RESUMEN
BACKGROUND: Obligate pollination mutualisms (OPMs) are specialized interactions in which female pollinators transport pollen between the male and female flowers of a single plant species and then lay eggs into those same flowers. The pollinator offspring hatch and feed upon some or all of the developing ovules pollinated by their mothers. Strong trait matching between plants and their pollinators in OPMs is expected to result in reciprocal partner specificity i.e., a single pollinator species using a single plant species and vice versa, and strict co-speciation. These issues have been studied extensively in figs and fig wasps, but little in the more recently discovered co-diversification of Epicephala moths and their Phyllanthaceae hosts. OPMs involving Epicephala moths are believed occur in approximately 500 species of Phyllanthaceae, making it the second largest OPM group after the Ficus radiation (> 750 species). In this study, we used a mixture of DNA barcoding, genital morphology and behavioral observations to determine the number of Epicephala moth species inhabiting the fruits of Breynia oblongifolia, their geographic distribution, pollinating behavior and phylogenetic relationships. RESULTS: We found that B. oblongifolia hosts two species of pollinator that co-occurred at all study sites, violating the assumption of reciprocal specificity. Male and female genital morphologies both differed considerably between the two moth species. In particular, females differed in the shape of their ovipositors, eggs and oviposition sites. Phylogenetic analyses indicated that the two Epicephala spp. on B. oblongifolia likely co-exist due to a host switch. In addition, we discovered that Breynia fruits are also often inhabited by a third moth, an undescribed species of Herpystis, which is a non-pollinating seed parasite. CONCLUSIONS: Our study reveals new complexity in interactions between Phyllantheae and Epicephala pollinators and highlights that host switching, co-speciation and non-pollinating seed parasites can shape species interactions in OPMs. Our finding that co-occurring Epicephala species have contrasting oviposition modes parallels other studies and suggests that such traits are important in Epicephala species coexistence.
Asunto(s)
Malpighiaceae/parasitología , Parásitos/fisiología , Polinización/fisiología , Animales , Teorema de Bayes , Código de Barras del ADN Taxonómico , Femenino , Geografía , Masculino , Mariposas Nocturnas/anatomía & histología , Mariposas Nocturnas/fisiología , Mariposas Nocturnas/ultraestructura , Nueva Gales del Sur , Ovario/citología , Oviposición , Óvulo Vegetal/citología , Parásitos/anatomía & histología , Parásitos/ultraestructura , Filogenia , Especificidad de la EspecieRESUMEN
A study of papers on amyloid fibers suggested to us that cylindrical beta-sheets are the only structures consistent with some of the x-ray and electron microscope data. We then found that our own 7-year-old and hitherto enigmatic x-ray diagram of poly-L-glutamine fits a cylindrical sheet of 31 A diameter made of beta-strands with 20 residues per helical turn. Successive turns are linked by hydrogen bonds between both the main chain and side chain amides, and side chains point alternately into and out of the cylinder. Fibers of the exon-1 peptide of huntingtin and of the glutamine- and asparagine-rich region of the yeast prion Sup35 give the same underlying x-ray diagrams, which show that they have the same structure. Electron micrographs show that the 100-A-thick fibers of the Sup35 peptide are ropes made of three protofibrils a little over 30 A thick. They have a measured mass of 1,450 Da/A, compared with 1,426 Da/A for a calculated mass of three protofibrils each with 20 residues per helical turn wound around each other with a helical pitch of 510 A. Published x-ray diagrams and electron micrographs show that fibers of synuclein, the protein that forms the aggregates of Parkinson disease, consist of single cylindrical beta-sheets. Fibers of Alzheimer A beta fragments and variants are probably made of either two or three concentric cylindrical beta-sheets. Our structure of poly-L-glutamine fibers may explain why, in all but one of the neurodegenerative diseases resulting from extension of glutamine repeats, disease occurs when the number of repeats exceeds 37-40. A single helical turn with 20 residues would be unstable, because there is nothing to hold it in place, but two turns with 40 residues are stabilized by the hydrogen bonds between their amides and can act as nuclei for further helical growth. The A beta peptide of Alzheimer's disease contains 42 residues, the best number for nucleating further growth. All these structures are very stable; the best hope for therapies lies in preventing their growth.
Asunto(s)
Amiloide/química , Proteínas de Saccharomyces cerevisiae , Enfermedad de Alzheimer/metabolismo , Asparagina/química , Cristalografía por Rayos X , Exones , Análisis de Fourier , Proteínas Fúngicas/metabolismo , Glutamina/química , Humanos , Proteína Huntingtina , Enlace de Hidrógeno , Microscopía Electrónica , Proteínas del Tejido Nervioso/química , Proteínas Nucleares/química , Factores de Terminación de Péptidos , Péptidos/química , Priones/química , Estructura Secundaria de ProteínaRESUMEN
The type 1 human immunodeficiency virus (HIV-1) contains a conical capsid comprising approximately 1,500 CA protein subunits, which organizes the viral RNA genome for uncoating and replication in a new host cell. In vitro, CA spontaneously assembles into helical tubes and cones that resemble authentic viral capsids. Here we describe electron cryo-microscopy and image reconstructions of CA tubes from six different helical families. In spite of their polymorphism, all tubes are composed of hexameric rings of CA arranged with approximate local p6 lattice symmetry. Crystal structures of the two CA domains were 'docked' into the reconstructed density, which showed that the amino-terminal domains form the hexameric rings and the carboxy-terminal dimerization domains connect each ring to six neighbours. We propose a molecular model for the HIV-1 capsid that follows the principles of a fullerene cone, in which the body of the cone is composed of curved hexagonal arrays of CA rings and the ends are closed by inclusion of 12 pentagonal 'defects'.
Asunto(s)
Cápside/química , VIH-1/química , Cápside/ultraestructura , VIH-1/ultraestructura , Procesamiento de Imagen Asistido por Computador , Modelos Moleculares , Conformación ProteicaRESUMEN
Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutations, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.
Asunto(s)
Demencia/genética , Neuropéptidos/genética , Mutación Puntual , Serpinas/genética , Biopolímeros/genética , Biopolímeros/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Demencia/patología , Femenino , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Masculino , Neuropéptidos/metabolismo , Prolina , Serina , Serpinas/metabolismo , NeuroserpinaRESUMEN
Importin-alpha is a cytosolic receptor that recognizes classical Nuclear Localization Signals (NLSs) and mediates import into the nucleus. We have used a number of methods to investigate the aggregation state of Xenopus importin-alpha both as a recombinant, purified protein and in cytosolic extracts. We have found that recombinant importin-alpha aggregates at a protein concentration similar to that estimated to be present in the Xenopus cytoplasm, and that the importin-alpha aggregation is relieved by NLS peptide binding, with the importin-alpha then binding the NLS as a monomer. We have also found that in HeLa cytosolic extracts, importin-alpha is present in an aggregated form. Similarly to the purified importin-alpha aggregation, NLS peptides relieve the aggregation of importin-alpha in the cytosol. These observations indicate that aggregation of importin-alpha in the cytosol may be an intrinsic property of the import receptor and may be functionally related to NLS binding.Our results suggest a novel mechanism for NLS recognition, whereby NLSs mediate disassembly of importin-alpha aggregates in the cytosol.
Asunto(s)
Proteínas Nucleares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Citosol/química , Células HeLa , Humanos , Carioferinas , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Membrana Nuclear/metabolismo , Señales de Localización Nuclear , Proteínas Nucleares/genética , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Ultracentrifugación , XenopusRESUMEN
The genome of the human immunodeficiency virus (HIV) is packaged within an unusual conical core particle located at the center of the infectious virion. The core is composed of a complex of the NC (nucleocapsid) protein and genomic RNA, surrounded by a shell of the CA (capsid) protein. A method was developed for assembling cones in vitro using pure recombinant HIV-1 CA-NC fusion proteins and RNA templates. These synthetic cores are capped at both ends and appear similar in size and morphology to authentic viral cores. It is proposed that both viral and synthetic cores are organized on conical hexagonal lattices, which by Euler's theorem requires quantization of their cone angles. Electron microscopic analyses revealed that the cone angles of synthetic cores were indeed quantized into the five allowed angles. The viral core and most synthetic cones exhibited cone angles of approximately 19 degrees (the narrowest of the allowed angles). These observations suggest that the core of HIV is organized on the principles of a fullerene cone, in analogy to structures recently observed for elemental carbon.
Asunto(s)
Cápside/química , Productos del Gen gag/química , VIH-1/química , Modelos Biológicos , Nucleocápside/química , ARN Viral/química , Cápside/ultraestructura , VIH-1/ultraestructura , Matemática , Microscopía Electrónica , Nucleocápside/ultraestructura , Proteínas Recombinantes de Fusión/química , Moldes GenéticosRESUMEN
Possible stereochemical determinants of the orientation of TBP on the TATA box are discussed using the crystal coordinates of TBP-TATA complexes, which have been determined by other groups. The C-terminal half of the TBP beta-sheet interacts with the TATA site of the DNA, and the N-terminal half with the A-rich site, so that the two sites with distinct curvatures produce a unique fit. Although chemical contacts take place between one side of the beta-sheet and the DNA minor groove, the interaction seems to be facilitated indirectly by the characteristics of the other side of the beta-sheet and the DNA major groove. Thus, Ala71, Leu162 and Pro190 differentiate the curvature of the beta-sheet in the N- and C-halves. The methyl positions in the DNA major groove modulate the bendability of the two DNA sites by using differences in the rolling capacity of TA and AT compared with PyT, and in the shifting capacity of AT compared with TT. The deformations of the first steps (TA and PyT) in the two sites are the largest and thus are important for the overall bending of the DNA. The differences between the two DNA sites are greatest at the second steps (AT and TT) and so these are important for determining the orientation of TBP.
Asunto(s)
Proteínas de Unión al ADN/química , TATA Box , Factores de Transcripción/química , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Estructura Secundaria de Proteína , Proteína de Unión a TATA-Box , Factores de Transcripción/metabolismoRESUMEN
Sequence-specific conformational differences between dinucleotide steps are characterised using published crystal coordinates with special attention to steric hindrance of the methyl group of a T base to the neighbouring base, and, more importantly, to the sugar-phosphate backbone. The TT step is inflexible and B-like, as it has two methyl groups which interlock with each other and with the sugar-phosphate backbones. AT slides, or overtwists, so that the methyl groups move away from the backbones, both lead the step towards the A-conformation. TA is most flexible as it does not have such restriction. These characteristics are observed with other pyrimidine-pyrimidine, pyrimidine-purine, purine-pyrimidine steps, respectively, but to less extent, depending on the number of non-A:T basepairs in the steps.
Asunto(s)
ADN/química , Fosfatos de Dinucleósidos/química , Conformación de Ácido Nucleico , Secuencia de Bases , Cristalografía por Rayos X , Bases de Datos Factuales , Enlace de Hidrógeno , Modelos MolecularesRESUMEN
The Z (Glu342-->Lys) and Siiyama (Ser53-->Phe) deficiency variants of alpha 1-antitrypsin result in the retention of protein in the endoplasmic reticulum of the hepatocyte by loop-sheet polymerization in which the reactive center loop of one molecule is inserted into a beta-pleated sheet of a second. We show here that antitrypsin Mmalton (Phe52-deleted), which is associated with the same liver inclusions, is also retained at an endoglycosidase H-sensitive stage of processing in the Xenopus oocyte and spontaneously forms polymers in vivo. These polymers, obtained from the plasma of an Mmalton/QO (null) bolton heterozygote, were much shorter than other antitrypsin polymers and contained a reactive center loop-cleaved species. Monomeric mutant antitrypsin was also isolated from the plasma. The monomeric component had a normal unfolding transition on transverse urea gradient gel electrophoresis and formed polymers in vitro more readily than M, but less readily than Z, antitrypsin. The A beta-sheet accommodated a reactive center loop peptide much less readily than Z antitrypsin, which in turn was less receptive than native M antitrypsin. The nonreceptive conformation of the A sheet in antitrypsin Mmalton had little effect on kinetic parameters, the formation of SDS-stable complexes, the S to R transition, and the formation of the latent conformation. Comparison of the results with similar findings of short chain polymers associated with the antithrombin variant Rouen VI (Bruce, D., Perry, D., Borg, J.-Y., Carrell, R. W., and Wardell, M. R. (1994) J. Clin. Invest. 94, 2265-2274) suggests that polymerization is more complicated than the mechanism proposed earlier. The Z, Siiyama, and Mmalton mutations favor a conformational change in the antitrypsin molecule to an intermediate between the native and latent forms. This would involve a partial overinsertion of the reactive loop into the A sheet with displacement of strand 1C and consequent loop-C sheet polymerization.
Asunto(s)
Mutación , Polímeros/química , alfa 1-Antitripsina/química , Secuencia de Aminoácidos , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Conformación Proteica , alfa 1-Antitripsina/genéticaRESUMEN
To find conditions for obtaining diffraction-quality crystals of a hammerhead RNA rapidly and reproducibly, we employed a "double screening" procedure in which we screened six different RNA synthetic constructs against 48 crystallization conditions using a newly devised sparse matrix. We obtained crystals immediately and diffraction-quality crystals of the sixth RNA construct within six months of initiating the screening of additional RNA sequences. The best crystals diffract to 2.9 A resolution when flash-cooled at synchrotron X-ray sources. Solid-support chemical synthesis combined with sparse matrix screening should allow rapid production of diffraction-quality crystals of a variety of small RNAs, reducing the time commitment for initiating such crystallography projects from several years to several months. The synthetic approach also makes introduction of modified bases to prevent self-cleavage and to generate isomorphous heavy-atom derivative crystals a rapid and straightforward process.
Asunto(s)
Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/síntesis química , Secuencia de Bases , Cristalización , Datos de Secuencia MolecularRESUMEN
We have solved the crystal structure of an all-RNA hammerhead ribozyme having a single 2'-O-methyl cytosine incorporated at the active site to prevent cleavage. The conditions used differ from those in another recent solution in four significant ways: first, it is an all-RNA ribozyme rather than a DNA-RNA hybrid; second, the connectivity of the ribozyme backbone strands is different; third, the crystals were grown in the presence of a much lower concentration of salt; and fourth, the crystal packing scheme is very different. Nevertheless, the three-dimensional structure of the all-RNA hammerhead ribozyme is similar to the previous structure. Five potential Mg(II)-binding sites are identified, including one positioned near the ribozyme catalytic pocket. Upon this basis, as well as upon comparisons with the metal-binding sites in the structurally homologous uridine turn of tRNAPhe, we propose a mechanism for RNA catalytic cleavage.
Asunto(s)
Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/metabolismo , Secuencia de Bases , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Citidina/análogos & derivados , ADN/química , ADN/metabolismo , Enlace de Hidrógeno , Magnesio/metabolismo , Metales , Modelos Moleculares , Datos de Secuencia Molecular , ARN/química , ARN/metabolismo , ARN de Transferencia de Fenilalanina/química , ARN de Transferencia de Fenilalanina/metabolismoRESUMEN
We have solved the crystal structure of an all-RNA hammerhead ribozyme by isomorphous replacement which contains a single 2'-O-methyl-cytosine at the active site to prevent cleavage. We describe the hammerhead RNA structure from a point of view which shows how the structural elements are disposed to bring the active site nucleotide into the catalytic pocket of the ribozyme. Five potential Mg(II) binding sites can be identified in the hammerhead RNA electron density maps. Of these, one is a newly identified metal site positioned near the ribozyme catalytic pocket, and another site corresponds to a Mn(II) site identified in the previous hammerhead RNA structure. We propose a mechanism for RNA catalytic cleavage on the basis of this new metal-binding site, as well as upon comparisons between the catalytic pocket of the hammerhead RNA and the metal-binding sites in the structurally homologous uridine turn of the anticodon loop in tRNAPhe.
Asunto(s)
ARN Catalítico/química , Secuencia de Bases , Sitios de Unión , Catálisis , Cristalización , Citosina/análogos & derivados , Citosina/química , Magnesio/metabolismo , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN de Transferencia de Fenilalanina/metabolismo , Uridina/químicaRESUMEN
Four inherited neurodegenerative diseases are linked to abnormally expanded repeats of glutamine residues in the affected proteins. Molecular modeling followed by optical, electron, and x-ray diffraction studies of a synthetic poly(L-glutamine) shows that it forms beta-sheets strongly held together by hydrogen bonds. Glutamine repeats may function as polar zippers, for example, by joining specific transcription factors bound to separate DNA segments. Their extension may cause disease either by increased, nonspecific affinity between such factors or by gradual precipitation of the affected proteins in neurons.
Asunto(s)
Glutamina/química , Enfermedad de Huntington/etiología , Atrofia Muscular Espinal/etiología , Proteínas del Tejido Nervioso/química , Proteínas Nucleares/química , Receptores Androgénicos/química , Degeneraciones Espinocerebelosas/etiología , Secuencia de Aminoácidos , Genes Dominantes , Humanos , Proteína Huntingtina , Enlace de Hidrógeno , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Monoclonal antibodies against the 110-kD component of the yeast spindle pole body (SPB) were used to clone the corresponding gene SPC110. SPC110 is identical to NUF1 (Mirzayan, C., C. S. Copeland, and M. Synder. 1992. J. Cell Biol. 116:1319-1332). SPC110/NUF1 has an MluI cell cycle box consensus sequence in its putative promoter region, and we found that the transcript was cell cycle regulated in a similar way to other MluI-regulated transcripts. Spc110p/Nuflp has a long central region with a predicted coiled-coil structure. We expressed this region in Escherichia coli and showed by rotary shadowing that rods of the predicted length were present. The 110-kD component is localized in the SPB to the gap between the central plaque and the sealed ends of the nuclear microtubules near the inner plaque (Rout, M., and J. V. Kilmartin. 1990. J. Cell Biol. 111:1913-1927). We found that rodlike structures bridge this gap. When truncations of SPC110 with deletions in the coiled-coil region of the protein replaced the wild-type gene, the gap between the central plaque and the ends of the microtubules decreased in proportion to the size of the deletion. This suggests that Spc110p connects these two parts of the SPB together and that the coiled-coil domain acts as a spacer element.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Huso Acromático/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión a Calmodulina , Ciclo Celular/fisiología , Clonación Molecular , Proteínas del Citoesqueleto , Análisis Mutacional de ADN , Epítopos , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/inmunología , Proteínas Fúngicas/aislamiento & purificación , Microscopía Electrónica , Datos de Secuencia Molecular , Proteínas Nucleares/inmunología , Proteínas Nucleares/aislamiento & purificación , Fragmentos de Péptidos/biosíntesis , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Huso Acromático/ultraestructuraRESUMEN
The Cys2-His2 zinc-finger is the most widely occurring DNA-binding motif. The first structure of a zinc-finger/DNA complex revealed a fairly simple mechanism for DNA recognition suggesting that the zinc-finger might represent a candidate template for designing proteins to recognize DNA. Residues at three key positions in an alpha-helical 'reading head' play a dominant role in base-recognition and have been targets for mutagenesis experiments aimed at deriving a recognition code. Here we report the structure of a two zinc-finger DNA-binding domain from the protein Tramtrack complexed with DNA. The amino-terminal zinc-finger and its interaction with DNA illustrate several novel features. These include the use of a serine residue, which is semi-conserved and located outside the three key positions, to make a base contact. Its role in base-recognition correlates with a large, local, protein-induced deformation of the DNA helix at a flexible A-T-A sequence and may give insight into previous mutagenesis experiments. It is apparent from this structure that zinc-finger/DNA recognition is more complex than was originally perceived.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/metabolismo , Proteínas de Drosophila , Proteínas Represoras , Factores de Transcripción/química , Dedos de Zinc , Secuencia de Aminoácidos , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Factores de Transcripción/metabolismoRESUMEN
The nuclear hormone receptors are a superfamily of ligand-activated DNA-binding transcription factors. We have determined the crystal structure (at 2.4 A) of the fully specific complex between the DNA-binding domain from the estrogen receptor and DNA. The protein binds as a symmetrical dimer to its palindromic binding site consisting of two 6 bp consensus half sites with three intervening base pairs. This structure reveals how the protein recognizes its own half site sequence rather than that of the related glucocorticoid receptor, which differs by only two base pairs. Since all nuclear hormone receptors recognize one or the other of these two consensus half site sequences, this recognition mechanism applies generally to the whole receptor family.
Asunto(s)
ADN/química , Receptores de Estrógenos/química , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Cristalografía por Rayos X , Regulación de la Expresión Génica , Enlace de Hidrógeno , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Glucocorticoides/químicaRESUMEN
BACKGROUND: The steroid/nuclear hormone receptors are a large family of conserved ligand-activated transcription factors that regulate gene expression through binding to response elements upstream of their target genes. Most members of this family bind to DNA as homodimers or heterodimers and recognize the sequence, spacing and orientation of the two half-sites of their response elements. The recognition and discrimination of the sequence and arrangements of these half-sites are mediated primarily by a highly conserved DNA-binding domain. RESULTS: Here we describe the DNA-binding properties of the isolated DNA-binding domain of the oestrogen receptor, the ERDBD, and its refined NMR structure. This domain is monomeric in solution, but two molecules bind cooperatively to specific DNA sequences; this cooperativity determines the arrangement of half-sites that is recognized by the ERDBD. The 10 carboxy-terminal residues and a region of 15 residues within the domain are disordered in the solution structure, yet are important for DNA binding. CONCLUSION: The cooperative nature of ERDBD binding to DNA is important. The previously-determined X-ray structure of the ERDBD dimer bound to DNA shows that the 15 internal residues disordered in solution make contact both with DNA and with the corresponding region of the other monomer. These results suggest that these residues become ordered during the process of binding to DNA, forming the dimer interface and thus contributing to the cooperative interaction between monomers.
Asunto(s)
ADN/metabolismo , Receptores de Estrógenos/química , Receptores de Estrógenos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , ADN/química , Imagenología Tridimensional , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Antitrypsin Siiyama is a rare example of the deficiency variants of antitrypsin that accumulate in the endoplasmic reticulum of the hepatocyte. The common example is Z antitrypsin, which has a mutation (Glu342-->Lys) at the junction of the head of the fifth strand of the A sheet and the base of the reactive center loop. It was previously shown that Z antitrypsin spontaneously polymerizes due to the insertion of the reactive center loop of one molecule into the A sheet of a second. The mutation in antitrypsin Siiyama (Ser53-->Phe) affects a residue that provides a ridge for the sliding movement that opens the A sheet, and it had been predicted that this would result in the same type of loop-sheet polymerization observed with the Z variant. We confirm this here and show that virtually all the plasma antitrypsin in a homozygote for the Siiyama variant was polymerized due to non-covalent bonding with a loss of accessibility of the reactive center loop. The common basis of the polymerization of Z and Siiyama antitrypsin is supported by identical findings on electron microscopy. Taken together these results confirm that loop-sheet polymerization is a general mechanism and as such is likely to be responsible for the intracellular inclusions associated with liver pathology.
Asunto(s)
Mutación , Fenilalanina/química , Serina/química , alfa 1-Antitripsina/genética , Adulto , Animales , Bovinos , Quimotripsina/metabolismo , Homocigoto , Humanos , Hepatopatías/genética , Masculino , Microscopía Electrónica , Modelos Moleculares , Fenilalanina/metabolismo , Polímeros , Serina/genética , alfa 1-Antitripsina/química , alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/ultraestructuraRESUMEN
The structure of GH5, the globular domain of the linker histone H5, has been solved to 2.5 A resolution by multiwavelength anomalous diffraction on crystals of the selenomethionyl protein. The structure shows a striking similarity to the DNA-binding domain of the catabolite gene activator protein CAP, thereby providing a possible model for the binding of GH5 to DNA.