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1.
Nature ; 592(7853): 283-289, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33524990

RESUMEN

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).


Asunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Modelos Animales de Enfermedad , SARS-CoV-2/inmunología , Envejecimiento/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Vacuna BNT162 , COVID-19/sangre , COVID-19/terapia , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/química , Vacunas contra la COVID-19/genética , Línea Celular , Ensayos Clínicos como Asunto , Femenino , Humanos , Inmunización Pasiva , Internacionalidad , Macaca mulatta/inmunología , Macaca mulatta/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Multimerización de Proteína , ARN Viral/análisis , Sistema Respiratorio/inmunología , Sistema Respiratorio/virología , SARS-CoV-2/química , SARS-CoV-2/genética , Solubilidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T/inmunología , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/química , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Sueroterapia para COVID-19 , Vacunas de ARNm
2.
Biophys J ; 117(11): 2228-2239, 2019 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-31703801

RESUMEN

Although the three-dimensional structures of G-protein coupled receptors (GPCRs), the largest superfamily of drug targets, have enabled structure-based drug design, there are no structures available for 87% of GPCRs. This is due to the stiff challenge in purifying the inherently flexible GPCRs. Identifying thermostabilized mutant GPCRs via systematic alanine scanning mutations has been a successful strategy in stabilizing GPCRs, but it remains a daunting task for each GPCR. We developed a computational method that combines sequence-, structure-, and dynamics-based molecular properties of GPCRs that recapitulate GPCR stability, with four different machine learning methods to predict thermostable mutations ahead of experiments. This method has been trained on thermostability data for 1231 mutants, the largest publicly available data set. A blind prediction for thermostable mutations of the complement factor C5a receptor 1 retrieved 36% of the thermostable mutants in the top 50 prioritized mutants compared to 3% in the first 50 attempts using systematic alanine scanning.


Asunto(s)
Simulación de Dinámica Molecular , Mutación , Receptor de Anafilatoxina C5a/química , Análisis de Secuencia/métodos , Alanina/química , Alanina/genética , Sustitución de Aminoácidos , Células HEK293 , Humanos , Aprendizaje Automático , Dominios Proteicos , Estabilidad Proteica , Receptor de Anafilatoxina C5a/genética
3.
J Biol Chem ; 287(44): 37321-9, 2012 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-22961980

RESUMEN

Human plasma cholesteryl ester transfer protein (CETP) transports cholesteryl ester from the antiatherogenic high-density lipoproteins (HDL) to the proatherogenic low-density and very low-density lipoproteins (LDL and VLDL). Inhibition of CETP has been shown to raise human plasma HDL cholesterol (HDL-C) levels and is potentially a novel approach for the prevention of cardiovascular diseases. Here, we report the crystal structures of CETP in complex with torcetrapib, a CETP inhibitor that has been tested in phase 3 clinical trials, and compound 2, an analog from a structurally distinct inhibitor series. In both crystal structures, the inhibitors are buried deeply within the protein, shifting the bound cholesteryl ester in the N-terminal pocket of the long hydrophobic tunnel and displacing the phospholipid from that pocket. The lipids in the C-terminal pocket of the hydrophobic tunnel remain unchanged. The inhibitors are positioned near the narrowing neck of the hydrophobic tunnel of CETP and thus block the connection between the N- and C-terminal pockets. These structures illuminate the unusual inhibition mechanism of these compounds and support the tunnel mechanism for neutral lipid transfer by CETP. These highly lipophilic inhibitors bind mainly through extensive hydrophobic interactions with the protein and the shifted cholesteryl ester molecule. However, polar residues, such as Ser-230 and His-232, are also found in the inhibitor binding site. An enhanced understanding of the inhibitor binding site may provide opportunities to design novel CETP inhibitors possessing more drug-like physical properties, distinct modes of action, or alternative pharmacological profiles.


Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/química , Fluorocarburos/química , Quinolinas/química , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Proteínas de Transferencia de Ésteres de Colesterol/genética , Cristalografía por Rayos X , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína
4.
Protein Expr Purif ; 87(1): 27-34, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23069765

RESUMEN

The T helper cell-derived cytokine interleukin-17A (IL-17A) is a variably glycosylated disulfide-linked homodimer of 34-38 kDa. Its polypeptide monomer contains one canonical N-glycosylation site at Asn68, and human recombinant IL-17A was partly N-glycosylated when expressed in human kidney (HEK293) cells as a fusion protein with a melittin signal sequence and an N-terminal hexahistidine tag. Orbitrap mass analyses of the tryptic N-glycopeptide 63-69 indicated that the N-glycosylation was of the GalNAc-terminated type characteristic of cultured kidney cells. The mass spectrum of IL-17A monomer also included peaks shifted by +948 Da from the respective masses of unglycosylated and N-glycosylated polypeptides. These were caused by unpredicted partial O-glycosylation of Thr26 with the mucin-like structure -GalNAc(-NeuNAc)-Gal-NeuNAc. Identical O-glycosylation occurred in commercially sourced recombinant IL-17A also expressed in HEK293 cells but with a different N-terminal sequence. Therefore, the kidney host cell line not only imposed its characteristic pattern of N-glycosylation on recombinant IL-17A but additionally created an O-glycosylation not known to be present in the T cell-derived cytokine. Mammalian host cell lines for recombinant protein expression generally impose their characteristic patterns of N-glycosylation on the product, but this work exemplifies how a host may also unpredictably O-glycosylate a protein that is probably not normally O-glycosylated.


Asunto(s)
Interleucina-17/biosíntesis , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicosilación , Células HEK293 , Humanos , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/química , Interleucina-17/química , Meliteno/biosíntesis , Meliteno/química , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Señales de Clasificación de Proteína , Proteínas Recombinantes de Fusión/química , Espectrometría de Masas en Tándem
5.
Nat Struct Mol Biol ; 30(1): 22-30, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36522428

RESUMEN

Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.


Asunto(s)
Hígado , Membranas Mitocondriales , Humanos , Hígado/metabolismo , Membranas Mitocondriales/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/química , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Secuencia de Aminoácidos
6.
J Am Chem Soc ; 134(4): 1978-81, 2012 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-22280495

RESUMEN

The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.


Asunto(s)
Acetilgalactosamina/química , Receptor de Asialoglicoproteína/química , Acetilgalactosamina/análogos & derivados , Humanos , Ligandos , Estructura Molecular , Estereoisomerismo
7.
Bioorg Med Chem Lett ; 22(24): 7523-9, 2012 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-23153798

RESUMEN

Previous drug discovery efforts identified classical PYK2 kinase inhibitors such as 2 and 3 that possess selectivity for PYK2 over its intra-family isoform FAK. Efforts to identify more kinome-selective chemical matter that stabilize a DFG-out conformation of the enzyme are described herein. Two sub-series of PYK2 inhibitors, an indole carboxamide-urea and a pyrazole-urea have been identified and found to have different binding interactions with the hinge region of PYK2. These leads proved to be more selective than the original classical inhibitors.


Asunto(s)
Quinasa 2 de Adhesión Focal/antagonistas & inhibidores , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Urea/farmacología , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Quinasa 2 de Adhesión Focal/metabolismo , Células HEK293 , Humanos , Indoles/síntesis química , Indoles/química , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirazoles/síntesis química , Pirazoles/química , Ratas , Relación Estructura-Actividad , Urea/análogos & derivados , Urea/química
8.
Nat Struct Mol Biol ; 14(5): 413-9, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17435765

RESUMEN

Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.


Asunto(s)
Hipercolesterolemia/genética , Mutación Missense/fisiología , Serina Endopeptidasas/metabolismo , Sitios de Unión , Humanos , Concentración de Iones de Hidrógeno , Hipercolesterolemia/etiología , Proproteína Convertasa 9 , Proproteína Convertasas , Unión Proteica/genética , Conformación Proteica , Receptores de LDL/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/genética
9.
Nat Struct Mol Biol ; 14(2): 106-13, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17237796

RESUMEN

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.


Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/química , Ésteres del Colesterol/química , Modelos Moleculares , Fosfatidilcolinas/química , Triglicéridos/química , Animales , Sitios de Unión , Células CHO , Proteínas de Transferencia de Ésteres de Colesterol/genética , Cricetinae , Cricetulus , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Mutación Puntual , Unión Proteica , Conformación Proteica
10.
J Med Chem ; 65(12): 8208-8226, 2022 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-35647711

RESUMEN

Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited because they require injection. Herein, we describe the discovery of the orally bioavailable, small-molecule, GLP-1R agonist PF-06882961 (danuglipron). A sensitized high-throughput screen was used to identify 5-fluoropyrimidine-based GLP-1R agonists that were optimized to promote endogenous GLP-1R signaling with nanomolar potency. Incorporation of a carboxylic acid moiety provided considerable GLP-1R potency gains with improved off-target pharmacology and reduced metabolic clearance, ultimately resulting in the identification of danuglipron. Danuglipron increased insulin levels in primates but not rodents, which was explained by receptor mutagensis studies and a cryogenic electron microscope structure that revealed a binding pocket requiring a primate-specific tryptophan 33 residue. Oral administration of danuglipron to healthy humans produced dose-proportional increases in systemic exposure (NCT03309241). This opens an opportunity for oral small-molecule therapies that target the well-validated GLP-1R for metabolic health.


Asunto(s)
Receptor del Péptido 1 Similar al Glucagón , Hipoglucemiantes , Animales , Receptor del Péptido 1 Similar al Glucagón/agonistas , Humanos , Hipoglucemiantes/farmacología , Péptidos/química
11.
Nat Commun ; 11(1): 3031, 2020 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-32541785

RESUMEN

Chemokines are important protein-signaling molecules that regulate various immune responses by activating chemokine receptors which belong to the G protein-coupled receptor (GPCR) superfamily. Despite the substantial progression of our structural understanding of GPCR activation by small molecule and peptide agonists, the molecular mechanism of GPCR activation by protein agonists remains unclear. Here, we present a 3.3-Å cryo-electron microscopy structure of the human chemokine receptor CCR6 bound to its endogenous ligand CCL20 and an engineered Go. CCL20 binds in a shallow extracellular pocket, making limited contact with the core 7-transmembrane (TM) bundle. The structure suggests that this mode of binding induces allosterically a rearrangement of a noncanonical toggle switch and the opening of the intracellular crevice for G protein coupling. Our results demonstrate that GPCR activation by a protein agonist does not always require substantial interactions between ligand and the 7TM core region.


Asunto(s)
Quimiocina CCL20/metabolismo , Receptores CCR6/química , Receptores CCR6/metabolismo , Quimiocina CCL20/química , Quimiocina CCL20/genética , Microscopía por Crioelectrón , Humanos , Ligandos , Unión Proteica , Receptores CCR6/genética , Receptores Acoplados a Proteínas G , Transducción de Señal
12.
Sci Rep ; 10(1): 8974, 2020 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-32488071

RESUMEN

Translation of modulation of drug target activity to therapeutic effect is a critical aspect for all drug discovery programs. In this work we describe the profiling of a non-receptor tyrosine-protein kinase (TYK2) inhibitor which shows a functionally relevant potency shift between human and preclinical species (e.g. murine, dog, macaque) in both biochemical and cellular assays. Comparison of the structure and sequence homology of TYK2 between human and preclinical species within the ATP binding site highlights a single amino acid (I960 → V) responsible for the potency shift. Through TYK2 kinase domain mutants and a TYK2 980I knock-in mouse model, we demonstrate that this single amino acid change drives a functionally relevant potency difference that exists between human and all evaluated preclinical species, for a series of TYK2 inhibitors which target the ATP binding site.


Asunto(s)
Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , TYK2 Quinasa/antagonistas & inhibidores , TYK2 Quinasa/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Perros , Humanos , Janus Quinasa 1 , Macaca , Ratones , Mutación , Dominios Proteicos/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , TYK2 Quinasa/genética , TYK2 Quinasa/metabolismo
13.
Biochemistry ; 48(13): 2941-9, 2009 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-19222187

RESUMEN

Proprotein convertase subtilisin-kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLR) on target cells and lowers the level of receptor by impeding its recycling. PCSK9 is self-processed to a complex of its prodomain and catalytic domain like a typical protein convertase, but it does not develop normal proteolytic activity. Instead, its propeptide remains complexed with the catalytic domain, and the C-terminal Gln152 of the prodomain occupies the active site like a substrate for peptide synthesis. To probe its latent catalytic activity, PCSK9 and its complex with the soluble LDLR extracellular domain were separately transferred into H218O, and time point samples were analyzed by peptide mapping with mass spectrometry to measure the rate and extent of incorporation of 18O into the Gln152 carboxylate. In free wild-type or D374Y mutant PCSK9, the t1/2 for exchange of 18O for both oxygens was near 5 min. This slow process progressed to completion, with the distribution of oxygen isotopes in the Gln152 carboxylate finally matching that in solvent. In contrast, exchange reached its final state in <30 s in LDLR-complexed D374Y mutant PCSK9, but approximately 40% of the molecules gave data indicating the presence of only one 18O atom in Gln152. With support from further experiments, this was attributed to hydrolysis of acylenzyme in H216O during preparations for digestion and indicated that PCSK9 complexed with LDLR contains approximately 40% intramolecular acylenzyme at equilibrium. The synthetic EGF-A domain of LDLR induced similar effects as the full-length receptor. The data suggest the existence of distinct conformational states in free and receptor-bound PCSK9.


Asunto(s)
Biocatálisis , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Glutamina/metabolismo , Humanos , Isótopos , Espectrometría de Masas , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Péptidos/química , Péptidos/metabolismo , Proproteína Convertasa 9 , Proproteína Convertasas , Serina Endopeptidasas/química , Ciclo del Sustrato
14.
Bioorg Med Chem Lett ; 18(23): 6071-7, 2008 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-18951788

RESUMEN

The synthesis and SAR for a series of diaminopyrimidines as PYK2 inhibitors are described. Using a combination of library and traditional medicinal chemistry techniques, a FAK-selective chemical series was transformed into compounds possessing good PYK2 potency and 10- to 20-fold selectivity against FAK. Subsequent studies found that the majority of the compounds were positive in a reactive metabolite assay, an indicator for potential toxicological liabilities. Based on the proposed mechanism for bioactivation, as well as a combination of structure-based drug design and traditional medicinal chemistry techniques, a follow-up series of PYK2 inhibitors was identified that maintained PYK2 potency, FAK selectivity and HLM stability, yet were negative in the RM assay.


Asunto(s)
Quinasa 2 de Adhesión Focal/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirimidinas/farmacología , Animales , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Diseño de Fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Humanos , Conformación Molecular , Estructura Molecular , Osteoporosis/tratamiento farmacológico , Pirimidinas/química , Ratas , Relación Estructura-Actividad
15.
Nat Commun ; 4: 1888, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23695682

RESUMEN

The constituent polypeptides of the interleukin-17 family form six different homodimeric cytokines (IL-17A-F) and the heterodimeric IL-17A/F. Their interactions with IL-17 receptors A-E (IL-17RA-E) mediate host defenses while also contributing to inflammatory and autoimmune responses. IL-17A and IL-17F both preferentially engage a receptor complex containing one molecule of IL-17RA and one molecule of IL-17RC. More generally, IL-17RA appears to be a shared receptor that pairs with other members of its family to allow signaling of different IL-17 cytokines. Here we report crystal structures of homodimeric IL-17A and its complex with IL-17RA. Binding to IL-17RA at one side of the IL-17A molecule induces a conformational change in the second, symmetry-related receptor site of IL-17A. This change favors, and is sufficient to account for, the selection of a different receptor polypeptide to complete the cytokine-receptor complex. The structural results are supported by biophysical studies with IL-17A variants produced by site-directed mutagenesis.


Asunto(s)
Interleucina-17/química , Receptores de Interleucina-17/química , Regulación Alostérica , Secuencia de Aminoácidos , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Células HEK293 , Humanos , Interleucina-17/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Interleucina-17/metabolismo , Resonancia por Plasmón de Superficie
16.
Biochemistry ; 42(11): 3203-13, 2003 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-12641451

RESUMEN

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.


Asunto(s)
Catepsinas/metabolismo , Secuencia de Bases , Catepsinas/antagonistas & inhibidores , Catepsinas/química , Catepsinas/aislamiento & purificación , Cristalografía por Rayos X , Cartilla de ADN , Humanos , Modelos Moleculares , Inhibidores de Proteasas/farmacología , Conformación Proteica , Especificidad por Sustrato
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