RESUMO
Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Epitopos , Subunidade alfa1 de Receptor de Interleucina-13/química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação/imunologia , Cristalografia por Raios X , Dimerização , Humanos , Fragmentos Fab das Imunoglobulinas/química , Interleucina-13/imunologia , Interleucina-13/metabolismo , Subunidade alfa1 de Receptor de Interleucina-13/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Leucina/metabolismo , Camundongos , Camundongos Transgênicos , Estrutura Terciária de ProteínaRESUMO
The activation of the epidermal growth factor receptor (EGFR) kinase requires ligand binding to the extracellular domain (ECD). Previous reports demonstrate that the EGFR-ECD can be crystallized in two conformations - a tethered monomer or, in the presence of ligand, an untethered back-to-back dimer. We use Biosensor analysis to demonstrate that even in the monomeric state different C-terminal extensions of both truncated (EGFR(1-501))-ECD and full-length EGFR(1-621)-ECD can change the conformation of the ligand-binding site. The binding of a monoclonal antibody mAb806, which recognizes the dimer interface, to the truncated EGFR(1-501)-Fc fusion protein is reduced in the presence of ligand, consistent with a change in conformation. On the cell surface, the presence of erythroblastosis B2 (erbB2) increases the binding of mAb806 to the EGFR. The conformation of the erbB2: EGFR heterodimer interface changes when the cells are treated with epidermal growth factor (EGF). We propose that ligand induces kinase-inactive, pre-formed EGFR dimers and heterodimers to change conformation leading to kinase-active tetramers, where kinase activation occurs via an asymmetric interaction between EGFR dimers.
Assuntos
Receptores ErbB/química , Ligantes , Animais , Anticorpos Monoclonais/química , Técnicas Biossensoriais , Linhagem Celular , Dimerização , Epitopos/química , Corantes Fluorescentes/química , Células HEK293 , Humanos , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR(287-302) complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR(C271A/C283A) mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR(C271A/C283A). Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Receptores ErbB/imunologia , Proteínas de Neoplasias/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Complexo Antígeno-Anticorpo/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cristalografia por Raios X , Epitopos , Humanos , Camundongos , Camundongos Nus , Conformação Proteica , Desnaturação Proteica/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
gp130 is the shared signal-transducing receptor subunit for the large and important family of interleukin 6-like cytokines. Previous x-ray structures of ligand-receptor complexes of this family lack the three membrane-proximal domains that are essential for signal transduction. Here we report the crystal structure of the entire extracellular portion of human gp130 (domains 1-6, D1-D6) at 3.6 A resolution, in an unliganded form, as well as a higher resolution structure of the membrane-proximal fibronectin type III domains (D4-D6) at 1.9 A. This represents the first atomic resolution structure of the complete ectodomain of any "tall" cytokine receptor. These structures show that other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. They also reveal that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire tall receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C termini of the membrane-proximal fibronectin type III domains of the tall cytokine receptors in close proximity within the transmembrane complex, favorable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.
Assuntos
Interleucina-6/química , Moléculas de Adesão de Célula Nervosa/química , Contactinas , Cristalografia por Raios X/métodos , Citocinas/metabolismo , Dimerização , Fibronectinas/química , Humanos , Ligantes , Conformação Molecular , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Diabodies (scFv dimers) are small, bivalent antibody mimetics of approximately 55kDa in size that possess rapid in vivo targeting pharmacokinetics compared to the intact parent antibody, and may prove highly suitable for imaging and therapeutic applications. Here, we describe T84.66Di, the first diabody crystal structure in which the scFvs comprise V domains linked in the V(L)-to-V(H) orientation. The structure was determined by X-ray diffraction analysis to 2.6 A resolution. The T84.66Di scFv was constructed from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66 variable domains connected by an eight residue peptide linker to provide flexibility between Fv modules and promote dimer formation with bivalent affinity to the cell-surface target, CEA. Therefore, it was surprising to observe a close association of some Fv module complementarity-determining regions in the T84.66 diabody crystal, especially compared to other diabody structures all of which are linked in the opposite V(H)-to-V(L) orientation. The differences between the arrangement of Fv modules in the T84.66Di V(L)-to-V(H) linked diabody structure compared to the crystal structure of L5MK16 and other proposed V(H)-to-V(L) linked diabodies has been investigated and their potential for flexibility discussed. The comparison between V(H)-to-V(L) and V(L)-to-V(H) linked diabodies revealed in this study represents a limited repertoire of possible diabody Fv orientations, but one that reveals the potential flexibility of these molecules. This analysis therefore provides some signposts that may impact on future molecular designs for these therapeutic molecules with respect to diabody flexibility and avidity.
Assuntos
Antígenos de Neoplasias/imunologia , Antígeno Carcinoembrionário/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/imunologia , Anticorpos Biespecíficos , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/metabolismo , Cristalização , Cristalografia por Raios X , Humanos , Fragmentos Fc das Imunoglobulinas , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Isoenzimas/imunologia , Modelos Moleculares , Neuraminidase/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosfolipase C delta , Conformação Proteica , Proteínas Recombinantes de Fusão , Fosfolipases Tipo C/imunologiaRESUMO
The insulin receptor (IR) and epidermal growth factor receptor (EGFR) families are closely related members of the receptor tyrosine kinase superfamily, and are among the more intensively studied proteins in biology. Deregulated signaling by the type-1 insulin-like growth factor receptor (IGF-1R) or members of the EGFR family has been implicated in the progression of a variety of cancers. These receptors have thus emerged as validated therapeutic targets for the development of antitumor agents. In this review, recent progress in the elucidation of the three-dimensional structures of the extracellular domains of both receptor families is discussed. While the IGF-1R provided the first description of the extracellular domains of these two receptor families, it is the EGFR family in which greatest progress has been achieved. Over the past year, the field has progressed from having a complete absence of X-ray crystal structures to having eight such structures; ErbB-2 alone or complexed with the two monoclonal antibodies pertuzumab (Genentech Inc/Roche Holdings AG/Chugai Pharmaceutical Co Ltd) and trastuzamab, ErbB-3 without a ligand, EGFR with a ligand bound in an unactivated monomeric conformation, and EGFR with either epidermal growth factor (EGF) or transforming growth factor-alpha (TGFalpha) in a 2:2 dimeric complex. This review will discuss these developments and the opportunities they provide for the design of new therapeutic agents targeting their extracellular domains.
Assuntos
Receptores ErbB/química , Conformação Proteica , Receptor de Insulina/química , Desenho de Fármacos , Receptores ErbB/antagonistas & inibidores , Humanos , Modelos Moleculares , Receptor de Insulina/antagonistas & inibidoresRESUMO
Leukemia inhibitory factor (LIF) receptor is a cell surface receptor that mediates the actions of LIF and other IL-6 type cytokines through the formation of high-affinity signaling complexes with gp130. Here we present the crystal structure of a complex of mouse LIF receptor with human LIF at 4.0 A resolution. The structure is, to date, the largest cytokine receptor fragment determined by x-ray crystallography. The binding of LIF to its receptor via the central Ig-like domain is unlike other cytokine receptor complexes that bind ligand predominantly through their cytokine-binding modules. This structure, in combination with previous crystallographic studies, also provides a structural template to understand the formation and orientation of the high-affinity signaling complex between LIF, LIF receptor, and gp130.
Assuntos
Imunoglobulinas/química , Imunoglobulinas/metabolismo , Fator Inibidor de Leucemia/química , Fator Inibidor de Leucemia/metabolismo , Receptores de OSM-LIF/química , Receptores de OSM-LIF/metabolismo , Animais , Cristalografia por Raios X , Receptor gp130 de Citocina/química , Receptor gp130 de Citocina/metabolismo , Humanos , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Interleucina-6/química , Interleucina-6/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/imunologia , Ligantes , Camundongos , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de OSM-LIF/genética , Receptores de OSM-LIF/imunologia , Transdução de SinaisRESUMO
The X-ray structure of influenza virus neuraminidase (NA) isolated from whale, subtype N9, has been determined at 2.2 A resolution and contains a tetrameric protein in the asymmetric unit. In structures of NA determined previously, a calcium ion is observed to coordinate amino acids near the substrate-binding site. In three of the NA monomers determined here this calcium is absent, resulting in structural alterations near the substrate-binding site. These changes affect the conformation of residues that participate in several key interactions between the enzyme and substrate and provide at a molecular level the basis of the structural and functional role of calcium in substrate and inhibitor binding. Several sulfate ions were identified in complex with the protein. These are located in the active site, occupying the space reserved for the substrate (sialic acid) carboxylate, and in positions leading away from the substrate-binding site. These sites offer a new opportunity for the design of inhibitors of influenza virus NA.
Assuntos
Cálcio/química , Neuraminidase/química , Orthomyxoviridae/enzimologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Íons , Modelos Químicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Sulfatos/química , Baleias/virologia , Raios XRESUMO
The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1-CR-L2) of human IR at 2.3 A resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) beta-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more alpha-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R.
Assuntos
Fator de Crescimento Insulin-Like I/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor de Insulina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cristalografia por Raios X , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Alinhamento de SequênciaRESUMO
The epidermal growth factor (EGF) receptor (EGFR) is one of four homologous transmembrane proteins that mediate the actions of a family of growth factors including EGF, transforming growth factor-alpha, and the neuregulins. We review the structure and function of the EGFR, from ligand binding to the initiation of intracellular signalling pathways that lead to changes in the biochemical state of the cell. The recent crystal structures of different domains from several members of the EGFR family have challenged our concepts of these processes.
Assuntos
Receptores ErbB/metabolismo , Transdução de Sinais , Animais , Movimento Celular , Humanos , Modelos Moleculares , Conformação Proteica , Fatores de Crescimento Transformadores/metabolismoRESUMO
Recent crystallographic studies have provided significant new insight into how receptor tyrosine kinases from the EGF receptor or ErbB family are regulated by their growth factor ligands. EGF receptor dimerization is mediated by a unique dimerization arm, which becomes exposed only after a dramatic domain rearrangement is promoted by growth factor binding. ErbB2, a family member that has no ligand, has its dimerization arm constitutively exposed, and this explains several of its unique properties. We outline a mechanistic view of ErbB receptor homo- and heterodimerization, which suggests new approaches for interfering with these processes when they are implicated in human cancers.
Assuntos
Receptores ErbB/metabolismo , Células Eucarióticas/metabolismo , Substâncias de Crescimento/metabolismo , Proteínas Oncogênicas v-erbB/metabolismo , Animais , Transformação Celular Neoplásica/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Estrutura Terciária de ProteínaRESUMO
ErbB2 does not bind ligand, yet appears to be the major signaling partner for other ErbB receptors by forming heteromeric complexes with ErbB1, ErbB3, or ErbB4. The crystal structure of residues 1-509 of ErbB2 at 2.5 A resolution reveals an activated conformation similar to that of the EGFR when complexed with ligand and very different from that seen in the unactivated forms of ErbB3 or EGFR. The structure explains the inability of ErbB2 to bind known ligands and suggests why ErbB2 fails to form homodimers. Together, the data suggest a model in which ErbB2 is already in the activated conformation and ready to interact with other ligand-activated ErbB receptors.
Assuntos
Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Cricetinae , Cristalografia por Raios X , DNA Complementar/genética , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-3/química , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
Recent crystallographic data on the isolated extracellular domain of the epidermal growth factor receptor (EGFR) have suggested a model for its activation by ligand. We have tested this model in the context of the full-length EGFR displayed at the cell surface, by introducing mutations in two regions (CR1 and CR2) of the extracellular domain thought to be critical for regulation of receptor activation. Mutations in the CR1 and CR2 domains have opposing effects on ligand binding affinity, receptor dimerization, tyrosine kinase activation, and signaling competence. Tyr(246) is a critical residue in the CR1 loop, which is implicated in the positioning and stabilization of the receptor dimer interface after ligand binding; mutations of Tyr(246) impair or abolish receptor function. Mutations in CR2, which weaken the interaction that restricts the receptor to the tethered (inactive) state, enhance responsiveness to EGF by increasing affinity for the ligand. However, weakening of the CR1/CR2 interaction does not result in spontaneous activation of the receptors' kinase. We have used an antibody (mAb 806), which recognizes a transition state of the EGF receptor between the negatively constrained, tethered state and the fully active back-to-back dimer conformation, to follow conformational changes in the wild-type and mutant EGF receptors after ligand binding. Our results suggest that EGFR on the cell surface can be untethered, but this form is inactive; thus, untethering of the receptor is not sufficient for activation, and ligand binding is essential for the correct positioning of the two receptor subunits to achieve kinase activation.
Assuntos
Membrana Celular/metabolismo , Receptores ErbB/fisiologia , Animais , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , Dimerização , Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Cinética , Ligantes , Sistema de Sinalização das MAP Quinases , Camundongos , Modelos Biológicos , Modelos Moleculares , Mutação , Fosforilação , Mutação Puntual , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Transfecção , Tirosina/química , Tirosina/metabolismoRESUMO
We report the crystal structure, at 2.5 A resolution, of a truncated human EGFR ectodomain bound to TGFalpha. TGFalpha interacts with both L1 and L2 domains of EGFR, making many main chain contacts with L1 and interacting with L2 via key conserved residues. The results indicate how EGFR family members can bind a family of highly variable ligands. In the 2:2 TGFalpha:sEGFR501 complex, each ligand interacts with only one receptor molecule. There are two types of dimers in the asymmetric unit: a head-to-head dimer involving contacts between the L1 and L2 domains and a back-to-back dimer dominated by interactions between the CR1 domains of each receptor. Based on sequence conservation, buried surface area, and mutagenesis experiments, the back-to-back dimer is favored to be biologically relevant.