共查询到20条相似文献,搜索用时 15 毫秒
1.
Christian Löw Henning Tidow Caroline Haupt Christian Scholz Jochen Balbach 《Journal of molecular biology》2010,398(3):375-390
SlyD (sensitive to lysis D; product of the slyD gene) is a prolyl isomerase [peptidyl-prolyl cis/trans isomerase (PPIase)] of the FK506 binding protein (FKBP) type with chaperone properties. X-ray structures derived from three different crystal forms reveal that SlyD from Thermus thermophilus consists of two domains representing two functional units. PPIase activity is located in a typical FKBP domain, whereas chaperone function is associated with the autonomously folded insert-in-flap (IF) domain. The two isolated domains are stable and functional in solution, but the presence of the IF domain increases the PPIase catalytic efficiency of the FKBP domain by 2 orders of magnitude, suggesting that the two domains act synergistically to assist the folding of polypeptide chains. The substrate binding surface of SlyD from T. thermophilus was mapped by NMR chemical shift perturbations to hydrophobic residues of the IF domain, which exhibits significantly reduced thermodynamic stability according to NMR hydrogen/deuterium exchange and fluorescence equilibrium transition experiments. Based on structural homologies, we hypothesize that this is due to the absence of a stabilizing β-strand, suggesting in turn a mechanism for chaperone activity by ‘donor-strand complementation.’ Furthermore, we identified a conserved metal (Ni2+) binding site at the C-terminal SlyD-specific helical appendix of the FKBP domain, which may play a role in metalloprotein assembly. 相似文献
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Chih-Chia Su Feng Yang Feng Long Mathew D. Routh Adam K. Mokhtari Katherine L. Rabe Young Jin Lee Edward W. Yu 《Journal of molecular biology》2009,393(2):342-656
Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu+ and Ag+ ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly β-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the α-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first β-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu+ and Ag+ were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates. 相似文献
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Several recent genome-wide association studies have linked the human MSMB gene, encoding prostate secretory protein of 94 residues (PSP94), with prostate cancer susceptibility. PSP94 is one of the most abundant proteins from prostatic secretions and a primary constituent of human semen. PSP94 suppresses tumor growth and metastasis, and its expression gradually decreases during progression of the prostate cancer. It is a rapidly evolving protein with homologues present in several species with 10 conserved cysteine residues. PSP94 homologues show high-affinity binding with different proteins from the cysteine-rich secretory protein family, some of which have been shown to be ion channel blockers. Here, we report the crystal structure of human PSP94 at 2.3 Å resolution. The structure shows that the amino and the carboxyl ends of the polypeptide chain are held in close proximity facing each other. A strong hydrogen bond between these ends, which are located respectively on the first and the last β-strands, leads to formation of an almost straight edge in PSP94 structure. Crystal structure shows that these edges from two PSP94 monomers associate in antiparallel fashion, leading to formation of a dimer. Our studies further show that dimers dissociate into monomers at acidic pH, possibly through distortion of the straight edge. Further, based on several observations, we propose that PSP94 binds to cysteine-rich secretory proteins and immunoglobulin G through the same edge, which is involved in the formation of PSP94 dimeric interface. 相似文献
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Sialic acids are acidic sugars present mostly on vertebrate cell surfaces, which can be metabolized by bacteria and act as an inflammation signal. N-Acetylneuraminic acid, the most abundant sialic acid, can enter into Escherichia coli K12 through NanC, an N-acetylneuraminic acid-inducible outer-membrane channel. With its 215 residues, NanC belongs to the family of small monomeric KdgM-related porins. KdgM homologues are found in gammaproteobacteria, including major plant and human pathogens, and together they define a large family of putative acidic sugar/oligosaccharide transporters, which are as yet poorly characterized. Here, we present the first high-resolution structure of a KdgM family member. NanC folds into a 28-Å-high, 12-stranded β-barrel, resembling the β-domain of autotransporter NalP and defining an open pore with an average radius of 3.3 Å. The channel is lined by two strings of basic residues facing each other across the pore, a feature that appears largely conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides. 相似文献
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The 3-hydroxybenzoate hydroxylase (MHBH) from Comamonas testosteroni KH122-3s is a single-component flavoprotein monooxygenase, a member of the glutathione reductase (GR) family. It catalyzes the conversion of 3-hydroxybenzoate to 3,4-dihydroxybenzoate with concomitant requirements for equimolar amounts of NADPH and molecular oxygen. The production of dihydroxy-benzenoid derivative by hydroxylation is the first step in the aerobic degradation of various phenolic compounds in soil microorganisms. To establish the structural basis for substrate recognition, the crystal structure of MHBH in complex with its substrate was determined at 1.8 A resolution. The enzyme is shown to form a physiologically active homodimer with crystallographic 2-fold symmetry, in which each subunit consists of the first two domains comprising an active site and the C-terminal domain involved in oligomerization. The protein fold of the catalytic domains and the active-site architecture, including the FAD and substrate-binding sites, are similar to those of 4-hydroxybenzoate hydroxylase (PHBH) and phenol hydroxylase (PHHY), which are members of the GR family, providing evidence that the flavoprotein aromatic hydroxylases share similar catalytic actions for hydroxylation of the respective substrates. Structural comparison of MHBH with the homologous enzymes suggested that a large tunnel connecting the substrate-binding pocket to the protein surface serves for substrate transport in this enzyme. The internal space of the large tunnel is distinctly divided into hydrophilic and hydrophobic regions. The characteristically stratified environment in the tunnel interior and the size of the entrance would allow the enzyme to select its substrate by amphiphilic nature and molecular size. In addition, the structure of the Xe-derivative at 2.5 A resolution led to the identification of a putative oxygen-binding site adjacent to the substrate-binding pocket. The hydrophobic nature of the xenon-binding site extends to the solvent through the tunnel, suggesting that the tunnel could be involved in oxygen transport. 相似文献
8.
Pseudomonas aeruginosa encodes an enzyme (PA2794) that is annotated as a sialidase (or neuraminidase), as it possesses three bacterial neuraminidase repeats that are a signature of nonviral sialidases. A recent report showed that when the gene encoding this sialidase is knocked out, this led to a reduction in biofilm production in the lungs of mice, and it was suggested that the enzyme recognizes pseudaminic acid, a sialic acid analogue that decorates the flagella of Pseudomonas, Helicobacter, and Campylobacter species. Here, we present the crystal structure of the P. aeruginosa enzyme and show that it adopts a trimeric structure, partly held together by an immunoglobulin-like trimerization domain that is C-terminal to a classical β-propeller sialidase domain. The recombinant enzyme does not show any sialidase activity with the standard fluorogenic sialic-acid-based substrate. The proposed active site contains certain conserved features of a sialidase: a nucleophilic tyrosine with its associated glutamic acid, and two of the usual three arginines that interact with the carboxylic acid group of the substrate, but is missing the first arginine and the aspartic acid that acts as an acid/base in all sialidases studied to date. We show, by in silico docking, that the active site may accommodate pseudaminic acid but not sialic acid and that this is due, in part, to a phenylalanine in the hydrophobic pocket that selects for the alternative stereochemistry of pseudaminic acid at C5 compared to sialic acid. Mutation of this phenylalanine to an alanine converts the enzyme into a sialidase, albeit a poor one, which we confirm by kinetics and NMR, and this allowed us to probe the function of other amino acids. We propose that a histidine plays the role of the acid/base, whose state is altered through a charge-relay system involving a novel His-Tyr-Glu triad. The location of this relay system precludes the presence of one of the three arginines usually found in a sialidase active site. 相似文献
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Heat shock protein (Hsp) 70/Hsp90-organizing proteins (Hop/Sti1) are thought to function as adaptor proteins to link the two chaperone machineries Hsp70 and Hsp90 during the processing of substrate proteins in eukaryotes. Hop (Hsp70/Hsp90-organizing protein) is composed of three tetratricopeptide repeat (TPR) domains, of which the first (TPR1) binds to Hsp70, the second (TPR2A) binds to Hsp90, and the third (TPR2B) is of unknown function. Contrary to most other eukaryotes, the homologue closest to the Caenorhabditis elegans Hop homologue R09E12.3 (CeHop) lacks the TPR1 domain and the short linker region connecting it to TPR2A, questioning the reported function as an Hsp90/Hsp70 adaptor in vitro and in vivo. We observed high constitutive expression levels of CeHop and detected significant phenotypes upon knockdown, linking the protein to functions in gonad development. Interestingly, we observed physical interactions with both chaperones Hsp70 and Hsp90, albeit only the interaction with Hsp90 is strong and inhibition of the Hsp90 ATPase activity can be observed upon binding of CeHop. However, the formation of ternary complexes with both chaperone machineries is impaired, as Hsp70 and Hsp90 compete for CeHop interaction sites, in particular as Hsp90 binds to both TPR domains simultaneously and requires both TPR domains for ATPase regulation. These results imply that, at least in C. elegans, essential functions of Hop exist which apparently do not depend on the simultaneous binding of Hsp90 and Hsp70 to Hop. 相似文献
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Kida H Sugano Y Iizuka R Fujihashi M Yohda M Miki K 《Journal of molecular biology》2008,383(3):465-474
Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of α and β subunits and forms a “jellyfish-like” structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two α and two β subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of α (α1 and α2) and β subunits (β1 and β2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD β1 subunit at 1.9 Å resolution and its functional analysis. TsPFD β1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The β hairpin linkers of β1 subunits assemble to form a β barrel “body” around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the β1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric β1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD β1 subunits act as molecular chaperones in living cells of some archaea. 相似文献
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Yu Hirano Makoto Higuchi Hirozo Oh-oka Zheng-Yu Wang 《Journal of molecular biology》2010,397(5):1175-1187
In green sulfur photosynthetic bacteria, the cytochrome cz (cyt cz) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt cz subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt cz subunit (C-cyt cz) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt cz consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt cz supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt cz. 相似文献
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Hodak H Wohlkönig A Smet-Nocca C Drobecq H Wieruszeski JM Sénéchal M Landrieu I Locht C Jamin M Jacob-Dubuisson F 《Journal of molecular biology》2008,376(2):414-426
Proteins that pass through the periplasm in an unfolded state are highly sensitive to proteolysis and aggregation and, therefore, often require protection by chaperone-like proteins. The periplasm of Gram-negative bacteria is well equipped with ATP-independent chaperones and folding catalysts, including peptidyl-prolyl isomerases (PPIases). The filamentous hemagglutinin of Bordetella pertussis, which is secreted by the two-partner secretion pathway, crosses the periplasm in an unfolded conformation. By affinity chromatography, we identified a new periplasmic PPIase of the parvulin family, Par27, which binds to an unfolded filamentous hemagglutinin fragment. Par27 differs from previously characterized bacterial and eukaryotic parvulins. Its central parvulin-like domain is flanked by atypical N- and C-terminal extensions that are found in a number of putative PPIases present mostly in β proteobacteria. Par27 displays both PPIase and chaperone activities in vitro. In vivo, Par27 might function as a general periplasmic chaperone in B. pertussis. 相似文献
14.
Ruiguang Ge Xuesong SunDongxian Wang Qinglu ZhouHongzhe Sun 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2011,1813(8):1422-1427
Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections. 相似文献
15.
The Piwi genes have an important role in stem cell development, gametogenesis and RNA interference in diverse organisms. So far, most of the studies have focused on the function of Piwis in vertebrates, but their function during spermiogenesis in invertebrates still remains largely unclear. In order to investigate the function of Piwis during spermiogenesis in the crab Portunus trituberculatus, we use RT-PCR and RACE to identify three Piwi complete cDNA sequences from the total RNA of the testis in P. trituberculatus. The deduced amino acid sequences of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 showed that each contains a well-conserved PAZ domain and PIWI domain. RT-PCR analyzed the tissue expression pattern of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 in the testis, heart, muscle, hepatopancreas and gill. All of the Piwis are found in germ cells of adult testis in P. trituberculatus by in situ hybridization, suggesting that these genes may play function during spermiogenesis in this species. 相似文献
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Delbecq S Auguin D Yang YS Löhr F Arold S Schetters T Précigout E Gorenflot A Roumestand C 《Journal of molecular biology》2008,375(2):409-424
Babesia divergens is the Apicomplexa agent of the bovine babesiosis in Europe: this infection leads to growth and lactation decrease, so that economical losses due to this parasite are sufficient to require the development of a vaccine. The major surface antigen of B. divergens has been described as a 37 kDa protein glycosyl phosphatidyl inositol (GPI)-anchored at the surface of the merozoite. The immuno-prophylactic potential of Bd37 has been demonstrated, and we present here the high-resolution solution structure of the 27 kDa structured core of Bd37 (Δ-Bd37) using NMR spectroscopy. A model for the whole protein has been obtained using additional small angle X-ray scattering (SAXS) data. The knowledge of the 3D structure of Bd37 allowed the precise epitope mapping of antibodies on its surface. Interestingly, the geometry of Δ-Bd37 reveals an intriguing similarity with the exocyst subunit Exo84p C-terminal region, an eukaryotic protein that has a direct implication in vesicle trafficking. This strongly suggests that Apicomplexa have developed in parallel molecular machines similar in structure and function to the ones used for endo- and exocytosis in eukaryotic cells. 相似文献
19.
Nocek B Evdokimova E Proudfoot M Kudritska M Grochowski LL White RH Savchenko A Yakunin AF Edwards A Joachimiak A 《Journal of molecular biology》2007,372(2):456-469
F(420) is a flavin-like redox-active coenzyme commonly used by archaea and some eubacteria in a variety of biochemical reactions in methanogenesis, the formation of secondary metabolites, the degradation of nitroaromatic compounds, activation of nitroimidazofurans, and F(420)-dependent photolysis in DNA repair. Coenzyme F(420)-2 biosynthesis from 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) and lactaldehyde involves six enzymatic steps and five proteins (CofA, CofB, CofC, CofD, and CofE). CofE, a F(420)-0:gamma-glutamyl ligase, is responsible for the last two enzymatic steps; it catalyses the GTP-dependent addition of two L-glutamate residues to F(420)-0 to form F(420)-2. CofE is found in archaea, the aerobic actinomycetes, and cyanobacteria. Here, we report the first crystal structure of the apo-F(420)-0:gamma-glutamyl ligase (CofE-AF) from Archaeoglobus fulgidus and its complex with GDP at 2.5 A and 1.35 A resolution, respectively. The structure of CofE-AF reveals a novel protein fold with an intertwined, butterfly-like dimer formed by two-domain monomers. GDP and Mn(2+) are bound within the putative active site in a large groove at the dimer interface. We show that the enzyme adds a glutamate residue to both F(420)-0 and F(420)-1 in two distinct steps. CofE represents the first member of a new structural family of non-ribosomal peptide synthases. 相似文献
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Y-family DNA polymerases (DNAPs) are often required in cells to synthesize past DNA-containing lesions, such as [+ ta]-B[a]P-N2-dG, which is the major adduct of the potent mutagen/carcinogen benzo[a]pyrene. The current model for the non-mutagenic pathway in Escherichia coli involves DNAP IV inserting deoxycytidine triphosphate opposite [+ ta]-B[a]P-N2-dG and DNAP V doing the next step(s), extension. We are investigating what structural differences in these related Y-family DNAPs dictate their functional differences. X-ray structures of Y-family DNAPs reveal a number of interesting features in the vicinity of the active site, including (1) the “roof-amino acid” (roof-aa), which is the amino acid that lies above the nucleobase of the deoxynucleotide triphosphate (dNTP) and is expected to play a role in dNTP insertion efficiency, and (2) a cluster of three amino acids, including the roof-aa, which anchors the base of a loop, whose detailed structure dictates several important mechanistic functions. Since no X-ray structures existed for UmuC (the polymerase subunit of DNAP V) or DNAP IV, we previously built molecular models. Herein, we test the accuracy of our UmuC(V) model by investigating how amino acid replacement mutants affect lesion bypass efficiency. A ssM13 vector containing a single [+ ta]-B[a]P-N2-dG is transformed into E. coli carrying mutations at I38, which is the roof-aa in our UmuC(V) model, and output progeny vector yield is monitored as a measure of the relative efficiency of the non-mutagenic pathway. Findings show that (1) the roof-aa is almost certainly I38, whose β-carbon branching R-group is key for optimal activity, and (2) I38/A39/V29 form a hydrophobic cluster that anchors an important mechanistic loop, aa29-39. In addition, bypass efficiency is significantly lower both for the I38A mutation of the roof-aa and for the adjacent A39T mutation; however, the I38A/A39T double mutant is almost as active as wild-type UmuC(V), which probably reflects the following. Y-family DNAPs fall into several classes with respect to the [roof-aa/next amino acid]: one class has [isoleucine/alanine] and includes UmuC(V) and DNAP η (from many species), while the second class has [alanine (or serine)/threonine] and includes DNAP IV, DNAP κ (from many species), and Dpo4. Thus, the high activity of the I38A/A39T double mutant probably arises because UmuC(V) was converted from the V/η class to the IV/κ class with respect to the [roof-aa/next amino acid]. Structural and mechanistic aspects of these two classes of Y-family DNAPs are discussed. 相似文献