RESUMO
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.
Assuntos
LDL-Colesterol/sangue , Fragmentos Fab das Imunoglobulinas/farmacologia , Receptores de LDL/química , Serina Endopeptidases/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Fluorimunoensaio , Humanos , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Macaca mulatta , Masculino , Camundongos , Camundongos Transgênicos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Receptores de LDL/metabolismo , Serina Endopeptidases/químicaRESUMO
PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.
Assuntos
Anticorpos Monoclonais/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Substituição de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Células Hep G2 , Humanos , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/genética , Hipercolesterolemia/imunologia , Hipercolesterolemia/metabolismo , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Lipoproteínas LDL/genética , Lipoproteínas LDL/imunologia , Mutagênese Sítio-Dirigida , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de LDL/genética , Receptores de LDL/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologiaRESUMO
The design of a series of peptidomimetic inhibitors of the hepatitis C virus NS3 protease is described. These inhibitors feature an indoline-2-carboxamide as a novel heterocyclic replacement for the P3 amino acid residue and N-terminal capping group of tripeptide based inhibitors. The crystal structure of the ternary NS3/NS4A/inhibitor complex for the most active molecule in this series highlights its suitability as an N-terminal capping group of a dipeptide inhibitor of the NS3 protease.
Assuntos
Antivirais/síntese química , Hepacivirus/enzimologia , Indóis/síntese química , Oligopeptídeos/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Antivirais/química , Cristalografia por Raios X , Indóis/química , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Ligação Proteica , EstereoisomerismoRESUMO
Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 A resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.
Assuntos
Histona Desacetilases/química , Histona Desacetilases/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Ácido Aspártico/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have determined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3- and 2.4-Angstroms resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small alpha-helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas não Estruturais Virais/química , Sítio Alostérico , Sequência de Aminoácidos , Antivirais/farmacologia , Sítios de Ligação , Dados de Sequência Molecular , Conformação Proteica , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Transfer of the tumor-inducing plasmid in Agrobacterium tumefaciens is controlled by a quorum-sensing system whose main components are the transcriptional regulator TraR and its autoinducer. This system allows bacteria to synchronize infection of the host plant when a "quorum" of cells has been reached. TraM is an A. tumefaciens protein involved in the regulation of this system because it binds to TraR and prevents it from binding DNA. As a first step to understanding the molecular basis for the regulation of TraR by TraM, we have determined the crystal structure of TraM at 1.65 A resolution. This protein is packed as a dimer, with each monomer consisting mainly of two antiparallel alpha helices. Monomers are tightly associated, with a large hydrophobic area buried upon dimerization. Secondly, we characterized the TraR-TraM complex in vitro. TraM (11.4 kDa, monomer molecular mass) binds tightly TraR (27 kDa, monomer molecular mass) forming a stable oligomeric complex that likely accounts for two TraR and two TraM dimers.
Assuntos
Proteínas de Bactérias/química , Agrobacterium tumefaciens/metabolismo , Cromatografia , Cristalografia por Raios X , Dimerização , Elétrons , Cinética , Luz , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Espalhamento de RadiaçãoRESUMO
TraM is a 11.4 kDa protein involved in the control of the conjugal transfer of Agrobacterium tumefaciens Ti plasmids by quorum-sensing. TraM was overexpressed and purified from Escherichia coli. This protein binds to the transcriptional regulator TraR, abolishing its function. Size-exclusion chromatography and dynamic light scattering show that the recombinant protein has an apparent molecular weight of 30 kDa in solution. Crystals have been obtained of both native and selenomethionine-substituted TraM by the vapour-diffusion method. Crystals diffract to 1.67 A and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 76.43, b = 47.09, c = 47.46 A and two molecules in the asymmetric unit. A two-wavelength MAD data set for the selenomethionine-substituted form has been collected to a resolution of 2.0 A. The selenium substructure (five out of six possible sites) has been solved using direct methods.
Assuntos
Agrobacterium tumefaciens/química , Proteínas de Bactérias/química , Agrobacterium tumefaciens/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cromatografia em Gel , Clonagem Molecular , Cristalização , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Luz , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Espalhamento de Radiação , Selenometionina/química , Alinhamento de SequênciaRESUMO
An electrospray mass spectrometry-based methodology has been developed to have a fast and sensitive method for protein-cofactor stoichiometry determination. As model systems, we used two proteins which require the presence of cofactors for activity: TraR, a member of the LuxR family of quorum-sensing transcriptional regulators, which requires an acyl-homoserine lactone molecule called Agrobacterium autoinducer (AAI) as coinducer and the NS3 protease of hepatitis C virus which complexes with a NS4A cofactor peptide. Both TraR/AAI and NS3/NS4A are noncovalent complexes. Our method requires only nanomolar concentration of sample. A calibration curve of the cofactor is determined by high-performance liquid chromatography (HPLC) coupled on-line with an ion trap mass spectrometer operated in selected reaction monitoring mode. Subsequently, the complex is analyzed using the same experimental setup. During the HPLC run, the complex dissociates, and cofactor and protein elute at different retention times. The peak area of the cofactor is integrated and the molar concentration of cofactor in the complex is extrapolated from the calibration curve. The stoichiometry is consequently calculated by dividing the molar concentration of protein injected by that of cofactor measured. Both TraR/AAI and NS3/NS4A complexes have 1:1 stoichiometries, in line with those already reported in the literature.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Hepacivirus/enzimologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/isolamento & purificação , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Calibragem , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Homosserina/análogos & derivados , Homosserina/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/isolamento & purificaçãoRESUMO
TraR is an Agrobacterium tumefaciens transcriptional regulator which binds the pheromone N-3-oxooctanoyl-L-homoserine lactone (AAI) in response to the bacterial population density. The TraR-AAI complex dimerizes and interacts with a specific 18-base-pair DNA sequence (TraBox), activating promoters containing this site. TraR was overexpressed and purified from Escherichia coli. Crystals of the ternary complex, in which dimeric TraR-AAI is bound to the TraBox sequence, have been obtained by the vapour-diffusion method. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 66.99, b = 94.67, c = 209.66 A, with two (TraR-AAI)(2)-TraBox complexes in the asymmetric unit. A three-wavelength MAD data set for the seleno-L-methionine-substituted form has been collected to a resolution of 3 A. 20 of the 24 crystallographically independent selenium sites were located as part of the MAD-phasing process.
Assuntos
Proteínas de Bactérias/química , Homosserina/análogos & derivados , Agrobacterium tumefaciens/química , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Sítios de Ligação , Cristalização , Cristalografia por Raios X , DNA/química , Homosserina/química , Substâncias Macromoleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SelênioRESUMO
The quorum sensing system allows bacteria to sense their cell density and initiate an altered pattern of gene expression after a sufficient quorum of cells has accumulated. In Agrobacterium tumefaciens, quorum sensing controls conjugal transfer of the tumour- inducing plasmid, responsible for plant crown gall disease. The core components of this system are the transcriptional regulator TraR and its inducing ligand N-(3-oxo-octanoyl)-L-homoserine lactone. This complex binds DNA and activates gene expression. We have determined the crystal structure of TraR in complex with its autoinducer and target DNA (PDB code 1h0m). The protein is dimeric, with each monomer composed of an N-terminal domain, which binds the ligand in an enclosed cavity far from the dimerization region, and a C-terminal domain, which binds DNA via a helix-turn-helix motif. The structure reveals an asymmetric homodimer, with one monomer longer than the other. The N-terminal domain resembles GAF/PAS domains, normally fused to catalytic signalling domains. In TraR, the gene fusion is between a GAF/PAS domain and a DNA-binding domain, resulting in a specific transcriptional regulator involved in quorum sensing.
Assuntos
Agrobacterium tumefaciens/química , Proteínas de Bactérias/química , Homosserina/análogos & derivados , Fatores de Transcrição/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Conjugação Genética , Cristalografia por Raios X , DNA Bacteriano/metabolismo , Dimerização , Evolução Molecular , Homosserina/química , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transativadores/químicaRESUMO
Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single alpha/beta domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.