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The solution structure of the lantibiotic immunity protein NisI and its interactions with nisin
(2015)
Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.
Identification of a lysosomal peptide transport system induced during dendritic cell development
(2007)
The delivery of protein fragments to major histocompatibility complex (MHC)-loading compartments of professional antigen-presenting cells is essential in the adaptive immune response against pathogens. Apart from the crucial role of the transporter associated with antigen processing (TAP) for peptide loading of MHC class I molecules in the endoplasmic reticulum, TAP-independent translocation pathways have been proposed but not identified so far. Based on its overlapping substrate specificity with TAP, we herein investigated the ABC transporter ABCB9, also named TAP-like (TAPL). Remarkably, TAPL expression is strongly induced during differentiation of monocytes to dendritic cells and to macrophages. TAPL does not, however, restore MHC class I surface expression in TAP-deficient cells, demonstrating that TAPL alone or in combination with single TAP subunits does not form a functional transport complex required for peptide loading of MHC I in the endoplasmic reticulum. In fact, by using quantitative immunofluorescence and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome-associated membrane protein LAMP-2. By in vitro assays, we demonstrate a TAPL-specific translocation of peptides into isolated lysosomes, which strictly requires ATP hydrolysis. These results suggest a mechanism by which antigenic peptides have access to the lysosomal compartment in professional antigen-presenting cells.
The ubiquitin (Ub) code denotes the complex Ub architectures, including Ub chains of different length, linkage-type and linkage combinations, which enable ubiquitination to control a wide range of protein fates. Although many linkage-specific interactors have been described, how interactors are able to decode more complex architectures is not fully understood. We conducted a Ub interactor screen, in humans and yeast, using Ub chains of varying length, as well as, homotypic and heterotypic branched chains of the two most abundant linkage types – K48- and K63-linked Ub. We identified some of the first K48/K63 branch-specific Ub interactors, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4 and huntingtin-interacting protein HIP1. Furthermore, we revealed the importance of chain length by identifying interactors with a preference for Ub3 over Ub2 chains, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50 and p97-adaptor FAF1. Crucially, we compared datasets collected using two common DUB inhibitors – Chloroacetamide and N-ethylmaleimide. This revealed inhibitor-dependent interactors, highlighting the importance of inhibitor consideration during pulldown studies. This dataset is a key resource for understanding how the Ub code is read.
LIN-2/7 (L27) domains are protein interaction modules that preferentially hetero-oligomerize, a property critical for their function in directing specific assembly of supramolecular signaling complexes at synapses and other polarized cell-cell junctions. We have solved the solution structure of the heterodimer composed of the L27 domains from LIN-2 and LIN-7. Comparison of this structure with other L27 domain structures has allowed us to formulate a general model for why most L27 domains form an obligate heterodimer complex. L27 domains can be divided in two types (A and B), with each heterodimer comprising an A/B pair. We have identified two keystone positions that play a central role in discrimination. The residues at these positions are energetically acceptable in the context of an A/B heterodimer, but would lead to packing defects or electrostatic repulsion in the context of A/A and B/B homodimers. As predicted by the model, mutations of keystone residues stabilize normally strongly disfavored homodimers. Thus, L27 domains are specifically optimized to avoid homodimeric interactions.
The human growth factor receptor MET is a receptor tyrosine kinase involved in cell proliferation, migration, and survival. MET is also hijacked by the intracellular pathogen Listeria monocytogenes. Its invasion protein, internalin B (InlB), binds to MET and promotes the formation of a signaling dimer that triggers the internalization of the pathogen. Here, we use a combination of structural biology, modeling, molecular dynamics simulations, and in situ single-molecule Förster resonance energy transfer (smFRET) experiments to elucidate the early events in MET activation by Listeria. Simulations show that InlB binding stabilizes MET in a conformation that promotes dimer formation. smFRET identifies the organization of the in situ signaling dimer. Further MD simulations of the dimer model are in quantitative agreement with smFRET. We accurately describe the structural dynamics underpinning an important cellular event and introduce a powerful methodological pipeline applicable to studying the activation of other plasma membrane receptors.
Determining the structure and mechanisms of all individual functional modules of cells at high molecular detail has often been seen as equal to understanding how cells work. Recent technical advances have led to a flush of high-resolution structures of various macromolecular machines, but despite this wealth of detailed information, our understanding of cellular function remains incomplete. Here, we discuss present-day limitations of structural biology and highlight novel technologies that may enable us to analyze molecular functions directly inside cells. We predict that the progression toward structural cell biology will involve a shift toward conceptualizing a 4D virtual reality of cells using digital twins. These will capture cellular segments in a highly enriched molecular detail, include dynamic changes, and facilitate simulations of molecular processes, leading to novel and experimentally testable predictions. Transferring biological questions into algorithms that learn from the existing wealth of data and explore novel solutions may ultimately unveil how cells work.
The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.
Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).
The core of photosystem I (PS1) is composed of the two related integral membrane polypeptides, PsaA and PsaB, which bind two symmetrical branches of cofactors, each consisting of two chlorophylls and a phylloquinone, that potentially link the primary electron donor and the tertiary acceptor. In an effort to identify amino acid residues near the phylloquinone binding sites, all tryptophans and histidines that are conserved between PsaA and PsaB in the region of the 10th and 11th transmembrane alpha-helices were mutated in Chlamydomonas reinhardtii. The mutant PS1 reaction centers appear to assemble normally and possess photochemical activity. An electron paramagnetic resonance (EPR) signal attributed to the phylloquinone anion radical (A(1)(-)) can be observed either transiently or after illumination of reaction centers with pre-reduced iron-sulfur clusters. Mutation of PsaA-Trp(693) to Phe resulted in an inability to photo-accumulate A(1)(-), whereas mutation of the analogous tryptophan in PsaB (PsaB-Trp(673)) did not produce this effect. The PsaA-W693F mutation also produced spectral changes in the time-resolved EPR spectrum of the P(700)(+) A(1)(-) radical pair, whereas the analogous mutation in PsaB had no observable effect. These observations indicate that the A(1)(-) phylloquinone radical observed by EPR occupies the phylloquinone-binding site containing PsaA-Trp(693). However, mutation of either tryptophan accelerated charge recombination from the terminal Fe-S clusters.
The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.
The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports SQ bound in the vicinity of reduced bH heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR-silent SQ bound close to oxidized bH, we calculated a midpoint redox potential (Em) of approximately 90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ.bH(3+) complexes. This favors recycling of the electron coming from center P and also implies a >2.5-fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre-steady-state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ.bH(3+) formation without inhibition of Q binding by QH2.
We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc(1) complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2-3 nm shift of the visible absorbance spectrum of the b(L) heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the b(L) spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the b(L) spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the b(L) heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.
We previously proposed that the dimeric cytochrome bc(1) complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc(1) monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289-22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc(1) complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10-16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c(1) by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.
Highlights
• USP32 deubiquitinates the Ragulator complex subunit LAMTOR1 at lysine (K) 20
• LAMTOR1 K20 ubiquitination impairs its binding to the vacuolar H+-ATPase
• USP32 knockout reduces mTORC1 activity and elevates autophagic flux
• Depletion of USP32 in Caenorhabditis elegans inhibits mTOR and induces autophagy
Summary
The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain–containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63-, and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez. Our results indicate that UBACez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.
We have investigated the role of reactive oxygen species and thiol-oxidizing agents in the induction of cell death and have shown that adenocarcinoma gastric (AGS) cells respond differently to the oxidative challenge according to the signaling pathways activated. In particular, apoptosis in AGS cells is induced via the mitochondrial pathway upon treatment with thiol-oxidizing agents, such as diamide. Apoptosis is associated with persistent oxidative damage, as evidenced by the increase in carbonylated proteins and the expression/activation of DNA damage-sensitive proteins histone H2A.X and DNA-dependent protein kinase. Resistance to hydrogen peroxide is instead associated with Keap1 oxidation and rapid translocation of Nrf2 into the nucleus. Sensitivity to diamide and resistance to hydrogen peroxide are correlated with GSH redox changes, with diamide severely increasing GSSG, and hydrogen peroxide transiently inducing protein-GSH mixed disulfides. We show that p53 is activated in response to diamide treatment by the oxidative induction of the Trx1/p38(MAPK) signaling pathway. Similar results were obtained with another carcinoma cell line, CaCo2, indicating that these findings are not limited to AGS cells. Our data suggest that thiol-oxidizing agents could be exploited as inducers of apoptosis in tumor histotypes resistant to ROS-producing chemotherapeutics.
The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.
Resonance Raman and Fourier transform infrared spectroscopies have been used to study the aa(3)-type cytochrome c oxidase and the Y280H mutant from Paracoccus denitrificans. The stability of the binuclear center in the absence of the Tyr(280)-His(276) cross-link is not compromised since heme a(3) retains the same proximal environment, spin, and coordination state as in the wild type enzyme in both the oxidized and reduced states. We observe two C-O modes in the Y280H mutant at 1966 and 1975 cm(-1). The 1975 cm(-1) mode is assigned to a gamma-form and represents a structure of the active site in which Cu(B) exerts a steric effect on the heme a(3)-bound CO. Therefore, the role of the cross-link is to fix Cu(B) in a certain configuration and distance from heme a(3), and not to allow histidine ligands to coordinate to Cu(B) rather than to heme a(3), rendering the enzyme inactive, as proposed recently (Das, T. K., Pecoraro, C., Tomson, F. L., Gennis, R. B., and Rousseau, D. L. (1998) Biochemistry 37, 14471-14476). The results provide solid evidence that in the Y280H mutant the catalytic site retains its active configuration that allows O(2) binding to heme a(3). Oxygenated intermediates are formed by mixing oxygen with the CO-bound mixed-valence wild type and Y280H enzymes with similar Soret maxima at 438 nm.
The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.
Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O2 activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O2 with the mixed valence form (CuA(2+), heme a(3+), heme a3(2+)-CuB(1+)) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm(-1) at the peroxy oxidation level (PM). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the PM intermediate is also formed in the reaction of Y167F with O2. These results suggest that in the fully reduced enzyme, the proton pumping ν(Fe(IV)=O) = 804 cm(-1) to ν(Fe(IV)=O) = 790 cm(-1) transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a3 but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a3. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a3-Asp-399-H2O site and, thus, to the exit/output proton channel (H2O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a3 controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.
Background: Understanding the coupling of O2 reduction to proton pumping by CcO requires detection of reaction intermediates.
Results: We have detected two oxoferryl intermediates at the PM oxidation state.
Conclusion: The H-bonding properties of the proximal heme a3 His ligand control the strength of the oxoferryl species.
Significance: The role of His-411, Thr-389, Gly-386, and Asp-399 residues in the proton pumping P→F transition is outlined.
Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp, Gln, Ala), Asp804 (Glu, Asn, Ala), and Asp808 (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K+-stimulated stationary currents and pre-steady-state currents under electrogenic Na+/Na+ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na+/K+ pump currents, the other constructs could be classified according to the [K+] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K+. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na+/Na+ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn776 and Asp804, respectively, decreases the extracellular Na+ affinity.
We report the first evidence for the formation of the "607- and 580-nm forms" in the cytochrome oxidase aa3/H2O2 reaction without the involvement of tyrosine 280. The pKa of the 607-580-nm transition is 7.5. The 607-nm form is also formed in the mixed valence cytochrome oxidase/O2 reaction in the absence of tyrosine 280. Steady-state resonance Raman characterization of the reaction products of both the wild-type and Y280H cytochrome aa3 from Paracoccus denitrificans indicate the formation of six-coordinate low spin species, and do not support, in contrast to previous reports, the formation of a porphyrin pi-cation radical. We observe three oxygen isotope-sensitive Raman bands in the oxidized wild-type aa3/H2O2 reaction at 804, 790, and 358 cm-1. The former two are assigned to the Fe(IV)[double bond]O stretching mode of the 607- and 580-nm forms, respectively. The 14 cm-1 frequency difference between the oxoferryl species is attributed to variations in the basicity of the proximal to heme a3 His-411, induced by the oxoferryl conformations of the heme a3-CuB pocket during the 607-580-nm transition. We suggest that the 804-790 cm-1 oxoferryl transition triggers distal conformational changes that are subsequently communicated to the proximal His-411 heme a3 site. The 358 cm-1 mode has been found for the first time to accumulate with the 804 cm-1 mode in the peroxide reaction. These results indicate that the mechanism of oxygen reduction must be reexamined.
The human transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen. The functional unit of TAP is a heterodimer composed of the TAP1 and TAP2 subunits, both of which are members of the ABC-transporter family. ABC-transporters are ATP-dependent pumps, channels, or receptors that are composed of four modules: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Although the TMDs are rather divergent in sequence, the NBDs are conserved with respect to structure and function. Interestingly, the NBD of TAP1 contains mutations at amino acid positions that have been proposed to be essential for catalytic activity. Instead of a glutamate, proposed to act as a general base, TAP1 contains an aspartate and a glutamine instead of the conserved histidine, which has been suggested to act as the linchpin. We used this degeneration to evaluate the individual contribution of these two amino acids to the ATPase activity of the engineered TAP1-NBD mutants. Based on our results a catalytic hierarchy of these two fundamental amino acids in ATP hydrolysis of the mutated TAP1 motor domain was deduced.
The transporter associated with antigen processing (TAP) plays a key role in adaptive immunity by translocating proteasomal degradation products from the cytosol into the endoplasmic reticulum lumen for subsequent loading onto major histocompatibility (MHC) class I molecules. For functional and structural analysis of this ATP-binding cassette complex, we established the overexpression of TAP in the methylotrophic yeast Pichia pastoris. Screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex, yielding 30 mg of TAP/liter of culture. Detailed analysis of TAP function in the membrane, solubilized, purified, and reconstituted states revealed a direct influence of the native lipid environment on activity. TAP-associated phospholipids, essential for function, were profiled by liquid chromatography Fourier transform mass spectrometry. The antigen translocation activity is stimulated by phosphatidylinositol and -ethanolamine, whereas cholesterol has a negative effect on TAP activity.
The Na+-F1F0-ATPase operon ofAcetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunita and b, a gene encoding a 16-kDa proteolipid (subunit c 1), and two genes encoding 8-kDa proteolipids (subunits c 2 andc 3). Because subunits a,b, and c 1 were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a,b, and c 1 are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunitsa, c 2, c 3,b, δ, α, γ, β, and ε. Biochemical and immunological analyses revealed that subunitsc 1, c 2, andc 3 are all part of the c-oligomer, the first of a F1F0-ATPase that contains 8- and 16-kDa proteolipids.
Cytotoxic T lymphocytes eliminate infected cells upon surface display of antigenic peptides on major histocompatibility complex I molecules. To promote immune evasion, UL49.5 of several varicelloviruses interferes with the pathway of major histocompatibility complex I antigen processing. However, the inhibition mechanism has not been elucidated yet. Within the macromolecular peptide-loading complex we identified the transporter associated with antigen processing (TAP1 and TAP2) as the prime target of UL49.5. Moreover, we determined the active oligomeric state and crucial elements of the viral factor. Remarkably, the last two residues of the cytosolic tail of UL49.5 are essential for endoplasmic reticulum (ER)-associated proteasomal degradation of TAP. However, this process strictly requires additional signaling of an upstream regulatory element in the ER lumenal domain of UL49.5. Within this new immune evasion mechanism, we show for the first time that additive elements of a small viral factor and their signaling across the ER membrane are essential for targeted degradation of a multi-subunit membrane complex.
In the search for novel organic charge transfer salts with variable degrees of charge transfer we have studied the effects of two modifications of the recently synthesized donor–acceptor system [tetramethoxypyrene (TMP)]–[tetracyanoquinodimethane (TCNQ)]. One is of chemical nature by substituting the acceptor TCNQ molecules by F4TCNQ molecules. The second consists in simulating the application of uniaxial pressure along the stacking axis of the system. In order to test the chemical substitution, we have grown single crystals of the TMP–F4TCNQ complex and analyzed its electronic structure via electronic transport measurements, ab initio density functional theory (DFT) calculations and UV/VIS/IR absorption spectroscopy. This system shows an almost ideal geometrical overlap of nearly planar molecules stacked alternately (mixed stack) and this arrangement is echoed by a semiconductor-like transport behavior with an increased conductivity along the stacking direction. This is in contrast to TMP–TCNQ which shows a less pronounced anisotropy and a smaller conductivity response. Our band structure calculations confirm the one-dimensional behavior of TMP–F4TCNQ with pronounced dispersion only along the stacking axis. Infrared measurements illustrating the C[triple bond, length as m-dash]N vibration frequency shift in F4TCNQ suggest however no improvement in the degree of charge transfer in TMP–F4TCNQ with respect to TMP–TCNQ. In both complexes about 0.1e is transferred from TMP to the acceptor. Concerning the pressure effect, our DFT calculations on the designed TMP–TCNQ and TMP–F4TCNQ structures under different pressure conditions show that application of uniaxial pressure along the stacking axis of TMP–TCNQ may be the route to follow in order to obtain a much more pronounced charge transfer.
Vertebrate life depends on renal function to filter excess fluid and remove low-molecular-weight waste products. An essential component of the kidney filtration barrier is the slit diaphragm (SD), a specialized cell-cell junction between podocytes. Although the constituents of the SD are largely known, its molecular organization remains elusive. Here, we use super-resolution correlative light and electron microscopy to quantify a linear rate of reduction in albumin concentration across the filtration barrier under no-flow conditions. Next, we use cryo-electron tomography of vitreous lamellae from high-pressure frozen native glomeruli to analyze the molecular architecture of the SD. The resulting densities resemble a fishnet pattern. Fitting of Nephrin and Neph1, the main constituents of the SD, results in a complex interaction pattern with multiple contact sites between the molecules. Using molecular dynamics simulations, we construct a blueprint of the SD that explains its molecular architecture. Our architectural understanding of the SD reconciles previous findings and provides a mechanistic framework for the development of novel therapies to treat kidney dysfunction.
Vertebrate life depends on renal function to filter excess fluid and remove low-molecular-weight waste products. An essential component of the kidney filtration barrier is the slit diaphragm (SD), a specialized cell-cell junction between podocytes. Although the constituents of the SD are largely known, its molecular organization remains elusive. Here, we use super-resolution correlative light and electron microscopy to quantify a linear rate of reduction in albumin concentration across the filtration barrier. Next, we use cryo-electron tomography of vitreous lamellae from high-pressure frozen native glomeruli to analyze the molecular architecture of the SD. The resulting densities resemble a fishnet pattern. Fitting of Nephrin and Neph1, the main constituents of the SD, results in a complex interaction pattern with multiple contact sites between the molecules. Using molecular dynamics flexible fitting, we construct a blueprint of the SD, where we describe all interactions. Our architectural understanding of the SD reconciles previous findings and provides a mechanistic framework for the development of novel therapies to treat kidney dysfunction.
A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.
Macrophages ingesting apoptotic cells attenuate inflammatory responses, such as reactive oxygen species (ROS) generation. In atherosclerosis, ongoing inflammation and accumulation of apoptotic/necrotic material are observed, suggesting defects of phagocytes in recognizing or responding to dying cells. Modified lipoproteins such as oxidized LDL (oxLDL) are known to promote inflammation and to interfere with apoptotic cell clearance. Here, we studied the impact of cells exposed to oxLDL on their ability to interfere with the oxidative burst in phagocytes. In contrast to apoptotic cells, cells dying in response to or in the presence of oxLDL failed to suppress ROS generation despite efficiently being taken up by phagocytes. In addition, apoptotic cells, but not oxLDL-treated cells, inhibited phosphorylation of extracellular signal-regulated kinase, which is important for NADPH oxidase activation. oxLDL treatment did not interfere with activation of the antiinflammatory transcriptional regulator peroxisome proliferator-activated receptor gamma by apoptotic cells. Moreover, cells exposed to oxLDL failed to suppress lipopolysaccharide- induced proinflammatory cytokine expression, whereas apoptotic cells attenuated these phagocyte responses. Thus, the presence of oxLDL during cell death impaired the ability of apoptotic cells to act antiinflammatory with regard to oxidative burst inhibition and cytokine expression in phagocytes.
On the basis of the results obtained in a previous paper it is shown that in the thermodynamic limit the analogues of the Massieu-Plandc functions are linked with each other by means of the Legendre transformation. The existence of the limiting function φk(∞) implies the existence of the limiting function φl(∞) (l<k) under the same assumptions. Passage to the limit and derivation with respect to all independent variables commute. A statistical derivation of the thermodynamic stability condition in its most general form is given which leads naturally to a statistical interpretation of the concept of thermodynamic stability.
It is shown that, for all conceivable ensembles of statistical thermodynamics, at the thermodynamic limit, the frequency function of the fluctuations of macroscopic extensive parameters equals a Gaussian. The proof is based on a generalisation of Khinchin's method using the concept of "smoothed frequency functions."
The influence of temperatur and pressure on the fluorescence quantum yield of N-methylacridone (9,10-dihydro-9-oxo-10-methyl-acridine) in toluene in the range of 283-313 K and 1 bar to 2.5 kbar, respectively, has been investigated. Treatment of the data in terms of the Eyring transition-state theory leads to a consistent interpretation of the observed effect. The unusually large increase of the quantum yield with increasing pressure is attributed to a positive volume of activation, ⊿V≠, for the thermally activated S1-T2 intersystem crossing which is known to be the only deactivation process (of the Si-state) competing with fluorescence. Comparison of the values for ⊿H≠, the activation enthalpy of this process, determined at various pressures, indicates a decrease in ⊿H≠ at elevated pressures. Since ⊿H≠ can be associated with the S1-T2 energy gap involved in intersystem crossing, this result further confirms the conclusion that the change in Franck-Condon factors alone cannot account for the decrease in the intersystem crossing rate with increasing pressure.
A detailed analysis of the chemical constituents of a Caribbean specimen of Aiolochroia crassa was performed. Five brominated products (1 -5) were isolated and one of these was a new bromotyrosine metabolite. The structure of the new compound 1 has been established from spectral studies. Compounds 1 and 2, which are the major brominated metabolites and have not been previously identified in any Aiolochroia species, could be usefully employed as chemotaxonomic markers.
New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases
(1995)
Reactive coenzyme analogues ω-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of ω-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability at 0 °C. L actate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazonium pentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3α,20β-Hydroxysteroid dehydrogenase reacts at 0 °C with the ethyl homologue and slowly with the propyl compound. The butyl-and pentyl analogues do not inactivate at 0 °C. Tests with 14C -labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2 ′-phospho-adenosine diphosphate, a structural analogue of NADP +, was prepared by condensation of adenosine-2,3-cyclophospho-5′-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid ester with 2 ′:3′-cyclonucleotide-3′-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehyd rogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP +-dependent dehydrogenases the com pound has been show n to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.
The sesquiterpenoic alcohol nerolidol was separated into its 4 stereoisomers by MPLC of the diastereomeric (1 S, 4 R)-camphanoates.
An analytical GC method was found by which both the enantiomeric pairs of (Z)- and (E)-nerolidol are resolved on a chiral cyclodextrin stationary phase. The olfactoric properties of the nerolidol stereoisomers were investigated.
A non-radioactive cell-free assay was developed to quantitatively determine inhibition of plant-type phytoene desaturase by bleaching herbicides. An active desaturase was prepared from an appropriately cloned E. coli transformant. Another E. coli transformant was used to produce the required phytoene. Phytofluene and t-carotene, the products of the desaturase reaction, were either determined by HPLC or optical absorption spectra. Enzyme kinetics and inhibition data for the bleaching tetrazole herbicide WL110547 are presented as an example.
By means of differential thermoanalysis, the miscibility of the main polar tetraether lipid of Thermoplasma acidophilum with two ester lipids, dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol, resp., in the presence of excess water was studied. It is shown that with increasing fraction of tetraether lipid in the mixture, the transition range of dipalmitoyl phosphatidylcholine is broadened and the temperature of the maximum heat flow (Tm) is shifted to lower temperatures; furthermore, the enthaply change (ΔH) of the transition declines. Similar results were obtained with mixtures of tetraether lipid with dipalmitoyl phosphatidylglycerol. It is therefore concluded that the main polar tetraether lipid of Thermoplasma acidophilum , which essentially forms monomolecular layers, is able to form stable common phases with bilayer-forming ester lipids. Miscibility of the tetraether lipid with dipalmitoyl phosphatidylglycerol, which are both monovalent anions at neutral pH, is also observed in the presence of high proton or calcium ion concentrations.
The bipolar main tetraether lipid (MPL) of Thermoplasma acidophilum has been shown to form typical liquid expanded films at the air-water interface. The limiting molecular area at the collaps pressure is approximately Ac=73 Å2 per molecule. Monopolar aiphytanyl diether lipids were found to occupy the same area at high surface pressure as MPL. Thus, it was concluded that in the monofilm only one of the two polar headgroups of the MPL molecules is hydrated, i.e. that the single MPL molecules arc oriented upright. The packing properties of MPT. in the monofilm are determined by the properties of the branched alkyl chains only; the polar head groups do not contribute to the space requirement in the film. The collaps pressure of the MPL film is approximately 39 mN m-1 at 8°C. At a surface pressure of π = 30 mN m-1 and 20 °C the film is stable for many hours.
pH-titrations with NADH show two ionizable groups in mitochondrial and cytoplasmic malate dehydrogenase, the first with a pKa in the range 6.8 -8.3 for the mitochondrial and 6.4-7.8 for the cytoplasmic enzyme, the second with a lower limit at 10.2 resp. 11. Comparison with bis-(dihydronicotinamide)-dinucleotide and dihydronicotina-mide-ribosyl-P2-ribose-pyrophosphate instead of NADH indicates that the second alkaline ionization is caused by a residue placed near the adenine binding site of the active centre of the two isoenzymes. Binding studies with NADH and NAD+ give evidence for the participation of a group in the mitochondrial enzyme with pKa 6.8, deprotonation of which is necessary for detectable association of NAD+. In contrast the fixation of NAD+ to the cytoplasmic enzyme is independent of pH.
Studies on the transport of anions and zwitterions of acidic amino acids in Streptomyces hydrogenans
(1983)
n Streptomyces hydrogenans, acidic amino acfds are taken up either as anions by a specific transport system or as zwitterions via a nonspecific one. Variations in the zwitterion concentration caused by changes in pH influence the uptake and exchange diffusion by the nonspecific system. Differences in pH-optima for ʟ-glutamate and ʟ-aspartate transport are due to the different pK2-values of these amino acids. The anion transport by the specific system is accompanied by a short hyperpolarization of the membrane potential followed by a secondary influx of potassium ions into the cells.
The recently developed stereospecific sodium salt glycosylation procedure has been successfully applied to the synthesis of the β-ᴅ-2′-deoxyribofuranosides of benzimidazole, 5,6-dihalogeno benzimidazoles, and some 2-substituted analogues in high yield. The 5,6-dibromo analogue was obtained by bromination of the parent nucleoside. These have all been characterized by spectroscopic methods, including 1H NMR, which permitted analyses of their solution conformations and comparison with those of the corresponding ribofuranosides. Some biological aspects, including preliminary results on cytotoxicity and antiviral activity, are briefly considered.
Protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases co-regulate cellular processes. In pathogenic bacteria, they are frequently exploited to act as key virulence factors for human diseases. Mycobacterium tuberculosis, the causative organism of tuberculosis, secretes a low molecular weight PTP (LMW-PTP), MptpA, which is required for its survival upon infection of host macrophages. Although there is otherwise no sequence similarity of LMW-PTPs to other classes of PTPs, the phosphate binding loop (P-loop) CX5R and the loop containing a critical aspartic acid residue (D-loop), required for the catalytic activity, are well conserved. In most high molecular weight PTPs, ligand binding to the P-loop triggers a large conformational reorientation of the D-loop, in which it moves ∼10 Å, from an “open” to a “closed” conformation. Until now, there have been no ligand-free structures of LMW-PTPs described, and hence the dynamics of the D-loop have remained largely unknown for these PTPs. Here, we present a high resolution solution NMR structure of the free form of the MptpA LMW-PTP. In the absence of ligand and phosphate ions, the D-loop adopts an open conformation. Furthermore, we characterized the binding site of phosphate, a competitive inhibitor of LMW-PTPs, on MptpA and elucidated the involvement of both the P- and D-loop in phosphate binding. Notably, in LMW-PTPs, the phosphorylation status of two well conserved tyrosine residues, typically located in the D-loop, regulates the enzyme activity. PtkA, the kinase complementary to MptpA, phosphorylates these two tyrosine residues in MptpA. We characterized the MptpA-PtkA interaction by NMR spectroscopy to show that both the P- and D-loop form part of the binding interface.
Much of the research on Na+/H+ exchange has been done in prokaryotic models, mainly on the NhaA Na+/H+-exchanger from Escherichia coli (EcNhaA). Two conserved aspartate residues, Asp-163 and Asp-164, are essential for transport and are candidates for possible binding sites for the two H+ that are exchanged for one Na+ to make the overall transport process electrogenic. More recently, a proposed mechanism of transport for EcNhaA has suggested direct binding of one of the transported H+ to the conserved Lys-300 residue, a salt bridge partner of Asp-163. This contention is supported by a study reporting that substitution of the equivalent residue, Lys-305, of a related Na+/H+ antiporter, NapA from Thermus thermophilus, renders the transporter electroneutral. In this work, we sought to establish whether the Lys-300 residue and its partner Asp-163 are essential for the electrogenicity of EcNhaA. To that end, we replaced Lys-300 with Gln, either alone or together with the simultaneous substitution of Asp-163 with Asn, and characterized these transporter variants in electrophysiological experiments combined with H+ transport measurements and stability analysis. We found that K300Q EcNhaA can still support electrogenic Na+/H+ antiport in EcNhaA, but has reduced thermal stability. A parallel electrophysiological investigation of the K305Q variant of TtNapA revealed that it is also electrogenic. Furthermore, replacement of both salt bridge partners in the ion-binding site of EcNhaA produced an electrogenic variant (D163N/K300Q). Our findings indicate that alternative mechanisms sustain EcNhaA activity in the absence of canonical ion-binding residues and that the conserved lysines confer structural stability.
The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.
Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.
The MAM (meprin/A5-protein/PTPmu) domain is present in numerous proteins with diverse functions. PTPμ belongs to the MAM-containing subclass of protein-tyrosine phosphatases (PTP) able to promote cell-to-cell adhesion. Here we provide experimental evidence that the MAM domain is a homophilic binding site of PTPμ. We demonstrate that the MAM domain forms oligomers in solution and binds to the PTPμ ectodomain at the cell surface. The presence of two disulfide bridges in the MAM molecule was evidenced and their integrity was found to be essential for MAM homophilic interaction. Our data also indicate that PTPμ ectodomain forms oligomers and mediates the cellular adhesion, even in the absence of MAM domain homophilic binding. Reciprocally, MAM is able to interact homophilically in the absence of ectodomain trans binding. The MAM domain therefore contains independent cis and trans interaction sites and we predict that its main role is to promote lateral dimerization of PTPμ at the cell surface. This finding contributes to the understanding of the signal transduction mechanism in MAM-containing PTPs.
By a comparative thin layer chromatographic screening of the methanol-soluble leaf exudates from more than 400 Aloe plants (183 species), 5-hydroxyaloin A was identified in 20 species. Whilst 13 of the 20 species revealed interindividual variations concerning to the occurrence of 5-hydroxyaloin A, this anthrone-C-glucosyl was unambiguously detected in each individual of 6 Aloe species. In the leaf exudates from A. marlothii Berger 5-hydroxyaloin A was only traceable in the aloin-containing chemivars. The complete anthrone-C-glucosyl pattern of these 7 clearly characterized species has been determined additionally by qualitative and quantitative high performance liquid chromatography: The results obtained demonstrate that 5-hydroxyaloin only occurs in the more stable A-configuration (10 R, 1′S), thus being till now the only anthrone-C-glycosyl which has not been found as diastereomeric pair genuinely in plants. As well, 5-hydroxyaloin A characterizes a quantitatively significant hydroxylating pathway in biosynthesis of anthranoids. It is discussed as a chemotaxonomic marker of the genus Aloe, especially of the sections Pachydendron and Eualoe.
Membrane-Phloretin Interaction, Infrared Raman, ESR Spectroscopy The transport inhibitor phloretin was bound to human red cell membrane and the concomitant structural changes were observed by spectroscopic methods. By the spin labeling method a decrease in fluidity of the membrane was found at 1 and 10 |iM concentrations of the reagent. This result was obtained with the 2-(3-Carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, and the 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl lipid spin labels. Infrared spectroscopy of modified membranes revealed an intensity increase of the POO~ band at about 1250 cm-1. Moreover, a shift of the peak at 1050 cm -1 to 1100 cm-1 was observed in the presence of phloretin. Raman spectroscopy of the membranes did not contradict the results found with infrared and ESR spectroscopy: In the phloretin modified membrane we observed a lack of the band at 1085 cm-1, which leads to suggest that the POO" and/or C-C regions are less fluid. Changes of the extracted red cell membrane lipids were less characteristic, and the results differed from those found in red cell membrane.