RESUMEN
At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller.
RESUMEN
Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Rhabdoviridae/enzimología , Rhabdoviridae/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Baculoviridae/genética , Investigación Biomédica/organización & administración , Investigación Biomédica/tendencias , Enzimas/genética , Escherichia coli/genética , Unión Europea , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcripción Genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.
Asunto(s)
Antivirales/farmacología , Biología Computacional , Cristalografía , Diseño de Fármacos , Genómica , Proteómica , Virus ARN/efectos de los fármacos , ARN Polimerasa Dependiente del ARN , Replicación Viral/efectos de los fármacos , Antivirales/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Cooperación Internacional , Modelos Moleculares , Virus ARN/enzimología , Virus ARN/patogenicidad , Virus ARN/fisiología , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
An automatic data-collection system has been implemented and installed on seven insertion-device beamlines and a bending-magnet beamline at the ESRF (European Synchrotron Radiation Facility) as part of the SPINE (Structural Proteomics In Europe) development of an automated structure-determination pipeline. The system allows remote interaction with beamline-control systems and automatic sample mounting, alignment, characterization, data collection and processing. Reports of all actions taken are available for inspection via database modules and web services.
Asunto(s)
Genes/genética , Sincrotrones/estadística & datos numéricos , Recolección de Datos/métodos , Gestión de la Información , Estructura Molecular , Control de Calidad , Programas InformáticosRESUMEN
The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.
Asunto(s)
Infecciones Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteómica/métodos , Proteínas Virales/química , Virosis/metabolismo , Animales , Infecciones Bacterianas/microbiología , Humanos , Pliegue de Proteína , Virosis/virologíaRESUMEN
SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.
Asunto(s)
Biología Computacional/estadística & datos numéricos , Proteómica/estadística & datos numéricos , Cristalización , Interpretación Estadística de Datos , Gestión de la Información , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Programas InformáticosRESUMEN
A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.
Asunto(s)
Bacillus anthracis/genética , Proteómica/métodos , Bacillus cereus/genética , Proteínas Bacterianas , Clonación Molecular , Biología Computacional , Cristalización , Cristalografía por Rayos X , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos , Espectroscopía de Resonancia Magnética , ARN de Transferencia/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Robótica , Esporas Bacterianas/genética , SulfurtransferasasRESUMEN
X-ray and electron microscopy analysis of Bluetongue virus (BTV), the type species of the Orbivirus genus within the family Reoviridae, have revealed various aspects of the organisation and structure of the proteins that form the viral capsid. Orbiviruses have a segmented dsRNA genome, which imposes constraints on their structure and life cycle. The atomic structure of the BTV core particle, the key viral component which transcribes the viral mRNA within the cell cytoplasm, revealed the architecture and assembly of the major core proteins VP7 and VP3. In addition, these studies formed the basis for a plausible model for the organisation of the dsRNA viral genome and the arrangement of the viral transcriptase complex (composed of the RNA-dependent RNA polymerase, the viral capping enzyme and RNA helicase) that resides within the core particle. Electron cryo-microscopy of the viral particle has shown how the two viral proteins VP2 and VP5 are arranged to form the outer capsid, with distinct packing arrangements between them and the core protein VP7. By comparison of the outer capsid proteins of orbiviruses with those of other nonturreted members of the family Reoviridae, we are able to propose a more detailed model of these structures and possible mechanisms for cell entry. Further structural results are also discussed including the atomic structure of an N-terminal domain of nonstructural protein NS2, a protein involved in virus genome assembly and morphogenesis.
Asunto(s)
Orbivirus/química , Proteínas Virales/química , Virión/ultraestructura , Transporte Biológico , Proteínas de la Cápside/química , Genoma Viral , Orbivirus/genética , Transcripción Genética , Proteínas del Núcleo Viral/química , Proteínas no Estructurales Virales/químicaRESUMEN
The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly, Chaetocoelopa sydneyensis, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5' and 3' untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 A reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-A-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.
Asunto(s)
Dípteros/virología , Genoma Viral , Virus de Insectos/clasificación , Picornaviridae/clasificación , Secuencia de Aminoácidos , Animales , Cápside/química , Cápside/ultraestructura , Proteínas de la Cápside/genética , Virus de Insectos/genética , Virus de Insectos/ultraestructura , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Filogenia , Picornaviridae/genética , Picornaviridae/ultraestructura , ARN Polimerasa Dependiente del ARN/genética , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
Structural studies of foot-and-mouth disease virus (FMDV) have largely focused on the mature viral particle, providing atomic resolution images of the spherical protein capsid for a number of sero- and sub-types, structures of the highly immunogenic surface loop, Fab and GAG receptor complexes. Additionally, structures are available for a few non-structural proteins. The chapter reviews our current structural knowledge and its impact on our understanding of the virus life cycle proceeding from the mature virus through immune evasion/inactivation, cell-receptor binding and replication and alludes to future structural targets.
Asunto(s)
Virus de la Fiebre Aftosa/química , Virus de la Fiebre Aftosa/ultraestructura , Proteínas de la Cápside/química , Genoma Viral , Proteoglicanos de Heparán Sulfato/metabolismo , Integrinas/metabolismo , Receptores Virales/metabolismo , Proteínas no Estructurales Virales/química , Ensamble de VirusRESUMEN
Crystals of bacteriophage PRD1, a virus containing an internal lipid bilayer, have been grown in thin-walled quartz capillary tubes by vapour diffusion as a means of eliminating mechanical handling of the crystals during data collection. It has been found that the addition of polyethylene glycol 20 000 (PEG 20K) to the mother liquor that bathes the crystals allows far higher resolution diffraction intensities to be observed. Growing and treating the crystals in this way has produced a small number of crystals which are particularly amenable to X-ray diffraction analysis.
Asunto(s)
Bacteriófago PRD1/química , Cristalización , Membranas/química , Cuarzo , Salmonella enterica/virología , Difracción de Rayos XRESUMEN
The HIV-2 serotype of HIV is a cause of disease in parts of the West African population, and there is evidence for its spread to Europe and Asia. HIV-2 reverse transcriptase (RT) demonstrates an intrinsic resistance to non-nucleoside RT inhibitors (NNRTIs), one of two classes of anti-AIDS drugs that target the viral RT. We report the crystal structure of HIV-2 RT to 2.35 A resolution, which reveals molecular details of the resistance to NNRTIs. HIV-2 RT has a similar overall fold to HIV-1 RT but has structural differences within the "NNRTI pocket" at both conserved and nonconserved residues. The structure points to the role of sequence differences that can give rise to unfavorable inhibitor contacts or destabilization of part of the binding pocket at positions 101, 106, 138, 181, 188, and 190. We also present evidence that the conformation of Ile-181 compared with the HIV-1 Tyr-181 could be a significant contributory factor to this inherent drug resistance of HIV-2 to NNRTIs. The availability of a refined structure of HIV-2 RT will provide a stimulus for the structure-based design of novel non-nucleoside inhibitors that could be used against HIV-2 infection.
Asunto(s)
ADN Polimerasa Dirigida por ARN/química , Fármacos Anti-VIH/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Farmacorresistencia Viral , Transcriptasa Inversa del VIH , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , ADN Polimerasa Dirigida por ARN/genética , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-ActividadRESUMEN
Six structures of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing combinations of resistance mutations for zidovudine (AZT) (M41L and T215Y) or lamivudine (M184V) have been determined as inhibitor complexes. Minimal conformational changes in the polymerase or nonnucleoside RT inhibitor sites compared to the mutant RTMC (D67N, K70R, T215F, and K219N) are observed, indicating that such changes may occur only with certain combinations of mutations. Model building M41L and T215Y into HIV-1 RT-DNA and docking in ATP that is utilized in the pyrophosphorolysis reaction for AZT resistance indicates that some conformational rearrangement appears necessary in RT for ATP to interact simultaneously with the M41L and T215Y mutations.
Asunto(s)
Fármacos Anti-VIH/farmacología , Codón , Farmacorresistencia Viral/genética , Transcriptasa Inversa del VIH/química , Lamivudine/farmacología , Mutación , Zidovudina/farmacología , Adenosina Trifosfato/metabolismo , Cristalización , Transcriptasa Inversa del VIH/genética , Conformación ProteicaRESUMEN
Double-stranded RNA (dsRNA) viruses conceal their genome from the host to avoid triggering unfavorable cellular responses. The crystal structure of the core of one such virus, bluetongue virus, reveals an outer surface festooned with dsRNA. This may represent a deliberate strategy to sequester dsRNA released from damaged particles to prevent host cell shutoff.
Asunto(s)
Virus de la Lengua Azul/metabolismo , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Proteínas del Núcleo Viral/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , ARN Bicatenario/química , ARN Viral/química , Proteínas del Núcleo Viral/química , Difracción de Rayos XRESUMEN
The bluetongue virus core is a molecular machine that simultaneously and repeatedly transcribes mRNA from 10 segments of viral double-stranded RNA, packaged in a liquid crystalline array. To determine how the logistical problems of transcription within a sealed shell are solved, core crystals were soaked with various ligands and analysed by X-ray crystallography. Mg(2+) ions produce a slight expansion of the capsid around the 5-fold axes. Oligonucleotide soaks demonstrate that the 5-fold pore, opened up by this expansion, is the exit site for mRNA, whilst nucleotide soaks pinpoint a separate binding site that appears to be a selective channel for the entry and exit of substrates and by-products. Finally, nucleotides also bind to the outer core layer, providing a substrate sink.
Asunto(s)
Virus de la Lengua Azul/fisiología , Transcripción Genética , Secuencia de Bases , Sitios de Unión , Virus de la Lengua Azul/genética , Calcio/metabolismo , Cristalografía por Rayos X , Magnesio/metabolismo , Fosfatos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sulfatos/metabolismoRESUMEN
Fcgamma receptors (FcgammaRs) are expressed on all immunologically active cells. They bind the Fc portion of IgG, thereby triggering a range of immunological functions. We have used surface plasmon resonance to analyze the kinetic and thermodynamic properties of the interactions between the ectodomains of human low affinity FcgammaRs (FcgammaRIIa, FcgammaRIIb, and FcgammaRIIIb-NA2) and IgG1 or the Fc fragment of IgG1. All three receptors bind Fc or IgG with similarly low affinities (K(D) approximately 0.6-2.5 microm) and fast kinetics, suggesting that FcgammaR-mediated recognition of aggregated IgG and IgG-coated particles or cells is mechanistically similar to cell-cell recognition. Interestingly, the Fc receptors exhibit distinct thermodynamic properties. Whereas the binding of the FcgammaRIIa and FcgammaRIIb to Fc is driven by favorable entropic and enthalpic changes, the binding of FcgammaRIII is characterized by highly unfavorable entropic changes. Although the structural bases for these differences remain to be determined, they suggest that the molecular events coupled to the binding differ among the low affinity FcgammaRs.
Asunto(s)
Antígenos CD/metabolismo , Receptores de IgG/metabolismo , Antígenos CD/química , Entropía , Proteínas Ligadas a GPI , Humanos , Inmunoglobulina G/metabolismo , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de IgG/química , Resonancia por Plasmón de Superficie , Temperatura , Termodinámica , Factores de TiempoRESUMEN
Mutations at either Tyr181 or Tyr188 within HIV-1 reverse transcriptase (RT) give high level resistance to many first generation non-nucleoside inhibitors (NNRTIs) such as the anti-AIDS drug nevirapine. By comparison second generation inhibitors, for instance the drug efavirenz, show much greater resilience to these mutations. In order to understand the structural basis for these differences we have determined a series of seven crystal structures of mutant RTs in complexes with first and second generation NNRTIs as well as one example of an unliganded mutant RT. These are Tyr181Cys RT (TNK-651) to 2.4 A, Tyr181Cys RT (efavirenz) to 2.6 A, Tyr181Cys RT (nevirapine) to 3.0 A, Tyr181Cys RT (PETT-2) to 3.0 A, Tyr188Cys RT (nevirapine) to 2.6 A, Tyr188Cys RT (UC-781) to 2.6 A and Tyr188Cys RT (unliganded) to 2.8 A resolution. In the two previously published structures of HIV-1 reverse transcriptase with mutations at 181 or 188 no side-chain electron density was observed within the p66 subunit (which contains the inhibitor binding pocket) for the mutated residues. In contrast the mutated side-chains can be seen in the NNRTI pocket for all seven structures reported here, eliminating the possibility that disordering contributes to the mechanism of resistance. In the case of the second generation compounds efavirenz with Tyr181Cys RT and UC-781 with Tyr188Cys RT there are only small rearrangements of either inhibitor within the binding site compared to wild-type RT and also for the first generation compounds TNK-651, PETT-2 and nevirapine with Tyr181Cys RT. For nevirapine with the Tyr188Cys RT there is however a more substantial movement of the drug molecule. We conclude that protein conformational changes and rearrangements of drug molecules within the mutated sites are not general features of these particular inhibitor/mutant combinations. The main contribution to drug resistance for Tyr181Cys and Tyr188Cys RT mutations is the loss of aromatic ring stacking interactions for first generation compounds, providing a simple explanation for the resilience of second generation NNRTIs, as such interactions make much less significant contribution to their binding.
Asunto(s)
Codón/genética , Farmacorresistencia Microbiana/genética , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , Mutación Missense/genética , Inhibidores de la Transcriptasa Inversa/farmacología , Sitios de Unión , Cristalografía por Rayos X , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
A series of 2-amino-5-arylthiobenzonitriles (1) was found to be active against HIV-1. Structural modifications led to the sulfoxides (2) and sulfones (3). The sulfoxides generally showed antiviral activity against HIV-1 similar to that of 1. The sulfones, however, were the most potent series of analogues, a number having activity against HIV-1 in the nanomolar range. Structural-activity relationship (SAR) studies suggested that a meta substituent, particularly a meta methyl substituent, invariably increased antiviral activities. However, optimal antiviral activities were manifested by compounds where both meta groups in the arylsulfonyl moiety were substituted and one of the substituents was a methyl group. Such a disubstitution led to compounds 3v, 3w, 3x, and 3y having IC50 values against HIV-1 in the low nanomolar range. When gauged for their broad-spectrum antiviral activity against key non-nucleoside reverse transcriptase inhibitor (NNRTI) related mutants, all the di-meta-substituted sulfones 3u-z and the 2-naphthyl analogue 3ee generally showed single-digit nanomolar activity against the V106A and P236L strains and submicromolar to low nanomolar activity against strains E138K, V108I, and Y188C. However, they showed a lack of activity against the K103N and Y181C mutant viruses. The elucidation of the X-ray crystal structure of the complex of 3v (739W94) in HIV-1 reverse transcriptase showed an overlap in the binding domain when compared with the complex of nevirapine in HIV-1 reverse transcriptase. The X-ray structure allowed for the rationalization of SAR data and potencies of the compounds against the mutants.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Nitrilos/síntesis química , Sulfonas/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Sitios de Unión , Línea Celular Transformada , Cristalografía por Rayos X , Transcriptasa Inversa del VIH/química , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Nitrilos/química , Nitrilos/farmacología , Conformación Proteica , Relación Estructura-Actividad , Sulfonas/química , Sulfonas/farmacologíaRESUMEN
The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 A, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 A, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 A, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.
Asunto(s)
Antígeno B7-1/química , Antígeno B7-1/metabolismo , Inmunoconjugados , Abatacept , Antígenos CD , Antígenos de Diferenciación/metabolismo , Antígenos CD28/metabolismo , Antígeno CTLA-4 , Cristalización , Glicosilación , Humanos , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Selenometionina/metabolismo , Solubilidad , Resonancia por Plasmón de Superficie , Difracción de Rayos XRESUMEN
In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail. Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.