membrane remodeling into a perinuclear quality control compartment (ERQC) for the degradation of misfolded proteins. Subsequent return to homeostasis involves clearance of the ERQC by endolysosomes. However, the factors that control perinuclear ER integrity and dynamics remain unclear. Here, we identify vimentin intermediate filaments as perinuclear anchors for the ER and endolysosomes. We show that perinuclear vimentin filaments engage the ER-embedded RING finger protein 26 (RNF26) at the C-terminus of its RING domain. This restricts RNF26 to perinuclear ER subdomains and enables the corresponding spatial retention of endolysosomes through RNF26-mediated membrane contact sites (MCS). We find that both RNF26 and vimentin are required for the perinuclear coalescence of the ERQC and its juxtaposition with proteolytic compartments, which facilitates efficient recovery from ER stress via the Sec62-mediated ER-phagy pathway. Collectively, our findings reveal a scaffolding mechanism that underpins the spatiotemporal integration of organelles during cellular proteostasis.
Assuntos
Filamentos Intermediários , Estresse Proteotóxico , Filamentos Intermediários/metabolismo , Vimentina/genética , Vimentina/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , AutofagiaRESUMO
Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells.
Assuntos
Infecções por Citomegalovirus , Imunoconjugados , Neoplasias , Humanos , Epitopos , Peptídeos , Anticorpos , Peptídeo Hidrolases , Neoplasias/terapiaRESUMO
In the past two decades, drug candidates with a covalent binding mode have gained the interest of medicinal chemists, as several covalent anticancer drugs have successfully reached the clinic. As a covalent binding mode changes the relevant parameters to rank inhibitor potency and investigate structure-activity relationship (SAR), it is important to gather experimental evidence on the existence of a covalent protein-drug adduct. In this work, we review established methods and technologies for the direct detection of a covalent protein-drug adduct, illustrated with examples from (recent) drug development endeavors. These technologies include subjecting covalent drug candidates to mass spectrometric (MS) analysis, protein crystallography, or monitoring intrinsic spectroscopic properties of the ligand upon covalent adduct formation. Alternatively, chemical modification of the covalent ligand is required to detect covalent adducts by NMR analysis or activity-based protein profiling (ABPP). Some techniques are more informative than others and can also elucidate the modified amino acid residue or bond layout. We will discuss the compatibility of these techniques with reversible covalent binding modes and the possibilities to evaluate reversibility or obtain kinetic parameters. Finally, we expand upon current challenges and future applications. Overall, these analytical techniques present an integral part of covalent drug development in this exciting new era of drug discovery.
RESUMO
Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.
Assuntos
Enzimas Desubiquitinantes , Poliubiquitina , Ubiquitinação , Poliubiquitina/metabolismo , Enzimas Desubiquitinantes/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an α-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5'-hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes.
Assuntos
Adenosina Difosfato Ribose , Arginina , Adenosina Difosfato Ribose/química , Arginina/metabolismo , ADP-Ribosilação , Ubiquitina/química , Proteínas Ubiquitinadas/metabolismo , Peptídeos/químicaRESUMO
The multifunctional human Parkinson's disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson's disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors.
Assuntos
Doença de Parkinson , Corantes , Humanos , Proteína Desglicase DJ-1 , Regulação para CimaRESUMO
Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.
Assuntos
Aminoaciltransferases , Culicidae , Malária , Processamento de Proteína Pós-Traducional , Esporozoítos , Aminoaciltransferases/imunologia , Animais , Culicidae/imunologia , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Humanos , Malária/genética , Malária/imunologia , Malária/parasitologia , Plasmodium berghei/genética , Plasmodium berghei/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologiaRESUMO
Covalent inhibition has become more accepted in the past two decades, as illustrated by the clinical approval of several irreversible inhibitors designed to covalently modify their target. Elucidation of the structure-activity relationship and potency of such inhibitors requires a detailed kinetic evaluation. Here, we elucidate the relationship between the experimental read-out and the underlying inhibitor binding kinetics. Interactive kinetic simulation scripts are employed to highlight the effects of in vitro enzyme activity assay conditions and inhibitor binding mode, thereby showcasing which assumptions and corrections are crucial. Four stepwise protocols to assess the biochemical potency of (ir)reversible covalent enzyme inhibitors targeting a nucleophilic active site residue are included, with accompanying data analysis tailored to the covalent binding mode. Together, this will serve as a guide to make an educated decision regarding the most suitable method to assess covalent inhibition potency. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol I: Progress curve analysis of substrate association competition Basic Data Analysis Protocol 1A: Two-step irreversible covalent inhibition Basic Data Analysis Protocol 1B: One-step irreversible covalent inhibition Basic Data Analysis Protocol 1C: Two-step reversible covalent inhibition Basic Data Analysis Protocol 1D: Two-step irreversible covalent inhibition with substrate depletion Basic Protocol II: Incubation time-dependent potency IC50 (t) Basic Data Analysis Protocol 2: Two-step irreversible covalent inhibition Basic Protocol III: Preincubation time-dependent inhibition without dilution Basic Data Analysis Protocol 3: Preincubation time-dependent inhibition without dilution Basic Data Analysis Protocol 3Ai: Two-step irreversible covalent inhibition Alternative Data Analysis Protocol 3Aii: Two-step irreversible covalent inhibition Basic Data Analysis Protocol 3Bi: One-step irreversible covalent inhibition Alternative Data Analysis Protocol 3Bii: One-step irreversible covalent inhibition Basic Data Analysis Protocol 3C: Two-step reversible covalent inhibition Basic Protocol IV: Preincubation time-dependent inhibition with dilution/competition Basic Data Analysis Protocol 4: Preincubation time-dependent inhibition with dilution Basic Data Analysis Protocol 4Ai: Two-step irreversible covalent inhibition Alternative Data Analysis Protocol 4Aii: Two-step irreversible covalent inhibition Basic Data Analysis Protocol 4Bi: One-step irreversible covalent inhibition Alternative Data Analysis Protocol 4Bii: One-step irreversible covalent inhibition.
Assuntos
Ensaios Enzimáticos , Inibidores Enzimáticos , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Cinética , Relação Estrutura-AtividadeRESUMO
DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a 'Ku-adaptor' that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance.
Assuntos
Quebras de DNA de Cadeia Dupla , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , DNA/metabolismo , Humanos , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol-alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CDWT and catalytically inactive mutant USP16CDC205A. Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol-alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.
Assuntos
Alcinos/química , Cisteína Proteases/química , Pargilina/análogos & derivados , Compostos de Sulfidrila/química , Enzimas Desubiquitinantes/química , Células HEK293 , Humanos , Pargilina/química , Propilaminas/química , Ubiquitina Tiolesterase/químicaRESUMO
Legionnaires' disease is caused by infection with the intracellularly replicating Gram-negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr ) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole-linked UbADPr , and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr . The inhibitory effects of modified Ub on the canonical eukaryotic E1-enzyme Uba1 are investigated and rationalized in the context of a high-resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull-down overexpressed DupA from cell lysate.
Assuntos
ADP-Ribosilação , Legionella pneumophila/enzimologia , Doença dos Legionários/microbiologia , Ubiquitina/química , Ubiquitina/metabolismo , Proteínas de Bactérias/metabolismo , Células HEK293 , Humanos , Hidrolases/metabolismo , Legionella pneumophila/crescimento & desenvolvimento , Enzimas Ativadoras de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Stable NAD+ analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD+ -consuming enzymes. To investigate the potential of such compounds to inhibit the adenosine diphosphate ribosyl (ADPr) transferase activity of the Legionella SdeC enzyme, we prepared three NAD+ analogues, namely carbanicotinamide adenosine dinucleotide (c-NAD+ ), thionicotinamide adenosine dinucleotide (S-NAD+ ) and benzamide adenosine dinucleotide (BAD). We optimized the chemical synthesis of thionicotinamide riboside and for the first time used an enzymatic approach to convert all three ribosides into the corresponding NAD+ mimics. We thus expanded the known scope of substrates for the NRK1/NMNAT1 enzyme combination by turning all three modified ribosides into NAD+ analogues in a scalable manner. We then compared the three NAD+ mimics side-by-side in a single assay for enzyme inhibition on Legionella effector enzyme SdeC. The class of SidE enzymes to which SdeC belongs was recently identified to be important in bacterial virulence, and we found SdeC to be inhibited by S-NAD+ and BAD with IC50 values of 28 and 39â µM, respectively.
Assuntos
Legionella pneumophila/enzimologia , NAD/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Modelos Moleculares , Conformação Molecular , NAD/síntese química , NAD/química , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases/químicaRESUMO
Ubiquitin-specific proteases (USPs) are an important class of deubiquitinating enzymes (DUBs) that carry out critical roles in cellular physiology and are regulated at multiple levels. Quantitative characterization of USP activity is crucial for mechanistic understanding of USP function and regulation. This requires kinetic analysis using in vitro activity assays on minimal and natural substrates with purified proteins. In this chapter we give advice for efficient design of USP constructs and their optimal expression, followed by a series of purification strategies. We then present protocols for studying USP activity quantitatively on minimal and more natural substrates, and we discuss how to include possible regulatory elements such as internal USP domains or external interacting proteins. Lastly, we examine different binding assays for studying USP interactions and discuss how these can be included in full kinetic analyses.
Assuntos
Proteases Específicas de Ubiquitina/metabolismo , Animais , Ensaios Enzimáticos/métodos , Humanos , Cinética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Especificidade por Substrato , Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/isolamento & purificaçãoRESUMO
USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.
Assuntos
Processamento de Proteína Pós-Traducional , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Isótopos de Carbono/química , Domínio Catalítico/genética , Ensaios Enzimáticos/métodos , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Proteína Supressora de Tumor p53/química , Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/isolamento & purificaçãoRESUMO
Ubiquitin (Ub) conjugation is a critical signalling process in eukaryotic cells. The precise regulation of deubiquitination is an important component of this signalling cascade. Here, we discuss how USP7 (or Herpes-Associated Ubiquitin-Specific Protease, HAUSP), one of the most abundant deubiquitinating enzymes, is regulated by complex formation with regulatory proteins and targets. Full activity of USP7 requires that its C-terminal Ub-like domains fold back onto the catalytic domain, to allow the remodelling of the active site to a catalytically competent state by the very C-terminal peptide. This regulatory mode can be modulated by complex formation with other proteins. USP7 is found in a large number of relatively stable complexes with different possible functions. Complex formation can provide recruitment of a target, bring in an E3 Ub ligase, or modulate the activation of the deubiquitinating enzyme activity. These complexes make up potential cellular "switches", using their (de)ubiquitination ability to switch pathways on or off upon cellular signals. Here, we summarize what is known for USP7 complexes, focussing on the prevalence of E3 Ub ligases and how complex formation can affect Ub switches.
Assuntos
Células Eucarióticas/enzimologia , Multimerização Proteica , Ubiquitina Tiolesterase/metabolismo , Complexos Multienzimáticos/metabolismo , Dobramento de ProteínaRESUMO
Ubiquitin conjugation is an important signal in cellular pathways, changing the fate of a target protein, by degradation, relocalisation or complex formation. These signals are balanced by deubiquitinating enzymes (DUBs), which antagonize ubiquitination of specific protein substrates. Because ubiquitination pathways are critically important, DUB activity is often carefully controlled. USP7 is a highly abundant DUB with numerous targets that plays complex roles in diverse pathways, including DNA regulation, p53 stress response and endosomal protein recycling. Full-length USP7 switches between an inactive and an active state, tuned by the positioning of 5 Ubl folds in the C-terminal HUBL domain. The active state requires interaction between the last two Ubls (USP7(45)) and the catalytic domain (USP7(CD)), and this can be promoted by allosteric interaction from the first 3 Ubl domains of USP7 (USP7(123)) interacting with GMPS. Here we study the transition between USP7 states. We provide a crystal structure of USP7(CD123) and show that CD and Ubl123 are connected via an extended charged alpha helix. Mutational analysis is used to determine whether the charge and rigidity of this 'connector helix' are important for full USP7 activity.
Assuntos
Domínio Catalítico , Peptidase 7 Específica de Ubiquitina/química , Sítio Alostérico , Cristalografia por Raios X , Enzimas Desubiquitinantes/química , Humanos , Estrutura Molecular , Mutagênese Sítio-Dirigida , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
The recently discovered futalosine pathway is a promising target for the development of new antibiotics. The enzymes involved in this pathway are crucial for the biosynthesis of the essential prokaryotic respiratory compound menaquinone, and as the pathway is limited to few bacterial species such as the gastric pathogen Helicobacter pylori it is a potential target for specific antibiotics. In this report, the crystal structure of an H. pylori methylthioadenosine nucleosidase (MTAN; an enzyme with broad specificity and activity towards 6-amino-6-deoxyfutalosine), which is involved in the second step of menaquinone biosynthesis, has been elucidated at a resolution of 1.76â Å and refined with R factors of Rwork = 17% and Rfree = 21%. Activity studies on the wild type and active-site mutants show that the hydrolysis of 6-amino-6-deoxyfutalosine follows a mechanism similar to that of Escherichia coli MTAN. Further evidence for this mode of action is supplied by the crystal structures of active-site mutants. Through the use of reaction intermediates, the structures give additional evidence for the previously proposed nucleosidase mechanism. These structures and the confirmed reaction mechanism will provide a structural basis for the design of new inhibitors targeting the futalosine pathway.
Assuntos
Helicobacter pylori/enzimologia , Nucleosídeos/metabolismo , Purina-Núcleosídeo Fosforilase/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Helicobacter pylori/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Purina-Núcleosídeo Fosforilase/metabolismo , Transdução de SinaisRESUMO
The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61.