RESUMEN
Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/química , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Tiadiazoles/química , Tiadiazoles/farmacología , Secuencia de Aminoácidos , Membrana Celular/química , Cristalización/métodos , Cristalografía por Rayos X , Análisis Mutacional de ADN , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.7/genética , Percepción del Dolor/efectos de los fármacos , Ingeniería de Proteínas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Protein ubiquitination patterns are an important component of cellular signaling. The WD-repeat protein WDR48 (USP1-associated factor UAF-1) stimulates activity of ubiquitin-specific proteases USP1, USP12, and USP46. To understand how WDR48 exerts its effect on the USP scaffold, we determined structures of the ternary WDR48:USP46:ubiquitin complex. WDR48 interacts with the USP46 fingers subdomain via a relatively small, highly polar surface on the top center of the WDR48 ß propeller. In addition, WDR48 has a novel ancillary domain and a C-terminal SUMO-like domain encircling the USP46-bound ubiquitin. Mutation of residues involved in the WDR48:USP46 interaction abrogated both binding and deubiquitinase activity of the complex. An analogous mutation in USP1 similarly blocked WDR48-dependent activation. Our data suggest a possible mechanism of deubiquitinase stimulation via stabilization and prolonged residence time of substrate. The unprecedented mode of interaction between the USP fingers domain and the WD-repeat ß propeller serves as a prototypical example for this family of deubiquitinases.
Asunto(s)
Endopeptidasas/química , Proteínas/química , Secuencia de Aminoácidos , Sitios de Unión , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas/genética , Proteínas/metabolismoRESUMEN
Bacterial ATP-binding cassette (ABC) importers play critical roles in nutrient acquisition and are potential antibacterial targets. However, structural bases for their inhibition are poorly defined. These pathways typically rely on substrate binding proteins (SBPs), which are essential for substrate recognition, delivery, and transporter function. We report the crystal structure of a Staphylococcus aureus SBP for Mn(II), termed MntC, in complex with FabC1, a potent antibody inhibitor of the MntABC pathway. This pathway is essential and highly expressed during S. aureus infection and facilitates the import of Mn(II), a critical cofactor for enzymes that detoxify reactive oxygen species (ROS). Structure-based functional studies indicate that FabC1 sterically blocks a structurally conserved surface of MntC, preventing its interaction with the MntB membrane importer and increasing wild-type S. aureus sensitivity to oxidative stress by more than 10-fold. The results define an SBP blocking mechanism as the basis for ABC importer inhibition by an engineered antibody fragment.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Fragmentos de Inmunoglobulinas/farmacología , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/inmunología , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/inmunología , Sitios de Unión , Fragmentos de Inmunoglobulinas/química , Datos de Secuencia Molecular , Unión Proteica , Staphylococcus aureus/enzimologíaRESUMEN
Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family of nonreceptor tyrosine kinases, which are essential for proper signaling in immune responses and development. Here we present a 2.0-Å-resolution crystal structure of a receptor-binding fragment of human TYK2, encompassing the FERM and SH2 domains, in complex with a so-called 'box2'-containing intracellular peptide motif from the interferon-α receptor chain 1 (IFNAR1). The TYK2-IFNAR1 interface reveals an unexpected receptor-binding mode that mimics a SH2 domain-phosphopeptide interaction, with a glutamate replacing the canonical phosphotyrosine residue. This structure provides the first view, to our knowledge, of a JAK in complex with its cognate receptor and defines the molecular logic through which JAKs have evolved to interact with divergent receptor sequences.
Asunto(s)
Interferón-alfa/química , TYK2 Quinasa/química , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Interferón-alfa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , TYK2 Quinasa/metabolismoRESUMEN
Homotrimeric TNF superfamily ligands signal by inducing trimers of their cognate receptors. As a biologically active heterotrimer, Lymphotoxin(LT)α1ß2 is unique in the TNF superfamily. How the three unique potential receptor-binding interfaces in LTα1ß2 trigger signaling via LTß Receptor (LTßR) resulting in lymphoid organogenesis and propagation of inflammatory signals is poorly understood. Here we show that LTα1ß2 possesses two binding sites for LTßR with distinct affinities and that dimerization of LTßR by LTα1ß2 is necessary and sufficient for signal transduction. The crystal structure of a complex formed by LTα1ß2, LTßR, and the fab fragment of an antibody that blocks LTßR activation reveals the lower affinity receptor-binding site. Mutations targeting each potential receptor-binding site in an engineered single-chain variant of LTα1ß2 reveal the high-affinity site. NF-κB reporter assays further validate that disruption of receptor interactions at either site is sufficient to prevent signaling via LTßR.
Asunto(s)
Citocinas/química , Heterotrímero de Linfotoxina alfa1 y beta2/metabolismo , Receptor beta de Linfotoxina/metabolismo , Complejos Multiproteicos/inmunología , Transducción de Señal/inmunología , Cromatografía en Gel , Citocinas/inmunología , Dimerización , Humanos , Complejos Multiproteicos/metabolismoRESUMEN
Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Fragmentos Fab de Inmunoglobulinas/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The NF-κB inducing kinase (NIK) regulates the non-canonical NF-κB pathway downstream of important clinical targets including BAFF, RANKL, and LTß. Despite numerous genetic studies associating dysregulation of this pathway with autoimmune diseases and hematological cancers, detailed molecular characterization of this central signaling node has been lacking. We undertook a systematic cloning and expression effort to generate soluble, well-behaved proteins encompassing the kinase domains of human and murine NIK. Structures of the apo NIK kinase domain from both species reveal an active-like conformation in the absence of phosphorylation. ATP consumption and peptide phosphorylation assays confirm that phosphorylation of NIK does not increase enzymatic activity. Structures of murine NIK bound to inhibitors possessing two different chemotypes reveal conformational flexibility in the gatekeeper residue controlling access to a hydrophobic pocket. Finally, a single amino acid difference affects the ability of some inhibitors to bind murine and human NIK with the same affinity.
Asunto(s)
Proteínas Serina-Treonina Quinasas/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fosforilación , Inhibidores de Proteínas Quinasas/química , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Secundaria de Proteína , Homología Estructural de Proteína , Quinasa de Factor Nuclear kappa BRESUMEN
Tank-binding kinase (TBK)1 plays a central role in innate immunity: it serves as an integrator of multiple signals induced by receptor-mediated pathogen detection and as a modulator of IFN levels. Efforts to better understand the biology of this key immunological factor have intensified recently as growing evidence implicates aberrant TBK1 activity in a variety of autoimmune diseases and cancers. Nevertheless, key molecular details of TBK1 regulation and substrate selection remain unanswered. Here, structures of phosphorylated and unphosphorylated human TBK1 kinase and ubiquitin-like domains, combined with biochemical studies, indicate a molecular mechanism of activation via transautophosphorylation. These TBK1 structures are consistent with the tripartite architecture observed recently for the related kinase IKKß, but domain contributions toward target recognition appear to differ for the two enzymes. In particular, both TBK1 autoactivation and substrate specificity are likely driven by signal-dependent colocalization events.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Catálisis , Activación Enzimática , Humanos , Modelos Moleculares , Fosforilación , Conformación Proteica , Proteínas Serina-Treonina Quinasas/químicaRESUMEN
Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands. The docking adaptations of the IL-34 and CSF-1 quaternary complexes, when compared to the stem cell factor assembly, draw a common evolutionary theme for transmembrane signaling. In addition, the structure of IL-34 engaged by a Fab fragment reveals the mechanism of a neutralizing antibody that can help deconvolute IL-34 from CSF-1 biology, with implications for therapeutic intervention in diseases with myeloid pathogenic mechanisms.
Asunto(s)
Anticuerpos Neutralizantes/química , Interleucinas/química , Factor Estimulante de Colonias de Macrófagos/química , Receptor de Factor Estimulante de Colonias de Macrófagos/química , Homología Estructural de Proteína , Baculoviridae , Sitios de Unión , Cristalografía por Rayos X , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Interleucinas/antagonistas & inhibidores , Interleucinas/genética , Cinética , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Factor Estimulante de Colonias de Macrófagos/genética , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-kit/química , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transducción de Señal/genética , Factor de Células Madre/química , TermodinámicaRESUMEN
The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.
Asunto(s)
Nucleótidos/metabolismo , Proteínas Son Of Sevenless/metabolismo , Proteínas ras/metabolismo , Sitios de Unión , Línea Celular , Humanos , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas ras/químicaRESUMEN
Addition and removal of ubiquitin or ubiquitin chains to and from proteins is a tightly regulated process that contributes to cellular signaling and protein stability. Here we show that phosphorylation of the human deubiquitinase DUBA (OTUD5) at a single residue, Ser177, is both necessary and sufficient to activate the enzyme. The crystal structure of the ubiquitin aldehyde adduct of active DUBA reveals a marked cooperation between phosphorylation and substrate binding. An intricate web of interactions involving the phosphate and the C-terminal tail of ubiquitin cause DUBA to fold around its substrate, revealing why phosphorylation is essential for deubiquitinase activity. Phosphoactivation of DUBA represents an unprecedented mode of protease regulation and a clear link between two major cellular signal transduction systems: phosphorylation and ubiquitin modification.
Asunto(s)
Endopeptidasas/metabolismo , Procesamiento Proteico-Postraduccional , Serina/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Fosforilación , Conformación Proteica , Ubiquitina/metabolismoRESUMEN
A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs. Interestingly, only one of these ten classes consisted of peptides containing the DXL motif. Nevertheless, all classes of peptides prevented APRIL, but not BAFF, from binding BCMA, their shared receptor. Synthetic peptides based on selected sequences inhibited APRIL binding to BCMA with IC(50) values of 0.49-27 microM. An X-ray crystallographic structure of APRIL bound to one of the phage-derived peptides showed that the peptide, lacking the DXL motif, was nevertheless bound in the DXL pocket on APRIL. Our results demonstrate that even though a focused, highly conserved motif is required for APRIL-receptor interaction, remarkably, many novel and distinct classes of peptides are also capable of binding APRIL at the ligand receptor interface.
Asunto(s)
Biblioteca de Péptidos , Péptidos/clasificación , Péptidos/aislamiento & purificación , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/antagonistas & inhibidores , Alanina/metabolismo , Secuencia de Aminoácidos , Animales , Antígeno de Maduración de Linfocitos B/química , Antígeno de Maduración de Linfocitos B/metabolismo , Proteínas Inmovilizadas/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis/genética , Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Solubilidad , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/química , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
The pH-dependent binding of Igs to the neonatal FcR (FcRn) plays a critical role in the in vivo homeostasis of IgGs. Modulating the interaction between Fc and FcRn through protein engineering is one method for improving the pharmacokinetics of therapeutic Abs. Recent studies disputed the direct relationship between increasing FcRn affinity and improved pharmacokinetic properties. In this work, we studied the pharmacokinetics of two human IgG1 Fc variants in cynomolgus monkey to further clarify the affinity-pharmacokinetic relationship. First, we report a number of novel Fc point mutations and combination variants, including some with primate-specific FcRn-binding improvements. By studying these variants along with some previously described variants across a wide range of affinities, we discovered a direct correlation of pH 6 affinity improvements with neutral pH improvements, suggesting that all of the tested variants exhibit similar pH dependency in FcRn binding. We then evaluated the pharmacokinetics of variants N434A and N434W, which, respectively, gave approximately 4- and 80-fold improvements in pH 6-binding affinity to both human and nonhuman primate FcRn. Surprisingly, clearance of N434W was similar to that of wild type. N434W is the first variant studied in primates that exhibits significant binding to FcRn at pH 7.4, and its clearance substantiates the principle that too much affinity improvement, i.e., beyond that of N434W, does not yield improved pharmacokinetics. In contrast, N434A exhibited a approximately 2-fold decrease in clearance in cynomolgus monkey, supporting the notion that modest increases in pH 6 FcRn affinity can result in improved pharmacokinetics in primates.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Macaca fascicularis/inmunología , Receptores Fc/inmunología , Secuencia de Aminoácidos , Animales , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Ratones , Modelos Moleculares , Mutación/genética , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Removal of pathogenic B lymphocytes by depletion of monoclonal antibodies (mAbs) or deprivation of B-cell survival factors has demonstrated clinical benefit in both oncologic and immunologic diseases. Partial clinical responses and emerging data demonstrating incomplete B-cell depletion after immunotherapy fuels the need for improved therapeutic modalities. Lessons from the first generation of therapeutics directed against B-cell-specific antigens (CD20, CD22) are being applied to develop novel antibodies with additional functional attributes. We describe the generation of a novel class of B-cell-directed therapy (anti-BR3 mAbs) that combines the depleting capacity of a therapeutic mAb and blockade of B-cell-activating factor (BAFF)-BR3 B-cell survival. In mice, treatment with antagonistic anti-BR3 antibodies results in quantitatively greater reduction in some B-cell subsets and qualitatively different effects on bone marrow plasma cells compared with BR3-Fc BAFF blockade or with anti-CD20 treatment. Comparative analysis of BR3-Fc and anti-BR3 mAb reveals a lower B-cell dependence for BAFF-mediated survival in nonhuman primates than in mice. This novel class of B-cell-targeted therapies shows species characteristics in mice and primates that will guide translation to treatment of human disease.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Receptor del Factor Activador de Células B/antagonistas & inhibidores , Enfermedades del Sistema Inmune/tratamiento farmacológico , Inmunoterapia , Depleción Linfocítica , Neoplasias/tratamiento farmacológico , Células Plasmáticas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Factor Activador de Células B/antagonistas & inhibidores , Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/inmunología , Células de la Médula Ósea/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Enfermedades del Sistema Inmune/inmunología , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Neoplasias/inmunología , Especificidad de la EspecieRESUMEN
BR3, which is expressed on all mature B cells, is a specific receptor for the B-cell survival and maturation factor BAFF (B-cell-activating factor belonging to the tumor necrosis factor [TNF] family). In order to investigate the consequences of targeting BR3 in murine models and to assess the potential of BR3 antibodies as human therapeutics, synthetic antibody phage libraries were employed to identify BAFF-blocking antibodies cross-reactive to murine and human BR3, which share 52% identity in their extracellular domains. We found an antibody, CB1, which exhibits muM affinity for murine BR3 and very weak affinity for the human receptor. CB3s, an affinity-matured variant of CB1, has sub-nM affinity for BR3 from both species. Alanine scanning and crystallographic structural analysis of the CB3s/BR3 complex reveal that CB3s mimics BAFF by interacting with a similar region of the BR3 surface. Despite this similarity in binding epitopes, CB1 variants antagonize BAFF-dependent human B-cell proliferation in vitro and are effective at reducing murine B-cell populations in vivo, showing significant promise as therapeutics for human B-cell-mediated diseases.
Asunto(s)
Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/química , Sitios de Unión , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Activación de Linfocitos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing. We find that this shorter form is capable of ligand-induced cell signaling and that the second CRD alone (TACI_d2) contains full affinity for both ligands. Furthermore, we report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with co-crystal structures of APRIL.TACI_d2 and APRIL.BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single CRD, and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system.
Asunto(s)
Cisteína , Proteínas de la Membrana/química , Receptores del Factor de Necrosis Tumoral/química , Factor de Necrosis Tumoral alfa/química , Empalme Alternativo , Animales , Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Cristalización , Cristalografía por Rayos X , Humanos , Ligandos , Proteínas de la Membrana/genética , Ratones , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/genética , Transducción de Señal , Soluciones , Proteína Activadora Transmembrana y Interactiva del CAML , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis TumoralRESUMEN
A proliferation-inducing ligand (APRIL) is a TNF-like cytokine that stimulates tumor cell growth. Within the TNF ligand superfamily, APRIL is most similar to B-cell activation factor (BAFF) with which it shares 30% sequence identity. APRIL binds the receptors B-cell maturation antigen (BCMA) and TACI with high affinity; both of these receptors have also been shown to bind BAFF, although BCMA has significantly higher affinity for APRIL than BAFF. Determination of the crystal structure of APRIL from three crystallization conditions at resolutions of 1.8-2.4A over a pH range from 5.0 to 8.5 reveals a compact trimeric ligand with a backbone fold very similar to that of BAFF (1.1A RMSD over 122 structurally equivalent Calpha atoms), with the exception of differences in the AA' and DE loop regions. Whereas BAFF has been shown to form 20-trimer assemblies under certain conditions, the molecular determinants required for BAFF-like assemblies are absent in the APRIL structure. No crystal packing suggestive of the formation of higher-order assemblies is seen in any of the crystal forms nor does the structure vary significantly between pH 5.0 and 8.5. Modeling of the APRIL-BCMA complex shows the resulting interface is in agreement with mutagenesis data.
Asunto(s)
Proteínas de la Membrana/química , Estructura Terciaria de Proteína , Factor de Necrosis Tumoral alfa/química , Secuencia de Aminoácidos , Animales , Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Cristalografía por Rayos X , Humanos , Proteínas de la Membrana/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Cuaternaria de Proteína , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/genética , Alineación de Secuencia , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Two structurally distinct classes of peptides were recently identified by phage display that bind the high-affinity IgE receptor, FcepsilonRI, and block IgE binding and subsequent receptor activation. Both classes adopt highly stable structures in solution, one forming a beta hairpin, with the other forming a helical "zeta" structure. Despite these differences, the two classes bind competitively to the same site on the receptor. Structural analyses of both peptide-receptor complexes by NMR spectroscopy and/or X-ray crystallography reveal that the unrelated peptide scaffolds have nevertheless converged to present a similar three-dimensional surface to interact with FcepsilonRI and that their modes of interaction share a key feature of the IgE-FcepsilonRI complex, the proline/tryptophan sandwich.
Asunto(s)
Unión Competitiva , Inmunoglobulina E/metabolismo , Péptidos/química , Receptores de IgE/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Cristalografía por Rayos X , Humanos , Inmunoglobulina E/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Receptores de IgE/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
B cell maturation antigen (BCMA) is a tumor necrosis factor receptor family member whose physiological role remains unclear. BCMA has been implicated as a receptor for both a proliferation-inducing ligand (APRIL) and B cell-activating factor (BAFF), tumor necrosis factor ligands that bind to multiple tumor necrosis factor receptor and have been reported to play a role in autoimmune disease and cancer. The results presented herein provide a dual perspective analysis of BCMA binding to both APRIL and BAFF. First, we characterized the binding affinity of monomeric BCMA for its ligands; BAFF binding affinity (IC50 = 8 +/- 5 microm) is about 1000-fold reduced compared with the high affinity interaction of APRIL (IC50 = 11 +/- 3 nm). Second, shotgun alanine scanning of BCMA was used to map critical residues for either APRIL or BAFF binding. In addition to a previously described "DXL" motif (Gordon, N. C., Pan, B., Hymowitz, S. G., Yin, J., Kelley, R. F., Cochran, A. G., Yan, M., Dixit, V. M., Fairbrother, W. J., and Starovasnik, M. A. (2003) Biochemistry 42, 5977-5983), the alanine scanning results predicted four amino acid positions in BCMA (Tyr13, Ile22, Gln25, and Arg27) that could impart ligand specificity. Substitution of Tyr13 was tolerated for BAFF binding but not APRIL binding. Arg27 was required for high affinity binding to APRIL, whereas substitutions of this residue had minimal effect on affinity for BAFF. Further phage display experiments suggested the single mutations of I22K, Q25D, and R27Y as providing the greatest difference in APRIL versus BAFF binding affinity. Incorporation of the Q25D and R27Y substitutions into BCMA produced a dual specificity variant, since it has comparable binding affinity for both APRIL and BAFF, IC50 = 350 and 700 nm, respectively. Binding of the I22K mutant of monomeric BCMA to BAFF was undetectable (IC50 > 100 microm), but affinity for binding to APRIL was similar to wild-type BCMA. Based on these results, a BCMA-Fc fusion with the single I22K mutation was produced that binds APRIL, IC50 = 12 nm, and has no measurable affinity for BAFF. These results suggest that APRIL is the preferred ligand for BCMA and show that specificity can be further modified through amino acid substitutions.
Asunto(s)
Proteínas de la Membrana/metabolismo , Ingeniería de Proteínas , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/metabolismo , Sustitución de Aminoácidos , Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión/genética , Humanos , Ligandos , Proteínas de la Membrana/inmunología , Mutación , Mapeo Peptídico , Unión Proteica , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
EDA is a tumor necrosis factor family member involved in ectodermal development. Splice variants EDA-A1 and EDA-A2 differ only by the presence of Glu 308 and Val 309 in the expected receptor binding region of EDA-A1 but not EDA-A2. This two amino acid difference functions as a switch controlling receptor specificity. EDA-A1 binds only to EDAR, while EDA-A2 is specific for XEDAR. In order to understand the structural basis of this switch, we determined the X-ray crystal structures of the TNF domain of both EDA-A1 and EDA-A2 at 2.3 A and 2.2 A, respectively. While the backbone conformation around the splice difference is similar in both isoforms, the conformation of the following loop, the surface charge, and the shape of the expected receptor binding site differ significantly.