RESUMEN
Envelope glycoproteins of human and simian immunodeficiency virus (HIV and SIV) undergo a series of conformational changes when they interact with receptor (CD4) and co-receptor on the surface of a potential host cell, leading ultimately to fusion of viral and cellular membranes. Structures of fragments of gp120 and gp41 from the envelope protein are known, in conformations corresponding to their post-attachment and postfusion states, respectively. We report the crystal structure, at 4 A resolution, of a fully glycosylated SIV gp120 core, in a conformation representing its prefusion state, before interaction with CD4. Parts of the protein have a markedly different organization than they do in the CD4-bound state. Comparison of the unliganded and CD4-bound structures leads to a model for events that accompany receptor engagement of an envelope glycoprotein trimer. The two conformations of gp120 also present distinct antigenic surfaces. We identify the binding site for a compound that inhibits viral entry.
Asunto(s)
Glicoproteínas de Membrana/química , Virus de la Inmunodeficiencia de los Simios/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Sitios de Unión , Antígenos CD4/química , Antígenos CD4/metabolismo , Antígenos CD4/farmacología , Cristalización , Cristalografía por Rayos X , Glicosilación , Ligandos , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Polisacáridos/análisis , Unión Proteica , Estructura Cuaternaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Receptores Virales/química , Receptores Virales/metabolismo , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Virus de la Inmunodeficiencia de los Simios/fisiología , Proteínas del Envoltorio Viral/metabolismo , Virión/química , Virión/metabolismoRESUMEN
The alpha/beta T cell receptor (TCR) HA1.7 specific for the hemagglutinin (HA) antigen peptide from influenza A virus is HLA-DR1 restricted but cross-reactive for the HA peptide presented by the allo-major histocompatibility complex (MHC) class II molecule HLA-DR4. We report here the structure of the HA1.7/DR4/HA complex, determined by X-ray crystallography at a resolution of 2.4 A. The overall structure of this complex is very similar to the previously reported structure of the HA1.7/DR1/HA complex. Amino acid sequence differences between DR1 and DR4, which are located deep in the peptide binding groove and out of reach for direct contact by the TCR, are able to indirectly influence the antigenicity of the pMHC surface by changing the conformation of HA peptide residues at position P5 and P6. Although TCR HA1.7 is cross-reactive for HA presented by DR1 and DR4 and tolerates these conformational differences, other HA-specific TCRs are sensitive to these changes. We also find a dependence of the width of the MHC class II peptide-binding groove on the sequence of the bound peptide by comparing the HA1.7/DR4/HA complex with the structure of DR4 presenting a collagen peptide. This structural study of TCR cross-reactivity emphasizes how MHC sequence differences can affect TCR binding indirectly by moving peptide atoms.
Asunto(s)
Antígeno HLA-DR1/química , Antígeno HLA-DR4/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Alelos , Secuencia de Aminoácidos , Reacciones Cruzadas , Antígeno HLA-DR1/genética , Antígeno HLA-DR1/inmunología , Antígeno HLA-DR4/genética , Antígeno HLA-DR4/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunologíaRESUMEN
Human histocompatibility leukocyte antigen (HLA)-DM is a major histocompatibility complex (MHC)-like protein that catalyzes exchange of antigenic peptides from MHC class II molecules. To investigate the molecular details of this catalysis we created four covalent complexes between HLA-DM and the MHC class II allele DR1. We introduced a disulfide bond between the naturally occurring cysteine beta46 on HLA-DM and an engineered cysteine on the end of a linker attached to either the NH(2)- or the COOH terminus of an antigenic peptide that is tightly bound on DR1. We find that when DM is attached to the NH(2) terminus of the peptide, it can, for all linker lengths tested, catalyze exchange of the peptide with a half-life a few minutes (compared with uncatalyzed t(1/2) > 100 h). This rate, which is several orders of magnitude greater than the one we obtain in solution assays using micromolar concentrations of HLA-DM, is dominated by a concentration independent factor, indicating an intramolecular catalytic interaction within the complex. A similar complex formed at the COOH terminus of the peptide shows no sign of DM-specific intramolecular catalysis. Restrictions on the possible interaction sites imposed by the length of the linkers indicate that the face of DR1 that accommodates the NH(2) terminus of the antigenic peptide interacts with the lateral face of HLA-DM that contains cysteine beta46.
Asunto(s)
Antígenos HLA-D/metabolismo , Antígeno HLA-DR1/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Línea Celular , Cisteína/química , Antígenos HLA-D/química , Antígenos HLA-D/genética , Antígeno HLA-DR1/química , Antígeno HLA-DR1/genética , Humanos , Técnicas In Vitro , Cinética , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoAsunto(s)
Productos del Gen tax/historia , Antígeno HLA-A2/historia , Fragmentos de Péptidos/historia , Receptores de Antígenos de Linfocitos T alfa-beta/historia , Cristalografía por Rayos X/historia , Productos del Gen tax/química , Productos del Gen tax/metabolismo , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Historia del Siglo XX , Humanos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virologíaRESUMEN
HIV/SIV envelope glycoproteins mediate the first steps in viral infection. They are trimers of a membrane-anchored polypeptide chain, cleaved into two fragments known as gp120 and gp41. The structure of HIV gp120 bound with receptor (CD4) has been known for some time. We have now determined the structure of a fully glycosylated SIV gp120 envelope glycoprotein in an unliganded conformation by X-ray crystallography at 4.0 A resolution. We describe here our experimental and computational approaches, which may be relevant to other resolution-limited crystallographic problems. Key issues were attention to details of beam geometry mandated by small, weakly diffracting crystals, and choice of strategies for phase improvement, starting with two isomorphous derivatives and including multicrystal averaging. We validated the structure by analyzing composite omit maps, averaged among three distinct crystal lattices, and by calculating model-based, SeMet anomalous difference maps. There are at least four ordered sugars on many of the thirteen oligosaccharides.
Asunto(s)
Glicoproteínas de Membrana/química , Proteínas del Envoltorio Viral/química , Animales , Antígenos CD4/química , Antígenos CD4/metabolismo , Simulación por Computador , Cristalografía por Rayos X , Glicosilación , Ligandos , Glicoproteínas de Membrana/fisiología , Modelos Moleculares , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/fisiologíaRESUMEN
Class II major histocompatibility complex (MHC) proteins are essential for normal immune system function but also drive many autoimmune responses. They bind peptide antigens in endosomes and present them on the cell surface for recognition by CD4(+) T cells. A small molecule could potentially block an autoimmune response by disrupting MHC-peptide interactions, but this has proven difficult because peptides bind tightly and dissociate slowly from MHC proteins. Using a high-throughput screening assay we discovered a class of noble metal complexes that strip peptides from human class II MHC proteins by an allosteric mechanism. Biochemical experiments indicate the metal-bound MHC protein adopts a 'peptide-empty' conformation that resembles the transition state of peptide loading. Furthermore, these metal inhibitors block the ability of antigen-presenting cells to activate T cells. This previously unknown allosteric mechanism may help resolve how gold(I) drugs affect the progress of rheumatoid arthritis and may provide a basis for developing a new class of anti-autoimmune drugs.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Antígenos de Histocompatibilidad Clase II/química , Péptidos/química , Sitio Alostérico , Animales , Presentación de Antígeno , Enfermedades Autoinmunes/metabolismo , Cromatografía en Gel , Cisplatino/química , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Drosophila melanogaster , Ensayo de Inmunoadsorción Enzimática , Tiomalato Sódico de Oro/farmacología , Humanos , Cinética , Complejo Mayor de Histocompatibilidad , Modelos Estadísticos , Conformación Molecular , Unión Proteica , Hipoclorito de Sodio/farmacología , Factores de TiempoRESUMEN
Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fragmentos de Péptidos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular , Humanos , Fragmentos de Péptidos/química , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , SpodopteraRESUMEN
Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.
Asunto(s)
Receptores Virales/fisiología , Simplexvirus/fisiología , Simplexvirus/patogenicidad , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Dimerización , Herpes Simple/metabolismo , Ligandos , Estructura Terciaria de Proteína , Simplexvirus/metabolismo , Triptófano/metabolismoRESUMEN
Four glycoproteins (gD, gB, gH, and gL) are essential for herpes simplex virus (HSV) entry into cells. An early step of fusion requires gD to bind one of several receptors, such as nectin-1 or herpesvirus entry mediator (HVEM). We hypothesize that a conformational change in gD occurs upon receptor binding that triggers the other glycoproteins to mediate fusion. Comparison of the crystal structures of gD alone and gD bound to HVEM reveals that upon HVEM binding, the gD N terminus transitions from a flexible stretch of residues to a hairpin loop. To address the contribution of this transition to the ability of gD to trigger fusion, we attempted to "lock" the gD N terminus into a looped conformation by engineering a disulfide bond at its N and C termini. The resulting mutant (gD-A3C/Y38C) failed to trigger fusion in the absence of receptor, suggesting that formation of the loop is not the sole fusion trigger. Unexpectedly, although gD-A3C/Y38C bound HVEM, it failed to bind nectin-1. This was due to the key role played by Y38 in interacting with nectin-1. Since tyrosines are often "hot spot" residues at the center of protein-protein interfaces, we mutated residues that surround Y38 on the same face of gD and tested their binding and functional properties. Our results suggest that this region of gD is important for nectin-1 interaction and is distinct from but partially overlaps the site of HVEM binding. Unique gD mutants with altered receptor usage generated in this study may help dissect the roles played by various HSV receptors during infection.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas del Envoltorio Viral/química , Animales , Sitios de Unión , Mapeo Epitopo , Humanos , Fusión de Membrana , Ratones , Nectinas , Conformación Proteica , Receptores Virales/fisiologíaRESUMEN
Studies are described of the processing and structural modifications of haemagglutinin required for its membrane fusion activity.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/fisiología , Fusión de Membrana , Conformación ProteicaRESUMEN
The alpha/beta T cell receptor complex transmits signals from MHC/peptide antigens through a set of constitutively associated signaling molecules, including CD3-epsilon/gamma and CD3-epsilon/delta. We report the crystal structure at 1.9-A resolution of a complex between a human CD3-epsilon/delta ectodomain heterodimer and a single-chain fragment of the UCHT1 antibody. CD3-epsilon/delta and CD3-epsilon/gamma share a conserved interface between the Ig-fold ectodomains, with parallel packing of the two G strands. CD3-delta has a more electronegative surface and a more compact Ig fold than CD3-gamma; thus, the two CD3 heterodimers have distinctly different molecular surfaces. The UCHT1 antibody binds near an acidic region of CD3-epsilon opposite the dimer interface, occluding this region from direct interaction with the TCR. This immunodominant epitope may be a uniquely accessible surface in the TCR/CD3 complex, because there is overlap between the binding site of the UCHT1 and OKT3 antibodies. Determination of the CD3-epsilon/delta structure completes the set of TCR/CD3 globular ectodomains and contributes information about exposed CD3 surfaces.
Asunto(s)
Complejo CD3/química , Fragmentos de Inmunoglobulinas/química , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Complejos Multiproteicos , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/química , Proteínas Recombinantes/química , Electricidad EstáticaRESUMEN
The class II MHC homolog HLA-DM catalyzes exchange of peptides bound to class II MHC proteins, and is an important component of the Ag presentation machinery. The mechanism of HLA-DM-mediated catalysis is largely obscure. HLA-DM catalyzes exchange of peptides of varying sequence, suggesting that a peptide sequence-independent component of the MHC-peptide interaction could be involved in the catalytic process. Twelve conserved hydrogen bonds between the peptide backbone and the MHC are a prominent sequence-independent feature of the MHC-peptide interaction. To evaluate the relative importance of these hydrogen bonds toward HLA-DM action, we prepared peptide variants that lacked the ability to form one or more of the hydrogen bonds as a result of backbone amide N-methylation or truncation, and tested their ability to be exchanged by HLA-DM. We found that disruption of hydrogen bonds involving HLA-DR1 residues alpha51-53, a short extended segment at the N terminus of the alpha subunit helical region, led to heightened HLA-DM catalytic efficacy. We propose that those bonds are disrupted in the MHC conformation recognized by HLA-DM to allow structural transitions in that area during DM-assisted peptide release. These results suggest that peptides or compounds that bind MHC but cannot form these interactions would be preferentially edited out by HLA-DM.
Asunto(s)
Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Catálisis , Secuencia Conservada , Antígenos HLA-D/fisiología , Antígeno HLA-DR1/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Enlace de Hidrógeno , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Cinética , Datos de Secuencia Molecular , Conformación Proteica , Edición de ARN/inmunologíaRESUMEN
There are 15 subtypes of influenza A virus (H1-H15), all of which are found in avian species. Three caused pandemics in the last century: H1 in 1918 (and 1977), H2 in 1957 and H3 in 1968. In 1997, an H5 avian virus and in 1999 an H9 virus caused outbreaks of respiratory disease in Hong Kong. We have determined the three-dimensional structures of the haemagglutinins (HAs) from H5 avian and H9 swine viruses closely related to the viruses isolated from humans in Hong Kong. We have compared them with known structures of the H3 HA from the virus that caused the 1968 H3 pandemic and of the HA--esterase--fusion (HEF) glycoprotein from an influenza C virus. Structure and sequence comparisons suggest that HA subtypes may have originated by diversification of properties that affected the metastability of HAs required for their membrane fusion activities in viral infection.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Virus de la Influenza A/química , Orthomyxoviridae/clasificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cristalografía por Rayos X , Evolución Molecular , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/fisiología , Concentración de Iones de Hidrógeno , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Fusión de Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Rotación , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
We have determined the structure of the HA of an avian influenza virus, A/duck/Ukraine/63, a member of the same antigenic subtype, H3, as the virus that caused the 1968 Hong Kong influenza pandemic, and a possible progenitor of the pandemic virus. We find that structurally significant differences between the avian and the human HAs are restricted to the receptor-binding site particularly the substitutions Q226L and G228S that cause the site to open and residues within it to rearrange, including the conserved residues Y98, W153, and H183. We have also analyzed complexes formed by the HA with sialopentasaccharides in which the terminal sialic acid is in either alpha2,3- or alpha2,6-linkage to galactose. Comparing the structures of complexes in which an alpha2,3-linked receptor analog is bound to the H3 avian HA or to an H5 avian HA leads to the suggestion that all avian influenza HAs bind to their preferred alpha2,3-linked receptors similarly, with the analog in a trans conformation about the glycosidic linkage. We find that alpha2,6-linked analogs are bound by both human and avian HAs in a cis conformation, and that the incompatibility of an alpha2,6-linked receptor with the alpha2,3-linkage-specific H3 avian HA-binding site is partially resolved by a small change in the position and orientation of the sialic acid. We discuss our results in relation to the mechanism of transfer of influenza viruses between species.
Asunto(s)
Evolución Molecular , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Virus de la Influenza A/genética , Gripe Humana/epidemiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Patos/virología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Hong Kong/epidemiología , Humanos , Virus de la Influenza A/química , Gripe Humana/virología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Receptores Virales/metabolismo , Alineación de SecuenciaRESUMEN
Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular beta-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.
Asunto(s)
Receptores del Factor de Necrosis Tumoral/química , Receptores Virales/química , Simplexvirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Sitios de Unión , Línea Celular Transformada , Chlorocebus aethiops , Expresión Génica , Humanos , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Receptores Virales/genética , Receptores Virales/metabolismo , Simplexvirus/fisiología , Relación Estructura-Actividad , Células VeroRESUMEN
Gycoprotein D (gD) is a glycoprotein expressed on the surface of several human and animal alpha herpes viruses. Binding of gD to cell-surface receptors has been shown to be necessary for herpes simplex virus 1 and 2 (HSV-1 and HSV-2) cell entry. The gD ectodomain consists of 316 residues and has no sequence homology to any other proteins of known structure. Two fragments of the HSV-1 gD ectodomain (gD(22-260): residues 22-260 and gD(285): residues 1-285) have been crystallized in two crystal forms. The complex between gD(285) and the ectodomain of HveA, a gD cellular receptor member of the tumor necrosis factor (TNFR) superfamily, has also been crystallized. Moreover, insect-cell-expressed selenomethionine-substituted gD(285) has been purified and crystallized alone and in complex with HveA.
Asunto(s)
Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Virales/química , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Cristalización , Humanos , Sustancias Macromoleculares , Unión Proteica , Estructura Terciaria de Proteína , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Difracción de Rayos XRESUMEN
The influenza virus uses hemagglutinin (HA) to fuse the viral and cellular membranes. As part of an effort to study the membrane-interacting elements of HA, the fusion peptide, and the C-terminal transmembrane anchor, we have expressed in Escherichia coli the full-length HA(2) chain with maltose-binding protein fused at its N-terminus. The chimeric protein can be refolded in vitro in the presence of specific detergents to yield stable, homogeneous trimers, as determined by analytical ultracentrifugation. The trimers have the so-called "low pH" conformation-the rearranged HA(2) conformation obtained when intact HA(1)/HA(2) is induced to refold by exposure to low pH-as detected by electron microscopy and monoclonalantibody reactivity. These results provide further evidence for the notion that the neutral-pH structure of intact HA is metastable and that binding of protons lowers the kinetic barriers that prevent rearrangement to the minimum-free-energy conformation. The refolded chimeric protein described here is a suitable species for undertaking studies of how the fusion peptide inserts into membranes and assessing the nature of possible intermediates in the fusion process.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Hemaglutininas Virales/química , Pliegue de Proteína , Secuencia de Aminoácidos , Proteínas Portadoras/síntesis química , Proteínas Portadoras/genética , Proteínas Portadoras/ultraestructura , Detergentes/química , Ensayo de Inmunoadsorción Enzimática , Proteínas de Escherichia coli/síntesis química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestructura , Vectores Genéticos , Glicoproteínas Hemaglutininas del Virus de la Influenza/biosíntesis , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/ultraestructura , Hemaglutininas Virales/biosíntesis , Hemaglutininas Virales/genética , Hemaglutininas Virales/ultraestructura , Concentración de Iones de Hidrógeno , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/ultraestructura , Solubilidad , Relación Estructura-Actividad , UltracentrifugaciónRESUMEN
The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.