共查询到20条相似文献,搜索用时 15 毫秒
1.
Yurong Wen Ester Behiels Jan Felix Jonathan Elegheert Bjorn Vergauwen Bart Devreese Savvas N. Savvides 《Nucleic acids research》2014,42(15):10134-10147
Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin–antitoxin proteins HipASO and HipBSO from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin–antitoxin protein module. Here we show that phosphorylated HipASO can engage in an unexpected ternary complex with HipBSO and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipBSO in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipASO in the ternary complex, indicative of its role in binding HipASO to abolish its function in persistence. The structure of HipASO in complex with a non-hydrolyzable ATP analogue shows that HipASO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipASO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought. 相似文献
2.
Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence. 相似文献
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《Biophysical journal》2020,118(5):1221-1233
Voltage-gated proton channels (HV1) are essential for various physiological tasks but are strongly inhibited by Zn2+ cations. Some determinants of Zn2+ binding have been elucidated experimentally and in computational studies. However, the results have always been interpreted under the assumption that Zn2+ binds to monomeric HV1 despite evidence that HV1 expresses as a dimer and that the dimer has a higher affinity for zinc than the monomer and experimental data that suggest coordination in the dimer interface. The results of former studies are also controversial, e.g., supporting either one single or two binding sites. Some structural determinants of the binding are still elusive. We performed a series of molecular dynamics simulations to address different structures of the human proton channel, the monomer and two plausible dimer conformations, to compare their respective potential to interact with and bind Zn2+ via the essential histidines. The series consisted of several copies of the system to generate independent trajectories and increase the significance compared to a single simulation. The amount of time simulated totals 29.9 μs for 126 simulations of systems comprising ∼59,000 to ∼187,000 atoms. Our approach confirms the existence of two binding sites in monomeric and dimeric human HV1. The dimer interface is more efficient for attracting and binding Zn2+ via the essential histidines than the monomer or a dimer with the histidines in the periphery. The higher affinity is due to the residues in the dimer interface that create an attractive electrostatic potential funneling the zinc cations toward the binding sites. 相似文献
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Nishith Saurav Topno Muthu Kannan 《Journal of biomolecular structure & dynamics》2018,36(7):1834-1852
8.
Dimerization of transmembrane (TM) α helices of membrane receptors plays a key role in signaling. We show that molecular dynamics simulations yield models of integrin TM helix heterodimers, which agree well with available NMR structures. We use?a multiscale simulation approach, combining coarse-grained and subsequent atomistic simulation, to model the dimerization of wild-type (WT) and mutated sequences of the αIIb and β3 integrin TM helices. The WT helices formed a stable, right-handed dimer with the same helix-helix interface as in the published NMR structure (PDB: 2K9J). In contrast, the presence of disruptive mutations perturbed the interface between the helices, altering the conformational stability of the dimer. The αIIb/β3 interface was more flexible than that of, e.g., glycophorin A. This is suggestive of a role for alternative packing modes of the TM helices in transbilayer signaling. 相似文献
9.
Takao Naganuma Naoko Nomura Min Yao Masahiro Mochizuki Toshio Uchiumi Isao Tanaka 《The Journal of biological chemistry》2010,285(7):4747-4756
The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with eukaryotic pentameric stalk complex; it provides access to eukaryotic elongation factors at levels comparable to that of the eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)2(P1)2(P1)2) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome. 相似文献
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Fis is an abundant bacterial DNA binding protein that functions in many different reactions. We show here that Fis subunits rapidly exchange between dimers in solution by disulfide cross-linking mixtures of Fis mutants with different electrophoretic mobilities and by monitoring energy transfer between fluorescently labeled Fis subunits upon heterodimer formation. The effects of detergents and salt concentrations on subunit exchange imply that the dimer is predominantly stabilized by hydrophobic forces, consistent with the X-ray crystal structures. Specific and nonspecific DNA strongly inhibit Fis subunit exchange. In all crystal forms of Fis, the separation between the DNA recognition helices within the Fis dimer is too short to insert into adjacent major grooves on canonical B-DNA, implying that conformational changes within the Fis dimer and/or the DNA must occur upon binding. We therefore investigated the functional importance of dimer interface flexibility for Fis-DNA binding by studying the DNA binding properties of Fis mutants that were cross-linked at different positions in the dimer. Flexibility within the core dimer interface does not appear to be required for efficient DNA binding, Fis-DNA complex dissociation, or Fis-induced DNA bending. Moreover, FRET-based experiments provided no evidence for a change in the spatial relationship between the two helix-turn-helix motifs in the Fis dimer upon DNA binding. These results support a model in which the unusually short distance between DNA recognition helices on Fis is accommodated primarily through bending of the DNA. 相似文献
11.
Binding of the estrogen receptor to DNA. The role of waters. 总被引:2,自引:0,他引:2
Molecular dynamics simulations are carried out to investigate the binding of the estrogen receptor, a member of the nuclear hormone receptor family, to specific and non-specific DNA. Two systems have been simulated, each based on the crystallographic structure of a complex of a dimer of the estrogen receptor DNA binding domain with DNA. One structure includes the dimer and a consensus segment of DNA, ds(CCAGGTCACAGTGACCTGG); the other structure includes the dimer and a nonconsensus segment of DNA, ds(CCAGAACACAGTGACCTGG). The simulations involve an atomic model of the protein-DNA complex, counterions, and a sphere of explicit water with a radius of 45 A. The molecular dynamics package NAMD was used to obtain 100 ps of dynamics for each system with complete long-range electrostatic interactions. Analysis of the simulations revealed differences in the protein-DNA interactions for consensus and nonconsensus sequences, a bending and unwinding of the DNA, a slight rearrangement of several amino acid side chains, and inclusion of water molecules at the protein-DNA interface region. Our results indicate that binding specificity and stability is conferred by a network of direct and water mediated protein-DNA hydrogen bonds. For the consensus sequence, the network involves three water molecules, residues Glu-25, Lys-28, Lys-32, Arg-33, and bases of the DNA. The binding differs for the nonconsensus DNA sequence in which case the fluctuating network of hydrogen bonds allows water molecules to enter the protein-DNA interface. We conclude that water plays a role in furnishing DNA binding specificity to nuclear hormone receptors. 相似文献
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Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO2 donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 Å resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca2+ ions or two ADP molecules and one Mg2+ ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca2+ ion and the Mg2+ ion are associated with the ADP molecule in the active site, and the other Ca2+ ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC. 相似文献
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《Biophysical journal》2022,121(19):3730-3744
Ras dimers have been proposed as building blocks for initiating the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cellular signaling pathway. To better examine the structure of possible dimer interfaces, the dynamics of Ras dimerization, and its potential signaling consequences, we performed molecular dynamics simulations totaling 1 ms of sampling, using an all-atom model of two full-length, farnesylated, guanosine triphosphate (GTP)-bound, wild-type KRas4b proteins diffusing on 29%POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine)-mixed POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membranes. Our simulations unveil an ensemble of thermodynamically weak KRas dimers spanning multiple conformations. The most stable conformations, having the largest interface areas, involve helix α2 and a hypervariable region (HVR). Among the dimer conformations, we found that the HVR of each KRas has frequent interactions with various parts of the dimer, thus potentially mediating the dimerization. Some dimer configurations have one KRas G-domain elevated above the lipid bilayer surface by residing on top of the other G-domain, thus likely contributing to the recruitment of cytosolic Raf kinases in the context of a stably formed multi-protein complex. We identified a variant of the α4-α5 KRas-dimer interface that is similar to the interfaces obtained with fluorescence resonance energy transfer (FRET) data of HRas on lipid bilayers. Interestingly, we found two arginine fingers, R68 and R149, that directly interact with the beta-phosphate of the GTP bound in KRas, in a manner similar to what is observed in a crystal structure of GAP-HRas complex, which can facilitate the GTP hydrolysis via the arginine finger of GTPase-activating protein (GAP). 相似文献
14.
DNA strand arrangement within the SfiI-DNA complex: atomic force microscopy analysis 总被引:2,自引:0,他引:2
The SfiI restriction enzyme binds to DNA as a tetramer holding two usually distant DNA recognition sites together before cleavage of the four DNA strands. To elucidate structural properties of the SfiI-DNA complex, atomic force microscopy (AFM) imaging of the complexes under noncleaving conditions (Ca2+ instead of Mg2+ in the reaction buffer) was performed. Intramolecular complexes formed by protein interaction between two binding sites in one DNA molecule (cis interaction) as well as complexes formed by the interaction of two sites in different molecules (trans interaction) were analyzed. Complexes were identified unambiguously by the presence of a tall spherical blob at the DNA intersections. To characterize the path of DNA within the complex, the angles between the DNA helices in the proximity of the complex were systematically analyzed. All the data show clear-cut bimodal distributions centered around peak values corresponding to 60 degrees and 120 degrees. To unambiguously distinguish between the crossed and bent models for the DNA orientation within the complex, DNA molecules with different arm lengths flanking the SfiI binding site were designed. The analysis of the AFM images for complexes of this type led to the conclusion that the DNA recognition sites within the complex are crossed. The angles of 60 degrees or 120 degrees between the DNA helices correspond to a complex in which one of the helices is flipped with respect to the orientation of the other. Complexes formed by five different recognition sequences (5'-GGCCNNNNNGGCC-3'), with different central base pairs, were also analyzed. Our results showed that complexes containing the two possible orientations of the helices were formed almost equally. This suggests no preferential orientation of the DNA cognate site within the complex, suggesting that the central part of the DNA binding site does not form strong sequence specific contacts with the protein. 相似文献
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Jia J Tarabykina S Hansen C Berchtold M Cygler M 《Structure (London, England : 1993)》2001,9(4):267-275
BACKGROUND: The Ca2+ binding apoptosis-linked gene-2 (ALG-2) protein acts as a proapoptotic factor in a variety of cell lines and is required either downstream or independently of caspases for apoptosis to occur. ALG-2 belongs to the penta-EF-hand (PEF) protein family and has two high-affinity and one low-affinity Ca2+ binding sites. Like other PEF proteins, its N terminus contains a Gly/Pro-rich segment. Ca2+ binding is required for the interaction with the target protein, ALG-2 interacting protein 1 (AIP1). RESULTS: We present the 2.3 A resolution crystal structure of Ca2+-Ioaded des1-20ALG-2 (aa 21-191), which was obtained by limited proteolysis of recombinant ALG-2 with elastase. The molecule contains eight alpha helices that fold into five EF-hands, and, similar to other members of this protein family, the molecule forms dimers. Ca2+ ions bind to EF1, EF3, and, surprisingly, to EF5. In the related proteins calpain and grancalcin, the EF5 does not bind Ca2+ and is thought to primarily facilitate dimerization. Most importantly, the conformation of des1-20ALG-2 is significantly different from that of calpain and grancalcin. This difference can be described as a rigid body rotation of EF1-2 relative to EF4-5 and the dimer interface, with a hinge within the EF3 loop. An electron density, which is interpreted as a hydrophobic Gly/Pro-rich decapeptide that is possibly derived from the cleaved N terminus, was found in a hydrophobic cleft between these two halves of the molecule. CONCLUSIONS: A different relative orientation of the N- and C-terminal halves of des1-20ALG-2 in the presence of Ca2+ and the peptide as compared to other Ca2+loaded PEF proteins changes substantially the shape of the molecule, exposing a hydrophobic patch on the surface for peptide binding and a large cleft near the dimer interface. We postulate that the binding of a Gly/ Pro-rich peptide in the presence of Ca2+ induces a conformational rearrangement in ALG-2, and that this mechanism is common to other PEF proteins. 相似文献
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《Journal of Asia》2022,25(3):101949
Bacteria have a particular strategy to invade the host immune system by forming an undetectable dormant state that may resuscitate and cause disease even after inhabiting for years in a host body. Several mechanisms are known to be responsible for bacterial dormancy, among them the hipBA toxin-antitoxin (TA) system which was initially identified in Escherichia coli. Here we explore the genomic distribution and functional association of hipBA TA homologs from an entomopathogenic bacterium Xenorhabdus nematophila. This bacterium is a symbiotic model with the nematode Steinernema carpocapsae. We found that HipA toxin homologs are more closely related than HipB antitoxins and have satisfactory adenine (for HipA homologs) and nucleic acid (for HipB homologs) ligand partners with a typical TA interaction network that may promote the X. nematophila towards a stringent response to form the dormant state. Such homologs distribution is an inclusion in the current TA repertoire of X. nematophila. 相似文献
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《Journal of molecular biology》2009,385(2):368-29346
Regulatory inactivation of DnaA is dependent on Hda (homologous to DnaA), a protein homologous to the AAA+ (ATPases associated with diverse cellular activities) ATPase region of the replication initiator DnaA. When bound to the sliding clamp loaded onto duplex DNA, Hda can stimulate the transformation of active DnaA-ATP into inactive DnaA-ADP. The crystal structure of Hda from Shewanella amazonensis SB2B at 1.75 Å resolution reveals that Hda resembles typical AAA+ ATPases. The arrangement of the two subdomains in Hda (residues 1-174 and 175-241) differs dramatically from that of DnaA. A CDP molecule anchors the Hda domains in a conformation that promotes dimer formation. The Hda dimer adopts a novel oligomeric assembly for AAA+ proteins in which the arginine finger, crucial for ATP hydrolysis, is fully exposed and available to hydrolyze DnaA-ATP through a typical AAA+ type of mechanism. The sliding clamp binding motifs at the N-terminus of each Hda monomer are partially buried and combine to form an antiparallel β-sheet at the dimer interface. The inaccessibility of the clamp binding motifs in the CDP-bound structure of Hda suggests that conformational changes are required for Hda to form a functional complex with the clamp. Thus, the CDP-bound Hda dimer likely represents an inactive form of Hda. 相似文献
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TraR is a LuxR-type quorum-sensing protein encoded by the tumour-inducing plasmid of Agrobacterium tumefaciens . TraR requires the pheromone N-3-oxooctanoyl- l -homoserine lactone (OOHL) for biological activity, and is dimeric both in solution and when bound to DNA. Dimerization is mediated primarily by two α-helices, one in the N-terminal OOHL binding domain, and the other in the C-terminal DNA binding domain. Each of these helices forms a parallel coiled coil with the identical helix of the opposite subunit. We have previously shown that OOHL is essential for resistance to proteolysis, and here we asked whether dimerization is also required for protease resistance. We constructed a series of site-directed mutations at the dimer interface, and tested these mutants for activity in vivo . Alteration of residues A149, A150, A153, A222 and I229 completely abolished activity, while alteration of three other residues also caused significant defects. All mutants were tested for dimerization as well as for specific DNA binding. The cellular abundance of these proteins in A. tumefaciens was measured using Western immunoblots and OOHL sequestration, while the half-life was measured by pulse-chase radiolabelling. We found a correlation between defects in in vivo activity, in vitro dimerization, DNA binding and protein half-life. We conclude that dimerization of TraR enhances resistance to cellular proteases. 相似文献
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Tommaso Eliseo Ignacio E. Sánchez Alejandro D. Nadra Mariano Dellarole Gonzalo de Prat Gay Daniel O. Cicero 《Journal of molecular biology》2009,388(2):327-344
DNA sequence recognition by the homodimeric C-terminal domain of the human papillomavirus type 16 E2 protein (E2C) is known to involve both direct readout and DNA-dependent indirect readout mechanisms, while protein-dependent indirect readout has been deduced but not directly observed. We have investigated coupling between specific DNA binding and the dynamics of the unusual E2C fold, using pH as an external variable. Nuclear magnetic resonance and isothermal titration calorimetry show that pH titration of His318 in the complex interface and His288 in the core of the domain is coupled to both binding and the dynamics of the β-barrel core of E2C, with a tradeoff between dimer stability and function. Specific DNA binding is, in turn, coupled to the slow dynamics and amide hydrogen exchange in the entire β-barrel, reaching residues far apart from the DNA recognition elements but not affecting the two helices of each monomer. The changes are largest in the dimerization interface, suggesting that the E2C β-barrel acts as a hinge that regulates the relative position of the DNA recognition helices. In conclusion, the cooperative dynamics of the human papillomavirus type 16 E2C β-barrel is coupled to sequence recognition in a protein-dependent indirect readout mechanism. The patterns of residue substitution in genital papillomaviruses support the importance of the protonation states of His288 and His318 and suggest that protein-dependent indirect readout and histidine pH titration may regulate DNA binding in the cell. 相似文献