RESUMEN
The recent emergence of highly pathogenic avian influenza A virus strains with subtype H5N1 pose a global threat to human health. Elucidation of the underlying mechanisms of viral replication is critical for development of anti-influenza virus drugs. The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer has crucial roles in viral RNA replication and transcription. It contains three proteins: PA, PB1 and PB2. PB1 harbours polymerase and endonuclease activities and PB2 is responsible for cap binding; PA is implicated in RNA replication and proteolytic activity, although its function is less clearly defined. Here we report the 2.9 ångström structure of avian H5N1 influenza A virus PA (PA(C), residues 257-716) in complex with the PA-binding region of PB1 (PB1(N), residues 1-25). PA(C) has a fold resembling a dragon's head with PB1(N) clamped into its open 'jaws'. PB1(N) is a known inhibitor that blocks assembly of the polymerase heterotrimer and abolishes viral replication. Our structure provides details for the binding of PB1(N) to PA(C) at the atomic level, demonstrating a potential target for novel anti-influenza therapeutics. We also discuss a potential nucleotide binding site and the roles of some known residues involved in polymerase activity. Furthermore, to explore the role of PA in viral replication and transcription, we propose a model for the influenza RdRp heterotrimer by comparing PA(C) with the lambda3 reovirus polymerase structure, and docking the PA(C) structure into an available low resolution electron microscopy map.
Asunto(s)
Aves/virología , Subtipo H5N1 del Virus de la Influenza A/enzimología , ARN Polimerasa Dependiente del ARN/química , Proteínas Virales/química , Proteínas Virales/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Nucleótidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , ARN Polimerasa Dependiente del ARN/metabolismo , Replicación ViralRESUMEN
Thyroid hormone responsive protein (Thrsp, also known as Spot 14 and S14) is a carbohydrate-inducible and thyroid-hormone-inducible nuclear protein specific to liver, adipose and lactating mammary tissues. Thrsp functions to activate genes encoding fatty-acid synthesis enzymes. Recent studies have shown that in some cancers human Thrsp (hS14) localizes to the nucleus and is amplified, suggesting that it plays a role in the regulation of lipogenic enzymes during tumourigenesis. Thrsp, a member of the Spot 14 superfamily, is an acidic homodimeric protein with no sequence similarity to other mammalian gene products and its biochemical function is elusive. To shed light on the structure-function relationship of this protein, human Thrsp was crystallized. Recombinant human Thrsp (hThrsp), the N-terminally truncated human Thrsp(10-146) (hThrsp9) and their selenomethionyl (SeMet) derivatives were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction-quality crystals were grown at 293â K using Li(2)SO(4) as a precipitant. Using synchrotron radiation, data for the hThrsp SeMet derivative, hThrsp9 and its SeMet derivative were collected to 4.0, 3.0 and 3.6â Å resolution, respectively, at 100â K. The crystals of full-length hThrsp and its SeMet derivative belonged to space group P4(1)2(1)2, with approximate unit-cell parameters a = b = 123.9, c = 242.1â Å, α = ß = γ = 90.0°. In contrast, the crystals of the truncated hThrsp9 and its SeMet derivative belonged to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 91.6, b = 100.8, c = 193.7â Å, α = ß = γ = 90.0°. A molecular-replacement solution calculated using a murine Spot 14 structure as a search model indicated the presence of six molecules per asymmetric unit, comprising three hThrsp homodimers.
Asunto(s)
Proteínas Nucleares/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Cristalización , Cristalografía por Rayos X , Expresión Génica , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Estructura Cuaternaria de Proteína , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
2-Substituted 3-nitropropanoic acids were designed and synthesized as inhibitors against carboxypeptidase A (CPA). (R)-2-Benzyl- 3-nitropropanoic acid showed a potent inhibition against CPA (K(i)=0.15 microM). X-ray crystallography discloses that the nitro group well mimics the transition state occurred in the hydrolysis catalyzed by CPA, that is, an O,O'-bidentate coordination to the zinc ion and the two respective hydrogen bonds with Glu-270 and Arg-127. Because the nitro group is a planar species, we proposed (R)-2-benzyl-3-nitropropanoic acid as a pseudo-transition-state analog inhibitor against CPA.
Asunto(s)
Carboxipeptidasas A/antagonistas & inhibidores , Carboxipeptidasas A/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Nitrógeno/química , Zinc/química , Catálisis , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Gadd45, MyD118 and CR6 (also termed Gadd45alpha, Gadd45beta and Gadd45gamma, respectively) comprise a family of proteins that play important roles in negative growth control, maintenance of genomic stability, DNA repair, cell-cycle control and apoptosis. Recombinant human Gadd45gamma and its selenomethionine derivative were expressed in an Escherichia coli expression system and purified; they were then crystallized using the hanging-drop vapour-diffusion method. Diffraction-quality crystals were grown at 291 K using PEG 3350 as precipitant. Using synchrotron radiation, the best diffraction data were collected to 2.3 A resolution for native crystals at 100 K; selenomethionyl derivative data were collected to 3.3 A resolution. All the crystals belonged to space group I2(1)3, with approximate unit-cell parameters a = b = c = 126 A.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Cristalización , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Selenometionina/química , Sincrotrones , Difracción de Rayos X , Proteinas GADD45RESUMEN
The number of atom-atom contacts in long distance can fit to the experimental binding energies in a dataset containing 151 experimental data with the correlation coefficient about 0.68. Based on this factor, a set of distance-dependent empirical potentials for various types of short-distance (2.4 A-5 A) contacts was obtained by guided fitting, i.e. a set of two parameters fitting. Incorporation of these short-distance potentials improved the correlation coefficients to 0.881.
Asunto(s)
Conformación Proteica , Proteínas , Bases de Datos de Proteínas , Modelos Teóricos , Datos de Secuencia Molecular , Unión Proteica , Proteínas/química , Proteínas/metabolismo , Estadística como Asunto , TermodinámicaRESUMEN
Staphylococcal nuclease (SNase) is a well-established model for protein folding studies. Its three-dimensional structure has been determined. The enzyme, Ca2+, and DNA or RNA substrate form a ternary complex. Glycine 20 is the second position of the first beta-turn of SNase, which may serve as the folding initiation site for the SNase polypeptide. To study the role of Gly20 in the conformational stability and catalysis of SNase, three mutants, in which Gly20 was replaced by alanine, valine, or isoleucine, were constructed and studied by using circular dichroism spectra, intrinsic and ANS-binding fluorescence spectra, stability and activity assays. The mutations have little effect on the conformational integrity of the mutants. However, the catalytic activity is reduced drastically by the mutations, and the stability of the protein is progressively decreased in the order G20AAsunto(s)
Sustitución de Aminoácidos
, Glicina/química
, Nucleasa Microcócica/química
, Nucleasa Microcócica/genética
, Nucleasa Microcócica/metabolismo
, Alanina/metabolismo
, Calcio/química
, Catálisis
, Dicroismo Circular
, Estabilidad de Enzimas
, Isoleucina/metabolismo
, Cinética
, Nucleasa Microcócica/efectos de los fármacos
, Nucleasa Microcócica/aislamiento & purificación
, Modelos Moleculares
, Conformación Proteica
, Desnaturalización Proteica
, Pliegue de Proteína
, Espectrometría de Fluorescencia
, Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
, Relación Estructura-Actividad
, Especificidad por Sustrato
, Urea/farmacología
, Valina/metabolismo
RESUMEN
When amino acid residues are represented by parameters describing their side chain lengths and polarities, a sequence function defined as the sum of the first two sequence autocorrelation functions is found to be negatively and linearly correlated with the logarithms of folding rates of beta-proteins. The new function reveals new features in beta-protein folding: larger residues slow down the folding while alternative distribution of polar-non-polar residues accelerates the folding.
Asunto(s)
Conformación Proteica , Pliegue de Proteína , Proteínas/química , Secuencia de Aminoácidos , Matemática , Estructura Secundaria de ProteínaRESUMEN
It is found that the helix parameter (HP), which favors clustering of non-polar residues, is linearly correlated with the logarithms of rate constants of folding of small two-state alpha-helical proteins. The definition is HP = N(H)(-1) sigma [f(i)+ (f(i-1)+f(i+1))/2], where f(i)=1 or -1, if the i'th residue is hydrophobic or hydrophilic, respectively, N(H) is the number of hydrophobic residues and the summation is taken over the hydrophobic residues.
Asunto(s)
Conformación Proteica , Pliegue de Proteína , Proteínas/química , Algoritmos , Aminoácidos/química , Animales , Humanos , Modelos QuímicosRESUMEN
The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45γ [corrected], revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45γ [corrected] identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45γ [corrected] is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Secuencias de Aminoácidos , Animales , Apoptosis/efectos de la radiación , Proteína Quinasa CDC2 , Ciclo Celular , Supervivencia Celular , Cristalografía por Rayos X , Ciclina B/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Mutación Missense , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Rayos UltravioletaRESUMEN
The relations of the binding free energies in a dataset of 69 protein complexes with the numbers of interfacial atom pairs, as well as with the atomic distances of the pairs, are analyzed. It is found that the interfacial main-chain atom pairs contribute more to the correlation than the interfacial side chain atom pairs do, and the polar atom pairs contribute more than the non-polar atom pairs do. Interfacial atom pairs with atomic distance in the range of 6-12 A are the most important to explain the differences in binding free energies in the datasets.
Asunto(s)
Complejos Multiproteicos/química , Interpretación Estadística de Datos , Unión ProteicaRESUMEN
How protein molecules pack into a crystal remains problematic. Packing units are direct materials for packing into crystals. The group generator method is introduced for automatically identifying the packing unit. By introducing deviations into the nucleation stage of crystallization, we proved that these deviations diminish in further packing. This process illustrates how translation symmetries are achieved by the growing of nuclei. Two effects, the size effect and the close up effect, are found to behave differently in this process.
Asunto(s)
Núcleo Celular/metabolismo , Biosíntesis de Proteínas , Proteínas/metabolismo , Cristalización , Modelos Biológicos , Proteínas/química , Proteínas/genéticaRESUMEN
The crystal structure of pokeweed antiviral protein from seeds of Phytolacca americana (PAP-S) was solved at 1.8A. PAP-S is a one-chain ribosome-inactivating protein (RIP) and distinctively contains three well-defined N-acetylglucosamines, each covalently linked to an asparagine residue at positions, 10, 44, and 255, respectively. The high-resolution structure clearly shows the three mono-sugars to have either an alpha- or a beta-conformation. Two of sugars are located on the same side of the molecule with the active pocket. Except one hydrogen bond, there are no intermolecular interactions between the polypeptide chain and the sugars. Instead the sugar conformations appear to be stabilized by intermolecular interactions. The sugar structure defined at high resolution provides a structural basis for understanding their possible biological activity. The structural comparisons of PAP-S with other PAPs reveal that the major disparity of these homologous molecules is the different charge distribution on the upper right side of the front side near the active pocket. Based on the available structure of the 50S ribosomal subunit, the possible interactions between PAPs and the ribosome are discussed.
Asunto(s)
Carbohidratos/química , N-Glicosil Hidrolasas , Proteínas de Plantas/química , Proteínas de Plantas/ultraestructura , Semillas/ultraestructura , Acetilglucosamina/química , Secuencia de Aminoácidos , Asparagina/química , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/química , Ribosomas/ultraestructura , Homología de Secuencia de AminoácidoRESUMEN
Thioredoxin, DsbA, the N-terminal active-site domain a and the non-active-site domain b of protein-disulfide isomerase are all monomeric with a thioredoxin fold, and each exhibits low or no isomerase and chaperone activity. We have linked the N terminus of the above four monomers, individually, to the C terminus of the N-terminal domain of DsbC via the flexible linker helix of the latter to produce four domain hybrids, DsbCn-Trx, DsbCn-DsbA, DsbCn-PDIa, and DsbCn-PDIb. These four hybrid proteins form homodimers, and except for DsbCn-PDIb they exhibit new or greatly elevated isomerase as well as chaperone activity. Three-dimensional structure prediction indicates that all the four domain hybrids adopt DsbC-like V-shaped structure with a broad uncharged cleft between the two arms for binding of non-native protein folding intermediates. The results provide strong evidence that dimerization creates chaperone and isomerase activity for monomeric thiol-protein oxidases or reductases, and suggesting a pathway for proteins to acquire new functions and/or higher biological efficiency during evolution.