RESUMO
Trehalose dimycolate, an unusual glycolipid in the outer membrane of Mycobacterium tuberculosis, stimulates macrophages by binding to the macrophage receptor mincle. This stimulation plays an important role both in infection by mycobacteria and in the use of derivatives of mycobacteria as adjuvants to enhance the immune response. The mechanism of trehalose dimycolate binding to the C-type carbohydrate-recognition domain in human mincle has been investigated using a series of synthetic analogs of trehalose dimycolate and site-directed mutagenesis of the human protein. The results support a mechanism of binding acylated trehalose derivatives to human mincle that is very similar to the mechanism of binding to bovine mincle, in which one glucose residue in the trehalose headgroup of the glycolipid is ligated to the principle Ca(2+)-binding site in the carbohydrate-recognition domain, with specificity for the disaccharide resulting from interactions with the second glucose residue. Acyl chains attached to the 6-OH groups of trehalose enhance affinity, with the affinity dependent on the length of the acyl chains and the presence of a hydrophobic groove adjacent to the sugar-binding sites. The results indicate that the available crystal structure of the carbohydrate-recognition domain of human mincle is unlikely to be in a fully active conformation. Instead, the ligand-binding conformation probably resembles closely the structure observed for bovine mincle in complex with trehalose. These studies provide a basis for targeting human mincle as a means of inhibiting interactions with mycobacteria and as an approach to harnessing the ability of mincle to stimulate the immune response.
Assuntos
Fatores Corda/química , Lectinas Tipo C/química , Mycobacterium tuberculosis/química , Receptores Imunológicos/química , Animais , Sítios de Ligação , Cálcio/química , Bovinos , Fatores Corda/síntese química , Humanos , Concentração de Íons de Hidrogênio , Lectinas Tipo C/metabolismo , Modelos Moleculares , Conformação Proteica , Receptores Imunológicos/metabolismoRESUMO
The MUS81-EME1 endonuclease maintains metazoan genomic integrity by cleaving branched DNA structures that arise during the resolution of recombination intermediates. In humans, MUS81 also forms a poorly characterized complex with EME2. Here, we identify and determine the structure of a winged helix (WH) domain from human MUS81, which binds DNA. WH domain mutations greatly reduce binding of the isolated domain to DNA and impact on incision activity of MUS81-EME1/EME2 complexes. Deletion of the WH domain reduces the endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes, and incisions made by MUS81-EME2 are made closer to the junction on substrates containing a downstream duplex, such as fork structures and nicked Holliday junctions. WH domain mutation or deletion in Schizosaccharomyces pombe phenocopies the DNA-damage sensitivity of strains deleted for mus81. Our results indicate an important role for the WH domain in both yeast and human MUS81 complexes.
Assuntos
Proteínas de Ligação a DNA/química , Endodesoxirribonucleases/metabolismo , Endonucleases/química , Sequência de Aminoácidos , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Estrutura Terciária de ProteínaRESUMO
The structure-specific endonuclease activity of the human XPF-ERCC1 complex is essential for a number of DNA processing mechanisms that help to maintain genomic integrity. XPF-ERCC1 cleaves DNA structures such as stem-loops, bubbles or flaps in one strand of a duplex where there is at least one downstream single strand. Here, we define the minimal substrate requirements for cleavage of stem-loop substrates allowing us to develop a real-time fluorescence-based assay to measure endonuclease activity. Using this assay, we show that changes in the sequence of the duplex upstream of the incision site results in up to 100-fold variation in cleavage rate of a stem-loop substrate by XPF-ERCC1. XPF-ERCC1 has a preference for cleaving the phosphodiester bond positioned on the 3'-side of a T or a U, which is flanked by an upstream T or U suggesting that a T/U pocket may exist within the catalytic domain. In addition to an endonuclease domain and tandem helix-hairpin-helix domains, XPF has a divergent and inactive DEAH helicase-like domain (HLD). We show that deletion of HLD eliminates endonuclease activity and demonstrate that purified recombinant XPF-HLD shows a preference for binding stem-loop structures over single strand or duplex alone, suggesting a role for the HLD in initial structure recognition. Together our data describe features of XPF-ERCC1 and an accepted model substrate that are important for recognition and efficient incision activity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endonucleases/metabolismo , DNA/química , Clivagem do DNA , Proteínas de Ligação a DNA/química , Ensaios Enzimáticos/métodos , Fluorometria/métodos , Humanos , Cinética , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.
Assuntos
Proteínas de Homeodomínio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Movimento Celular/genética , Movimento Celular/fisiologia , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Immunoblotting , Proteínas com Domínio LIM , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)(2) domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3' flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes.
Assuntos
DNA Arqueal/química , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Aeropyrum/genética , Aeropyrum/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Reparo do DNA , DNA Arqueal/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Substâncias Macromoleculares , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
The xeroderma pigmentosa group F protein (XPF) is a founding member of a family of 3'-flap endonucleases that play an essential role in nucleotide-excision repair, DNA replication and recombination. The XPF gene has been cloned from Aeropyrum pernix, encoding a 254-residue protein (apXPF). Recombinant protein was produced in Escherichia coli and purified by three chromatographic steps. Three different crystal forms of apXPF were grown in trigonal, monoclinic and triclinic systems. The trigonal crystals diffracted to 2.8 A and were grown in the presence of double-stranded DNA. Monoclinic crystals were grown without DNA and diffracted to 3.2 A. Triclinic crystals were grown from a truncated apXPF protein lacking the tandem helix-hairpin-helix motifs and diffracted to 2.1 A.
Assuntos
Aeropyrum/enzimologia , Endonucleases/química , Xeroderma Pigmentoso/enzimologia , Aeropyrum/genética , Cristalização , Cristalografia por Raios X , DNA/química , Primers do DNA , Endonucleases/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Peso Molecular , Plasmídeos/genética , Proteínas RecombinantesRESUMO
Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.
Assuntos
Galectinas/classificação , Galectinas/genética , Filogenia , Vertebrados/genética , Animais , Cromossomos/genética , Evolução Molecular , HumanosRESUMO
The Kupffer cell receptor for glycoproteins has been reported to have a role in clearance of galactose- and fucose-terminated glycoproteins from circulation. Although the gene and a cDNA encoding the receptor have been described, there has been little study of the receptor protein. To address some questions about possible ligands and functions for this receptor, fragments representing portions of the extracellular domain have been expressed and characterized. The extracellular domain consists of a trimer stabilized by an extended coiled-coil of alpha-helices. The receptor displays monosaccharide-binding characteristics similar to the hepatic asialoglycoprotein receptor, but with somewhat less selectivity. The two best monosaccharide ligands are GalNAc and galactose. alpha-Methyl fucoside is a particularly poor ligand. Analysis of Kupffer cell receptor binding to glycoproteins and oligosaccharides released from them reveals highest affinity for desialylated, complex N-linked glycans. The best glycoprotein ligands contain multiple highly branched oligosaccharides. A human ortholog of the rat receptor gene does not encode a full-length protein and is not expressed in liver. These characteristics suggest that the receptor may have functions parallel to those of the hepatocyte asialoglycoprotein receptor in some (but not all) mammalian species.
Assuntos
Glicoproteínas/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Receptor de Asialoglicoproteína , Glicolipídeos/metabolismo , Humanos , Dados de Sequência Molecular , Monossacarídeos/metabolismo , Neuraminidase , Oligossacarídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores Imunológicos/genética , Receptores Imunológicos/isolamento & purificação , Proteínas Recombinantes , Alinhamento de SequênciaRESUMO
Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains. At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell-cell signalling, glycoprotein turnover and innate immunity. Genome-wide analysis of potential carbohydrate-binding domains is now possible. Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only. At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates. The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena. An overall picture of the lectins present in humans can now be proposed. Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome. Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.