共查询到20条相似文献,搜索用时 31 毫秒
1.
Tiago Baeta Karine Giandoreggio-Barranco Isabel Ayala Elisabete C.C.M. Moura Paola Sperandeo Alessandra Polissi Jean-Pierre Simorre Cedric Laguri 《The Journal of biological chemistry》2021,297(6)
Lipopolysaccharide (LPS) is an essential glycolipid that covers the surface of gram-negative bacteria. The transport of LPS involves a dedicated seven-protein transporter system called the lipopolysaccharide transport system (Lpt) machinery that physically spans the entire cell envelope. The LptB2FG complex is an ABC transporter that hydrolyzes ATP to extract LPS from the inner membrane for transport to the outer membrane. Here, we extracted LptB2FG directly from the inner membrane with its original lipid environment using styrene-maleic acid polymers. We found that styrene-maleic acid polymers–LptB2FG in nanodiscs display not only ATPase activity but also a previously uncharacterized adenylate kinase (AK) activity, as it catalyzed phosphotransfer between two ADP molecules to generate ATP and AMP. The ATPase and AK activities of LptB2FG were both stimulated by the interaction on the periplasmic side with the periplasmic LPS transport proteins LptC and LptA and inhibited by the presence of the LptC transmembrane helix. We determined that the isolated ATPase module (LptB) had weak AK activity in the absence of transmembrane proteins LptF and LptG, and one mutation in LptB that weakens its affinity for ADP led to AK activity similar to that of fully assembled complex. Thus, we conclude that LptB2FG is capable of producing ATP from ADP, depending on the assembly of the Lpt bridge, and that this AK activity might be important to ensure efficient LPS transport in the fully assembled Lpt system. 相似文献
2.
Shivani Ahuja Nicole Jahr Sang-Choul Im Subramanian Vivekanandan Nataliya Popovych Stéphanie V. Le Clair Rui Huang Ronald Soong Jiadi Xu Kazutoshi Yamamoto Ravi P. Nanga Angela Bridges Lucy Waskell Ayyalusamy Ramamoorthy 《The Journal of biological chemistry》2013,288(30):22080-22095
Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. 相似文献
3.
Maryam Faridounnia Hans Wienk Lidija Kova?i? Gert E. Folkers Nicolaas G. J. Jaspers Robert Kaptein Jan H. J. Hoeijmakers Rolf Boelens 《The Journal of biological chemistry》2015,290(33):20541-20555
The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe231, Leu231 lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. 相似文献
4.
George Mermelekas Ekaterini Georgopoulou Alexander Kallis Maria Botou Vassilios Vlantos Stathis Frillingos 《The Journal of biological chemistry》2010,285(45):35011-35020
Bacterial and fungal members of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family use the NAT signature motif, a conserved 11-amino acid sequence between amphipathic helices TM9a and TM9b, to define function and selectivity of the purine binding site. To examine the role of flanking helices TM9a, TM9b, and TM8, we employed Cys-scanning analysis of the xanthine-specific homolog YgfO from Escherichia coli. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequences 259FLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAV314 and 342TIAVMLVILGLFP354 including these TMs (underlined) was replaced individually with Cys, except the irreplaceable Glu-272 and Asp-304, which had been studied previously. Of 67 single Cys mutants, 55 accumulate xanthine to 35–140% of the steady state observed with C-less, five (I265C, D276C, I277C, G299C, L350C) accumulate to low levels (10–20%) and seven (T278C, A279C, T280C, A281C, G305C, G351C, P354C) show negligible expression in the membrane. Extensive mutagenesis reveals that a carboxyl group is needed at Asp-276 for high activity and that D276E differs from wild type as it recognizes 8-methylxanthine (Ki 79 μm) but fails to recognize 2-thioxanthine, 3-methylxanthine or 6-thioxanthine; bulky replacements of Ala-279 or Thr-280 and replacements of Gly-305, Gly-351, or Pro-354 impair activity or expression. Single Cys mutants V261C, A273C, G275C, and S284C are sensitive to inactivation by N-ethylmaleimide and sensitivity of G275C (IC50 15 μm) is enhanced in the presence of substrate. The data suggest that residues crucial for the transport mechanism cluster in two conserved motifs, at the cytoplasmic end of TM8 (EXXGDXXAT) and in TM9a (GXXXDG). 相似文献
5.
Jung-Eun Gil Eui Kim Il-Shin Kim Bonsu Ku Wei Sun Park Byung-Ha Oh Sung Ho Ryu Wonhwa Cho Won Do Heo 《The Journal of biological chemistry》2012,287(49):41268-41276
Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth. 相似文献
6.
Jin-Long Gao Yanling Lu Gina Browne Benjamin C.-M. Yap Jill Trewhella Neil Hunter Ky-Anh Nguyen 《The Journal of biological chemistry》2012,287(50):42243-42258
The widely expressed DNA-protective protein from starved-cells (Dps) family proteins are considered major contributors to prokaryotic resistance to stress. We show here that Porphyromonas gingivalis Dps (PgDps), previously described as an iron-storage and DNA-binding protein, also mediates heme sequestration. We determined that heme binds strongly to PgDps with an apparent Kd of 3.7 × 10−8
m and is coordinated by a single surface-located cysteine at the fifth axial ligand position. Heme and iron sequestered in separate sites by PgDps provide protection of DNA from H2O2-mediated free radical damage and were found to be important for growth of P. gingivalis under excess heme as the only iron source. Conservation of the heme-coordinating cysteine among Dps isoforms from the Bacteroidales order suggests that this function may be a common feature within these anaerobic bacteria. 相似文献
7.
Brian J. Hawkins Mark D. Levin Patrick J. Doonan Nataliya B. Petrenko Christiana W. Davis Vickas V. Patel Muniswamy Madesh 《The Journal of biological chemistry》2010,285(34):26494-26505
Mitochondrial membrane potential loss has severe bioenergetic consequences and contributes to many human diseases including myocardial infarction, stroke, cancer, and neurodegeneration. However, despite its prominence and importance in cellular energy production, the basic mechanism whereby the mitochondrial membrane potential is established remains unclear. Our studies elucidate that complex II-driven electron flow is the primary means by which the mitochondrial membrane is polarized under hypoxic conditions and that lack of the complex II substrate succinate resulted in reversible membrane potential loss that could be restored rapidly by succinate supplementation. Inhibition of mitochondrial complex I and F0F1-ATP synthase induced mitochondrial depolarization that was independent of the mitochondrial permeability transition pore, Bcl-2 (B-cell lymphoma 2) family proteins, or high amplitude swelling and could not be reversed by succinate. Importantly, succinate metabolism under hypoxic conditions restores membrane potential and ATP levels. Furthermore, a reliance on complex II-mediated electron flow allows cells from mitochondrial disease patients devoid of a functional complex I to maintain a mitochondrial membrane potential that conveys both a mitochondrial structure and the ability to sequester agonist-induced calcium similar to that of normal cells. This finding is important as it sets the stage for complex II functional preservation as an attractive therapy to maintain mitochondrial function during hypoxia. 相似文献
8.
Roman Martin Philipp Hackert Maike Ruprecht Stefan Simm Lukas Brüning Oliver Mirus Katherine E. Sloan Grzegorz Kudla Enrico Schleiff Markus T. Bohnsack 《RNA (New York, N.Y.)》2014,20(8):1173-1182
Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis. 相似文献
9.
Kathy Wong Guennadi Kozlov Yinglu Zhang Kalle Gehring 《The Journal of biological chemistry》2015,290(41):24727-24737
Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaires disease. Here, we crystallized three nucleotide binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term the KLAMP domain due to the presence of homologous domains in bacterial histidine kinase-like ATP binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3′,5′-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3′,5′-cAMP reveals the contribution of Tyr-61 and Tyr-69 that produces π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide binding domains associated with bacterial virulence. 相似文献
10.
Shin-ichi Yokobori Aya Kitamura Henri Grosjean Yoshitaka Bessho 《Nucleic acids research》2013,41(13):6531-6543
In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNAArgICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNAArgCCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNAArgCCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNAArg and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNAArgACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNAArgACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNAArgCCG and tadA, and then acquired a new tRNAArgUCG. This permitted further tRNAArgACG mutations with tRNAArgGCG or its disappearance, leaving a single tRNAArgUCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy. 相似文献
11.
Sandro Vivona Daniel J. Cipriano Seán O'Leary Ye Henry Li Timothy D. Fenn Axel T. Brunger 《The Journal of biological chemistry》2013,288(34):24984-24991
Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism. 相似文献
12.
Juliana Mu?oz-Escobar Edna Matta-Camacho Guennadi Kozlov Kalle Gehring 《The Journal of biological chemistry》2015,290(37):22841-22850
E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C
terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity. 相似文献
13.
WNK4 (with-no-lysine (K) kinase-4) is present in the distal nephron of the kidney and plays an important role in the regulation of renal ion transport. The epithelial Ca2+ channel TRPV5 (transient receptor potential vanilloid 5) is the gatekeeper of transcellular Ca2+ reabsorption in the distal nephron. Previously, we reported that activation of protein kinase C (PKC) increases cell-surface abundance of TRPV5 by inhibiting caveola-mediated endocytosis of the channel. Here, we report that WNK4 decreases cell-surface abundance of TRPV5 by enhancing its endocytosis. Deletion analysis revealed that stimulation of endocytosis of TRPV5 involves amino acids outside the kinase domain of WNK4. We also investigated interplay between WNK4 and PKC regulation of TRPV5. The maximal level of TRPV5 current density stimulated by the PKC activator 1-oleoyl-acetyl-sn-glycerol (OAG) is the same with or without WNK4. The relative increase of TRPV5 current stimulated by OAG, however, is greater in the presence of WNK4 compared with that without WNK4 (∼215% increase versus 60% increase above the level without OAG). Moreover, the rate of increase of TRPV5 by OAG is faster with WNK4 than without WNK4. The enhanced increase of TRPV5 in the presence of WNK4 is also observed when PKC is activated by parathyroid hormones. Thus, WNK4 exerts tonic inhibition of TRPV5 by stimulating caveola-mediated endocytosis. The lower basal TRPV5 level in the presence of WNK4 allows amplification of the stimulation of channel by PKC. This interaction between WNK4 and PKC regulation of TRPV5 may be important for physiological regulation of renal Ca2+ reabsorption by parathyroid hormones or the tissue kallikrein in vivo. 相似文献
14.
Anke Miriam Kr?hling Luis Alvarez Katharina Debowski Qui Van Monika Gunkel Stephan Irsen Ashraf Al-Amoudi Timo Strünker Elisabeth Kremmer Eberhard Krause Ingo Voigt Simone W?rtge Ari Waisman Ingo Weyand Reinhard Seifert Ulrich Benjamin Kaupp Dagmar Wachten 《PLoS genetics》2013,9(12)
The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca2+-regulated proteins that—in mature sperm—are involved in flagellar bending. 相似文献
15.
Stefan Düsterh?ft Katharina H?bel Mirja Oldefest Juliane Lokau Georg H. Waetzig Athena Chalaris Christoph Garbers Jürgen Scheller Stefan Rose-John Inken Lorenzen Joachim Gr?tzinger 《The Journal of biological chemistry》2014,289(23):16336-16348
A disintegrin and metalloprotease 17 (ADAM17) is a major sheddase involved in the regulation of a wide range of biological processes. Key substrates of ADAM17 are the IL-6 receptor (IL-6R) and TNF-α. The extracellular region of ADAM17 consists of a prodomain, a catalytic domain, a disintegrin domain, and a membrane-proximal domain as well as a small stalk region. This study demonstrates that this juxtamembrane segment is highly conserved, α-helical, and involved in IL-6R binding. This process is regulated by the structure of the preceding membrane-proximal domain, which acts as molecular switch of ADAM17 activity operated by a protein-disulfide isomerase. Hence, we have termed the conserved stalk region “Conserved ADAM seventeen
dynamic interaction sequence” (CANDIS). Finally, we identified the region in IL-6R that binds to CANDIS. In contrast to the type I transmembrane proteins, the IL-6R, and IL-1RII, CANDIS does not bind the type II transmembrane protein TNF-α, demonstrating fundamental differences in the respective shedding by ADAM17. 相似文献
16.
Patrick D. Scheu Yun-Feng Liao Julia Bauer Holger Kneuper Thomas Basché Gottfried Unden Wolfgang Erker 《Journal of bacteriology》2010,192(13):3474-3483
DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system in Escherichia coli that responds to extracellular C4-dicarboxylates. The oligomeric state of full-length DcuS was investigated in vitro and in living cells by chemical cross-linking and by fluorescence resonance energy transfer (FRET) spectroscopy. The FRET results were quantified by an improved method using background-free spectra of living cells for determining FRET efficiency (E) and donor fraction {fD = (donor)/[(donor) + (acceptor)]}. Functional fusions of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) variants of green fluorescent protein to DcuS were used for in vivo FRET measurements. Based on noninteracting membrane proteins and perfectly interacting proteins (a CFP-YFP fusion), the results of FRET of cells coexpressing DcuS-CFP and DcuS-YFP were quantitatively evaluated. In living cells and after reconstitution of purified recombinant DcuS in proteoliposomes, DcuS was found as a dimer or higher oligomer, independent of the presence of an effector. Chemical cross-linking with disuccinimidyl suberate showed tetrameric, in addition to dimeric, DcuS in proteoliposomes and in membranes of bacteria, whereas purified DcuS in nondenaturing detergent was mainly monomeric. The presence and amount of tetrameric DcuS in vivo and in proteoliposomes was not dependent on the concentration of DcuS. Only membrane-embedded DcuS (present in the oligomeric state) is active in (auto)phosphorylation. Overall, the FRET and cross-linking data demonstrate the presence in living cells, in bacterial membranes, and in proteoliposomes of full-length DcuS protein in an oligomeric state, including a tetramer.The DcuSR (dicarboxylate uptake sensor and regulator) system of Escherichia coli is a typical two-component system consisting of a membranous sensor kinase (DcuS) and a cytoplasmic response regulator (DcuR) (11, 26, 48). DcuS responds to C4-dicarboxylates like fumarate, malate, or succinate (19). In the presence of the C4-dicarboxlates, the expression of the genes of anaerobic fumarate respiration (dcuB, fumB, and frdABCD) and of aerobic C4-dicarboxylate uptake (dctA) is activated. DcuS is a histidine protein kinase composed of two transmembrane helices with an intermittent sensory PAS domain in the periplasm (PASP) that was also termed the PDC domain (for PhoQ/DcuS/DctB/CitA domain or fold) (7, 20, 32, 48). The second transmembrane helix is followed by a cytoplasmic PAS domain (PASC) and the C-terminal transmitter domain. PASC functions in signal transfer from transmembrane helix 2 (TM2) to the kinase domain (9). The C-terminal part of the transmitter domain consists of a catalytic or HATPase (histidine kinase/ATPase) subdomain for autophosphorylation of DcuS (16). The N-terminal part of the transmitter contains two conserved α-helical regions, including a conserved His residue which is the site for autophosphorylation. The α-helices serve in dimerization and form a four-helix bundle in the kinase dimer (dimerization and histidine phosphotransfer [DHp] domain) (25, 35, 42, 44).The dimeric sensor kinases have been supposed to phosphorylate mutually, by the catalytic domain of one monomer, the His residue of the partner monomer (10). The oligomeric state of the membrane-bound sensor kinases EnvZ and VirA was also deduced from in vivo complementation studies (31, 46). In addition, signal transduction across the membrane and along cytoplasmic PAS domains appears to be a mechanical process requiring oligomeric proteins (9, 40). Therefore, His kinases are supposed to be dimeric in the functional state, but a higher oligomeric state has not been tested and is conceivable. Only a limited number of membrane-bound sensor kinases have been studied for their oligomerization in their membrane-bound state. Thus, the oligomeric state of the KdpD and TorS sensor kinases of E. coli have been shown to prevail in the detergent-solubilized state as oligomers, presumably dimers (14, 29). There was indirect information that functional DcuS is a dimer as well. Purified DcuS shows kinase activity only after reconstitution into liposomes, and phosphorylation is stimulated by C4-dicarboxylates (16, 19). Detergent-solubilized DcuS, on the other hand, shows no kinase activity, and it was assumed that reconstituted DcuS prevails as a dimer, whereas the inactivation of the detergent-solubilized form is due to monomerization. Recently, it was suggested that autophosphorylation in a sensor kinase of Thermotoga maritima proceeds by a cis mechanism on DHp and catalytic kinase domains within the same monomer (6). The sensor kinase is supposed to prevail as a dimer for reasons of signal transfer to the sensor domain, but the presence of cis phosphorylation principally brings into question the need for dimers for sensor kinase function.Overall, it appears that sensor kinases are oligomers for functional reasons. There is, however, no clear evidence for an oligomeric state of full-length sensor kinases in their membrane-embedded state. Moreover, the studies do not address the question of whether the sensor kinases are dimers or higher oligomers. Therefore, several aspects of the oligomeric state of sensor kinases in vivo in bacterial membranes, that is, before solubilization by detergent, are not clear. In this study, the oligomerization of full-length DcuS was examined in vivo in growing bacteria and in bacterial membranes and in vitro after isolation and reconstitution in liposomes by chemical cross-linking and fluorescence resonance energy transfer (FRET) spectroscopy. FRET techniques have been used widely to study intermolecular interactions of biological molecules (1, 4, 18, 21, 23, 34). The sensitivity of fluorescence allows experiments at low concentrations of native proteins, and genetically generated fusions of DcuS with fluorescent proteins ensure site-specific labeling of DcuS for noninvasive and nondestructive measurements in living cells. In particular, it was investigated whether dimers or higher oligomeric states can be detected for DcuS and whether the oligomerization state depends on function-related parameters. 相似文献
17.
Christos Gournas Thomas Evangelidis Alexandros Athanasopoulos Emmanuel Mikros Vicky Sophianopoulou 《The Journal of biological chemistry》2015,290(10):6141-6155
Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly56, Thr57), TMS3 (Glu138), and TMS6 (Phe248), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle. 相似文献
18.
Thomas Becker Susanne E. Horvath Lena B?ttinger Natalia Gebert Günther Daum Nikolaus Pfanner 《The Journal of biological chemistry》2013,288(23):16451-16459
The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex. 相似文献
19.
20.