共查询到20条相似文献,搜索用时 11 毫秒
1.
Searle MS Garner TP Strachan J Long J Adlington J Cavey JR Shaw B Layfield R 《Biochemical Society transactions》2012,40(2):404-408
UBDs [Ub (ubiquitin)-binding domains], which are typically small protein motifs of <50 residues, are used by receptor proteins to transduce post-translational Ub modifications in a wide range of biological processes, including NF-κB (nuclear factor κB) signalling and proteasomal degradation pathways. More than 20 families of UBDs have now been characterized in structural detail and, although many recognize the canonical Ile44/Val70-binding patch on Ub, a smaller number have alternative Ub-recognition sites. The A20 Znf (A20-like zinc finger) of the ZNF216 protein is one of the latter and binds with high affinity to a polar site on Ub centred around Asp58/Gln62. ZNF216 shares some biological function with p62, with both linked to NF-κB signal activation and as shuttle proteins in proteasomal degradation pathways. The UBA domain (Ub-associated domain) of p62, although binding to Ub through the Ile44/Val70 patch, is unique in forming a stable dimer that negatively regulates Ub recognition. We show that the A20 Znf and UBA domain are able to form a ternary complex through independent interactions with a single Ub molecule, supporting functional models for Ub as a 'hub' for mediating multi-protein complex assembly and for enhancing signalling specificity. 相似文献
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Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process. 相似文献
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Several reports from mammals indicate that an increase in the mutation rate in late-replicating regions may, in part, be responsible for the observed genomic heterogeneity in neutral substitution rates and levels of diversity, although the mechanisms for this remain poorly understood. Recent evidence also suggests that late replication is associated with high mutability in yeast. This then raises the question as to whether a similar effect is operating across all eukaryotes. Limited evidence from one chromosome arm in Drosophila melanogaster suggests the opposite pattern, with regions overlapping early-firing origins showing increased levels of diversity and divergence. Given the availability of genome-wide replication timing profiles for D. melanogaster, we now return to this issue. Consistent with what is seen in other taxa, we find that divergence at synonymous sites in exon cores, as well as divergence at putatively unconstrained intronic sites, is elevated in late-replicating regions. Analysis of genes with low codon usage bias suggests a ~30% difference in mutation rate between the earliest and the latest replicating sequence. Intronic sequence suggests a more modest difference. We additionally show that an increase in diversity in late-replicating sequences is not owing to replication timing covarying with the local recombination rate. If anything, the effects of recombination mask the impact of replication timing. We conclude that, contrary to prior reports and consistent with what is seen in mammals and yeast, there is indeed a relationship between rates of nucleotide divergence and diversity and replication timing that is consistent with an increase in the mutation rate during late S-phase in D. melanogaster. It is therefore plausible that such an effect might be common among eukaryotes. The result may have implications for the inference of positive selection. 相似文献
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Although smallpox has been eradicated from the human population, it is presently feared as a possible agent of bioterrorism. The smallpox virus codes for its own topoisomerase enzyme that differs from its cellular counterpart by requiring a specific DNA sequence for activation of catalysis. Here we present crystal structures of the smallpox virus topoisomerase enzyme bound both covalently and noncovalently to a specific DNA sequence. These structures reveal the basis for site-specific DNA recognition, and they explain how catalysis is likely activated by formation of a specific enzyme-DNA interface. Unexpectedly, the poxvirus enzyme uses a major groove binding alpha helix that is not present in the human enzyme to recognize part of the core recognition sequence and activate the enzyme for catalysis. The topoisomerase-DNA complex structures also provide a three-dimensional framework that may facilitate the rational design of therapeutic agents to treat poxvirus infections. 相似文献
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Welin M Skovgaard T Knecht W Zhu C Berenstein D Munch-Petersen B Piskur J Eklund H 《The FEBS journal》2005,272(14):3733-3742
The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3'-modified nucleoside analogs like 3'-azidothymidine (AZT) was nearly unchanged. Here, we identify the mutation N64D as being responsible for these changes. Furthermore, we crystallized the mutant enzyme in the presence of one of its substrates, thymidine, and the feedback inhibitor, dTTP. The introduction of the charged Asp residue appears to destabilize the LID region (residues 167-176) of the enzyme by electrostatic repulsion and no hydrogen bond to the 3'-OH is made in the substrate complex by Glu172 of the LID region. This provides a binding space for more bulky 3'-substituents like the azido group in AZT but influences negatively the interactions between Dm-dNK, substrates and feedback inhibitors based on deoxyribose. The detailed picture of the structure-function relationship provides an improved background for future development of novel mutant suicide genes for Dm-dNK-mediated gene therapy. 相似文献
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Structural basis of membrane invagination by F-BAR domains 总被引:1,自引:0,他引:1
Frost A Perera R Roux A Spasov K Destaing O Egelman EH De Camilli P Unger VM 《Cell》2008,132(5):807-817
BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination. 相似文献
8.
Kuhn H 《Prostaglandins & other lipid mediators》2000,62(3):255-270
The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of mammalian lipoxygenases but, unfortunately, the structural reasons for lipoxygenase specificity are not well understood. Although there are no direct structural data on lipoxygenase/substrate interaction, experiments with modified fatty acid substrates and mutagenesis studies suggest that for 12- and 15-lipoxygenases, arachidonic acid slides into the substrate-binding pocket with its methyl end ahead. For arachidonate 5- and/or 8-lipoxygenation two alternative models for the enzyme/substrate interaction have been developed: 1) The orientation-determined model and 2) the space-determined model. This review explores the experimental data available on the mechanistic reasons for lipoxygenase specificity and concludes that each of the above-mentioned hypotheses may be valid for arachidonate 5-lipoxygenation under certain circumstances. 相似文献
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Structural similarities and functional diversity of eukaryotic discoidin-like domains 总被引:1,自引:0,他引:1
Kiedzierska A Smietana K Czepczynska H Otlewski J 《Biochimica et biophysica acta》2007,1774(9):1069-1078
The discoidin domain is a approximately 150 amino acid motif common in both eukaryotic and prokaryotic proteins. It is found in a variety of extracellular, intracellular and transmembrane multidomain proteins characterized by a considerable functional diversity, mostly involved in developmental processes. The biological role of the domain depends on its interactions with different molecules, including growth factors, phospholipids and lipids, galactose or its derivatives, and collagen. The conservation of the motif, as well as the serious physiological consequences of discoidin domain disorders underscore the importance of the fold, while the ability to accommodate such an extraordinarily broad range of ligand molecules makes it a fascinating research target. In present review we characterize the distinctive features of discoidin domains and briefly outline the biological role of this module in various eukaryotic proteins. 相似文献
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Kervinen J Abad M Crysler C Kolpak M Mahan AD Masucci JA Bayoumy S Cummings MD Yao X Olson M de Garavilla L Kuo L Deckman I Spurlino J 《The Journal of biological chemistry》2008,283(1):427-436
Divergence of substrate specificity within the context of a common structural framework represents an important mechanism by which new enzyme activity naturally evolves. We present enzymological and x-ray structural data for hamster chymase-2 (HAM2) that provides a detailed explanation for the unusual hydrolytic specificity of this rodent alpha-chymase. In enzymatic characterization, hamster chymase-1 (HAM1) showed typical chymase proteolytic activity. In contrast, HAM2 exhibited atypical substrate specificity, cleaving on the carboxyl side of the P1 substrate residues Ala and Val, characteristic of elastolytic rather than chymotryptic specificity. The 2.5-A resolution crystal structure of HAM2 complexed to the peptidyl inhibitor MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone revealed a narrow and shallow S1 substrate binding pocket that accommodated only a small hydrophobic residue (e.g. Ala or Val). The different substrate specificities of HAM2 and HAM1 are explained by changes in four S1 substrate site residues (positions 189, 190, 216, and 226). Of these, Asn(189), Val(190), and Val(216) form an easily identifiable triplet in all known rodent alpha-chymases that can be used to predict elastolytic specificity for novel chymase-like sequences. Phylogenetic comparison defines guinea pig and rabbit chymases as the closest orthologs to rodent alpha-chymases. 相似文献
11.
Hayakawa A Hayes SJ Lawe DC Sudharshan E Tuft R Fogarty K Lambright D Corvera S 《The Journal of biological chemistry》2004,279(7):5958-5966
The FYVE domain is a conserved protein motif characterized by its ability to bind with high affinity and specificity to phosphatidylinositol 3-phosphate (PI3P), a phosphoinositide highly enriched in early endosomes. The PI3P polar head group contacts specific amino acid residues that are conserved among FYVE domains. Despite full conservation of these residues, the ability of different FYVE domains to bind to endosomes in cells is highly variable. Here we show that the endosomal localization in intact cells absolutely requires structural features intrinsic to the FYVE domain in addition to the PI3P binding pocket. These features are involved in FYVE domain dimerization and in interaction with the membrane bilayer. These interactions, which are determined by non-conserved residues, are likely to be essential for the temporal and spatial control of protein associations at the membrane-cytosol interface within the endocytic pathway. 相似文献
12.
Edelmann MJ Iphöfer A Akutsu M Altun M di Gleria K Kramer HB Fiebiger E Dhe-Paganon S Kessler BM 《The Biochemical journal》2009,418(2):379-390
OTUB (otubain) 1 is a human deubiquitinating enzyme that is implicated in mediating lymphocyte antigen responsiveness, but whose molecular function is generally not well defined. A structural analysis of OTUB1 shows differences in accessibility to the active site and in surface properties of the substrate-binding regions when compared with its close homologue, OTUB2, suggesting variations in regulatory mechanisms and substrate specificity. Biochemical analysis reveals that OTUB1 has a preference for cleaving Lys(48)-linked polyubiquitin chains over Lys(63)-linked polyubiquitin chains, and it is capable of cleaving NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), but not SUMO (small ubiquitin-related modifier) 1/2/3 and ISG15 (interferon-stimulated gene 15) conjugates. A functional comparison of OTUB1 and OTUB2 indicated a differential reactivity towards ubiquitin-based active-site probes carrying a vinyl methyl ester, a 2-chloroethyl or a 2-bromoethyl group at the C-terminus. Mutational analysis suggested that a narrow P1' site, as observed in OTUB1, correlates with its ability to preferentially cleave Lys(48)-linked ubiquitin chains. Analysis of cellular interaction partners of OTUB1 by co-immunoprecipitation and MS/MS (tandem mass spectrometry) experiments demonstrated that FUS [fusion involved in t(12;6) in malignant liposarcoma; also known as TLS (translocation in liposarcoma) or CHOP (CCAAT/enhancer-binding protein homologous protein)] and RACK1 [receptor for activated kinase 1; also known as GNB2L1 (guanine-nucleotide-binding protein beta polypeptide 2-like 1)] are part of OTUB1-containing complexes, pointing towards a molecular function of this deubiquitinating enzyme in RNA processing and cell adhesion/morphology. 相似文献
13.
Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system. 相似文献
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RNase II is a member of the widely distributed RNR family of exoribonucleases, which are highly processive 3'-->5' hydrolytic enzymes that play an important role in mRNA decay. Here, we report the crystal structure of E. coli RNase II, which reveals an architecture reminiscent of the RNA exosome. Three RNA-binding domains come together to form a clamp-like assembly, which can only accommodate single-stranded RNA. This leads into a narrow, basic channel that ends at the putative catalytic center that is completely enclosed within the body of the protein. The putative path for RNA agrees well with biochemical data indicating that a 3' single strand overhang of 7-10 nt is necessary for binding and hydrolysis by RNase II. The presence of the clamp and the narrow channel provides an explanation for the processivity of RNase II and for why its action is limited to single-stranded RNA. 相似文献
15.
Wu H Dombrovsky L Tempel W Martin F Loppnau P Goodfellow GH Grant DM Plotnikov AN 《The Journal of biological chemistry》2007,282(41):30189-30197
The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9-A resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes. 相似文献
16.
King GF Shih YL Maciejewski MW Bains NP Pan B Rowland SL Mullen GP Rothfield LI 《Nature structural biology》2000,7(11):1013-1017
Correct positioning of the division septum in Escherichia coli depends on the coordinated action of the MinC, MinD and MinE proteins. Topological specificity is conferred on the MinCD division inhibitor by MinE, which counters MinCD activity only in the vicinity of the preferred midcell division site. Here we report the structure of the homodimeric topological specificity domain of Escherichia coli MinE and show that it forms a novel alphabeta sandwich. Structure-directed mutagenesis of conserved surface residues has enabled us to identify a spatially restricted site on the surface of the protein that is critical for the topological specificity function of MinE. 相似文献
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Iovane E Giabbai B Muzzolini L Matafora V Fornili A Minici C Giannese F Degano M 《Biochemistry》2008,47(15):4418-4426
Enzymes with nucleoside hydrolase activity (NHs) belonging to homology group I either are markedly specific for pyrimidine nucleoside substrates or hydrolyze with comparable efficiencies the N-glycosidic bond in all common nucleosides. The biochemical and structural basis for these differences in substrate specificity is still unknown. Here we characterize the binding interactions between the slowly hydrolyzed substrate inosine and the Escherichia coli pyrimidine-specific NH YeiK using cryotrapping and X-ray crystallography. Guided by the structural features of the Michaelis complex, we show the synergic effect of two specific point mutations in YeiK that increase the catalytic efficiency toward purine nucleosides to values comparable to those of natural nonspecific NHs. We demonstrate that the integrity of an active-site catalytic triad comprised of two hydroxylated amino acids and one histidine residue is a requirement for the highly efficient hydrolysis of inosine by group I NHs. Instead, cleavage of the YeiK-preferred substrate uridine is not affected by mutations at the same locations, suggesting a different fine chemical mechanism for the hydrolysis of the two nucleoside substrates. Our study provides for the first time direct evidence that distinct subsets of amino acid residues are involved in the hydrolysis of purine or pyrimidine nucleosides in group I NHs. 相似文献
19.
Structural basis for ubiquitin recognition by SH3 domains 总被引:1,自引:0,他引:1
The SH3 domain is a protein-protein interaction module commonly found in intracellular signaling and adaptor proteins. The SH3 domains of multiple endocytic proteins have been recently implicated in binding ubiquitin, which serves as a signal for diverse cellular processes including gene regulation, endosomal sorting, and protein destruction. Here we describe the solution NMR structure of ubiquitin in complex with an SH3 domain belonging to the yeast endocytic protein Sla1. The ubiquitin binding surface of the Sla1 SH3 domain overlaps substantially with the canonical binding surface for proline-rich ligands. Like many other ubiquitin-binding motifs, the SH3 domain engages the Ile44 hydrophobic patch of ubiquitin. A phenylalanine residue located at the heart of the ubiquitin-binding surface of the SH3 domain serves as a key specificity determinant. The structure of the SH3-ubiquitin complex explains how a subset of SH3 domains has acquired this non-traditional function. 相似文献