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M le Maire

Publications and source records attributed to M le Maire.

At least 19 recordsLinked to original sources
PubMed2003

Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.

We have found that despite a markedly low calcium affinity the D813A/D818A mutant is capable, after complexation with Cr.ATP, of occluding Ca(2+) to the same extent (1-2 Ca(2+) per ATPase monomer) as wild- type ATPase. The inherent ability of the synthetic L6-7 loop peptide to bind Ca(2+) was demonstrated with murexide and mass spectrometry. NMR analysis indicated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum with coordination by all three aspartate residues D813/D815/D818 that resulted in an altered conformation of the peptide chain. Overall our observations suggest that, in addition to mediating contact between the intramembranous Ca(2+) binding sites and the cytosolic phosphorylation site as previously suggested, the L6-7 loop, in a preceding step, participates in the formation of an entrance port important for lodging Ca(2+) at a high-affinity binding site inside the membrane.

Adenosine Triphosphate↗
PubMed2002

Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5.

Binding of bacteriophage T5 to Escherichia coli cells is mediated by specific interactions between the receptor-binding protein pb5 (67.8 kDa) and the outer membrane iron-transporter FhuA. A histidine-tagged form of pb5 was overproduced and purified. Isolated pb5 is monomeric and organized mostly as beta-sheets (51%). pb5 functionality was attested in vivo by its ability to impair infection of E. coli cells by phage T5 and Phi80, and to prevent growth of bacteria on iron-ferrichrome as unique iron source. pb5 was functional in vitro, since addition of an equimolar concentration of pb5 to purified FhuA prevented DNA release from phage T5. However, pb5 alone was not sufficient for the conversion of FhuA into an open channel. Direct interaction of pb5 with FhuA was demonstrated by isolating a pb5/FhuA complex using size-exclusion chromatography. The stoichiometry, 1 mol of pb5/1 mol of FhuA, was deduced from its molecular mass, established by analytical ultracentrifugation after determination of the amount of bound detergent. SDS-PAGE and differential scanning calorimetry experiments highlighted the great stability of the complex: (i) it was not dissociated by 2% SDS even when the temperature was raised to 70 degrees C; (ii) thermal denaturation of the complex occurred at 85 degrees C, while pb5 and FhuA were denatured at 45 degrees C and 74 degrees C, respectively. The stability of the complex renders it suitable for high-resolution structural studies, allowing future analysis of conformational changes into both FhuA and pb5 upon adsorption of the virus to its host.

Bacterial Outer Membrane Proteins↗
PubMed2001

SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

Adult↗
PubMed2001

Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins.

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.

Allosteric Regulation↗
PubMed2000

Interaction of membrane proteins and lipids with solubilizing detergents.

Detergents are indispensable in the isolation of integral membrane proteins from biological membranes to study their intrinsic structural and functional properties. Solubilization involves a number of intermediary states that can be studied by a variety of physicochemical and kinetic methods; it usually starts by destabilization of the lipid component of the membranes, a process that is accompanied by a transition of detergent binding by the membrane from a noncooperative to a cooperative interaction already below the critical micellar concentration (CMC). This leads to the formation of membrane fragments of proteins and lipids with detergent-shielded edges. In the final stage of solubilization membrane proteins are present as protomers, with the membrane inserted sectors covered by detergent. We consider in detail the nature of this interaction and conclude that in general binding as a monolayer ring, rather than as a micelle, is the most probable mechanism. This mode of interaction is supported by neutron diffraction investigations on the disposition of detergent in 3-D crystals of membrane proteins. Finally, we briefly discuss the use of techniques such as analytical ultracentrifugation, size exclusion chromatography, and mass spectrometry relevant for the structural investigation of detergent solubilized membrane proteins.

Crystallography↗
PubMed2000

Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase.

Amphipols are short-chain amphipathic polymers designed to keep membrane proteins soluble in aqueous solutions. We have evaluated the effects of the interaction of amphipols with sarcoplasmic reticulum Ca(2+)-ATPase either in a membrane-bound or a soluble form. If the addition of amphipols to detergent-solubilized ATPase was followed by removal of detergent, soluble complexes formed, but these complexes retained poor ATPase activity, were not very stable upon long incubation periods, and at high concentrations they experienced aggregation. Nevertheless, adding excess detergent to diluted detergent-free ATPase-amphipol complexes incubated for short periods immediately restored full activity to these complexes, showing that amphipols had protected solubilized ATPase from the rapid and irreversible inactivation that otherwise follows detergent removal. Amphipols also protected solubilized ATPase from the rapid and irreversible inactivation observed in detergent solutions if the ATPase Ca(2+) binding sites remain vacant. Moreover, in the presence of Ca(2+), amphipol/detergent mixtures stabilized concentrated ATPase against inactivation and aggregation, whether in the presence or absence of lipids, for much longer periods of time (days) than detergent alone. Our observations suggest that mixtures of amphipols and detergents are promising media for handling solubilized Ca(2+)-ATPase under conditions that would otherwise lead to its irreversible denaturation and/or aggregation.

Binding, Competitive↗
PubMed2000

Hepatitis B virus-related insertional mutagenesis implicates SERCA1 gene in the control of apoptosis.

We have used the Hepatitis B Virus DNA genome as a probe to identify genes clonally mutated in vivo, in human liver cancers. In a tumor, HBV-DNA was found to be integrated into the gene encoding Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA), which pumps calcium, an important intracellular messenger for cell viability and growth, from the cytosol to the endoplasmic reticulum. The HBV X gene promoter cis-activates chimeric HBV X/SERCA1 transcripts, with splicing of SERCA1 exon 11, encoding C-terminally truncated SERCA1 proteins. Two chimeric HBV X/SERCA1 proteins accumulate in the tumor and form dimers. In vitro analyses have demonstrated that these proteins localize to the ER, determine its calcium depletion and induce cell death. We have also shown that these biological effects are related to expression of the SERCA, rather than of the viral moiety. This report involves for the first time the expression of mutated SERCA proteins in vivo in a tumor cell proliferation and in vitro in the control of cell viability. Oncogene (2000).

Aged↗
PubMed1999

NMR conformational study of the sixth transmembrane segment of sarcoplasmic reticulum Ca2+-ATPase.

In current topological models, the sarcoplasmic reticulum Ca2+-ATPase contains 10 putative transmembrane spans (M1-M10), with spans M4/M5/M6 and probably M8 participating in the formation of the membranous calcium-binding sites. We describe here the conformational properties of a synthetic peptide fragment (E785-N810) encompassing the sixth transmembrane span (M6) of Ca2+-ATPase. Peptide M6 includes three residues (N796, T799, and D800) out of the six membranous residues critically involved in the ATPase calcium-binding sites. 2D-NMR experiments were performed on the M6 peptide selectively labeled with 15N and solubilized in dodecylphosphocholine micelles to mimic a membrane-like environment. Under these conditions, M6 adopts a helical structure in its N-terminal part, between residues I788 and T799, while its C-terminal part (G801-N810) remains disordered. Addition of 20% trifluoroethanol stabilizes the alpha-helical N-terminal segment of the peptide, and reveals the propensity of the C-terminal segment (G801-L807) to form also a helix. This second helix is located at the interface or in the aqueous environment outside the micelles, while the N-terminal helix is buried in the hydrophobic core of the micelles. Furthermore, the two helical segments of M6 are linked by a flexible hinge region containing residues T799 and D800. These conformational features may be related to the transient formation of a Schellman motif (L797VTDGL802) encoded in the M6 sequence, which probably acts as a C-cap of the N-terminal helix and induces a bend with respect to the helix axis. We propose a model illustrating two conformations of M6 and its insertion in the membrane. The presence of a flexible region within M6 would greatly facilitate concomitant participation of all three residues (N796, T799, and D800) believed to be involved in calcium complexation.

Amino Acid Sequence↗
PubMed1998

Ligand binding to macromolecules or micelles: use of centrifugal ultrafiltration to measure low-affinity binding.

We describe a method for estimating ligand binding to a macromolecular sample under conditions where this binding is of low affinity and must be measured under equilibrium conditions, without removal of the unbound ligand. The method is based on centrifugal ultrafiltration through a membrane with a molecular mass cut-off intermediate between that of the ligand and that of the target, and the amount of bound ligand is calculated from the difference between the (total) ligand in the concentrated sample and the (free) ligand in the ultrafiltrate. Centrifugal ultrafiltration makes it possible to separate free ligand from bound ligand (without changing its concentration) and to simultaneously concentrate the target (such that the proportion of bound ligand becomes significant, even under low-affinity binding conditions). We applied this technique, using Centricon 10 (Amicon) devices, to several cases (soluble proteins, intact membranes, detergent-solubilized proteins, and pure detergent micelles) and assessed its value with respect to the common artifacts that occur in other protocols involving protein retention on nitrocellulose filters (nonspecific ligand adsorption and protein denaturation).

Adenosine Triphosphate↗
PubMed1998

Construction and use of a detergent-sensitive electrode to measure dodecyl sulfate activity and binding.

We describe the construction and use of a dodecyl sulfate-sensitive electrode cell to measure the activity of the detergent in biological samples. The electrode is based on the incorporation of a cetyltrimethylammonium/dodecyl sulfate complex in a siloxane polymer membrane. The cell records changes in the activity of SDS from 10(-6) to 10(-5) M SDS up to the critical micellar concentration. In aqueous solutions the cell follows Nernst' law with an electrometric response which is not affected by protein per se, but is modified by supporting electrolytes like NaCl. We demonstrate by comparison with equilibrium dialysis that the electrode can be used both to detect the high-affinity binding sites of serum albumin for SDS and to follow cooperative binding of the detergent to serum albumin, ovalbumin, and beta-lactoglobulin in the concentration interval 10(-4)-10(-3) M of unbound SDS. We conclude that the electrode has properties which should enable its use to monitor changes in SDS activity during interaction with biological material. The electrode may also be used to measure the activity of other detergents which, like SDS, form a sparingly soluble complex with cetyltrimethylammonium bromide.

Calibration↗
PubMed1998

Secondary structure of the membrane-bound domains of H+,K+-ATPase and Ca2+-ATPase, a comparison by FTIR after proteolysis treatment of the native membranes.

The sarcoplasmic reticulum Ca2+-ATPase and the gastric H+,K+-ATPase were cleaved under three different proteolysis conditions. After elimination of the protease and of the cleaved peptides, the vesicles containing the membrane-bound peptides of the ATPases were studied by Fourier transform attenuated total reflection infrared spectroscopy. In the harsher proteolysis conditions, the membrane-associated domain of the Ca2+-ATPase represented about 20% of the protein and was mainly constituted of alpha-helices. Polarized infrared spectroscopy showed that these alpha-helices were mainly oriented perpendicular to the membrane. However, only 10-20% of the H+,K+-ATPase was cleaved. The remaining, membrane-associated domain of the protein contained about 30% of alpha-helices and 30% of beta-sheet structures. The alpha-helices adopted a mainly transmembrane orientation. While the data on the Ca2+-ATPase are in general agreement with the current model of the protein, our results indicate that caution must be used in choosing this protein as a general structural model for all P-type ATPases. The protease-resistant, membrane-associated domain of the H+K+-ATPase is indeed much larger than predicted and also contained beta-sheet structures.

Adenosine Triphosphate↗
PubMed1998

Spectroscopic studies of the interaction of Ca2+-ATPase-peptides with dodecyl maltoside and its brominated analog.

The transmembrane sector of sarcoplasmic reticulum Ca2+-ATPase comprises ten putative transmembrane spans (M1-M10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca2+ binding: peptide M6 (amino acid residues 785-810), peptide M7-L (amino acid residues 808-847) corresponding to loop 6-7 and the majority of span M7, and peptide M7-S (amino acid residues 818-847) which contains a shorter version of loop 6-7 than M7-L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the alpha-helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7-L peptide, the L6-7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca2+-ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n-dodecyl beta-D-maltoside or quenching using the recently introduced brominated analog of n-dodecyl beta-D-maltoside: 7,8-dibromododecyl beta-maltoside [de Foresta, B., Legros, N., Plusquellec, D., le Maire, M. & Champeil, P. (1996) Eur J. Biochem. 241, 343-354]. Our results indicate that M7-L and M7-S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size-exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n-dodecyl beta-D-maltoside/7,8-dibromododecyl beta-maltoside, using tryptophan octyl ester and solubilized Ca2+-ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca2+-ATPase topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca2+-ATPase and the rather short alpha-helix span of M6 may lead to exposure of a significant part of the C-terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca2+ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site-directed mutagenesis, that this transmembrane segment should be essential in Ca2+ binding.

Amino Acid Sequence↗
PubMed1998

The cytoplasmic loop located between transmembrane segments 6 and 7 controls activation by Ca2+ of sarcoplasmic reticulum Ca2+-ATPase.

During active cation transport, sarcoplasmic reticulum Ca2+-ATPase, like other P-type ATPases, undergoes major conformational changes, some of which are dependent on Ca2+ binding to high affinity transport sites. We here report that, in addition to previously described residues of the transmembrane region (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478), the region located in the cytosolic L6-7 loop connecting transmembrane segments M6 and M7 has a definite influence on the sensitivity of the Ca2+-ATPase to Ca2+, i.e. on the affinity of the ATPase for Ca2+. Cluster mutation of aspartic residues in this loop results in a strong reduction of the affinity for Ca2+, as shown by the Ca2+ dependence of ATPase phosphorylation from either ATP or Pi. The reduction in Ca2+ affinity for phosphorylation from Pi is observed both at acidic and neutral pH, suggesting that these mutations interfere with binding of the first Ca2+, as proposed for some of the intramembranous residues essential for Ca2+ binding (Andersen, J. P. (1995) Biosci. Rep. 15, 243-261). Treatment of the mutated Ca2+-ATPase with proteinase K, in the absence or presence of various Ca2+ concentrations, leads to Ca2+-dependent changes in the proteolytic degradation pattern similar to those in the wild type but observed only at higher Ca2+ concentrations. This implies that these effects are not due to changes in the conformational state of Ca2+-free ATPase but that changes affecting the proteolytic digestion pattern require higher Ca2+ concentrations. We conclude that aspartic residues in the L6-7 loop might interact with Ca2+ during the initial steps of Ca2+ binding.

Biological Transport↗
PubMed1998

Characterization of a protease-resistant domain of the cytosolic portion of sarcoplasmic reticulum Ca2+-ATPase. Nucleotide- and metal-binding sites.

Treatment of rabbit sarcoplasmic reticulum Ca2+-ATPase with a variety of proteases, including elastase, proteinase K, and endoproteinases Asp-N and Glu-C, results in accumulation of soluble fragments starting close to the ATPase phosphorylation site Asp351 and ending in the Lys605-Arg615 region, well before the conserved sequences generally described as constituting the "hinge" region of this P-type ATPase (residues 670-760). These fragments, designated as p29/30, presumably originate from a relatively compact domain of the cytoplasmic head of the ATPase. They retain two structural characteristics of intact Ca2+-ATPase as follows: high sensitivity of peptidic bond Arg505-Ala506 to trypsin cleavage, and high reactivity of lysine residue Lys515 toward the fluorescent label fluorescein 5'-isothiocyanate. Regarding functional properties, these fragments retain the ability to bind nucleotides, although with reduced affinity compared with intact Ca2+-ATPase. The fragments also bind Nd3+ ions, leaving open the possibility that these fragments could contain the metal-binding site(s) responsible for the inhibitory effect of lanthanide ions on ATPase activity. The p29/30 soluble domain, like similar proteolytic fragments that can be obtained from other P-type ATPases, may be useful for obtaining three-dimensional structural information on the cytosolic portion of these ATPases, with or without bound nucleotides. From our findings we infer that a real hinge region with conformational flexibility is located at the C-terminal boundary of p29/30 (rather than in the conserved region of residues 670-760); we also propose that the ATP-binding cleft is mainly located within the p29/30 domain, with the phosphorylation site strategically located at the N-terminal border of this domain.

Adenosine Triphosphate↗
PubMed1998

The mechanism of detergent solubilization of liposomes and protein-containing membranes.

The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C12E8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and beta-D-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.

Animals↗
PubMed1998

Treatment with crystalline ultra-pure urea reduces the aggregation of integral membrane proteins without inhibiting N-terminal sequencing.

We have demonstrated that N-terminal sequencing can be performed successfully despite boiling protein samples in the presence of urea under precise conditions, before loading them onto SDS-PAGE and transfer to polyvinylidene difluoride membrane. Using myoglobin as a test protein, we found that its ability to undergo N-terminal sequencing was not affected by the presence of urea provided "ultra-pure" urea was used. Consistent with this result, we verified that urea did not carbamylate myoglobin since its molecular mass was measured by mass spectrometry after electroelution of the protein band from the gel. These observations are useful for the study of integral membrane proteins, in particular to study their topology from proteolysis experiments, since heating in the presence of urea before SDS-PAGE reduces membrane protein aggregation [Soulié, S., Mo/ller, J.V., Falson, P., and le Maire, M. (1996) Anal. Biochem. 236, 363-364]. We show that the sequencing yield of a hydrophobic peptide from reticulum Ca2+-ATPase was more than doubled in the presence of urea in accord with the quantification of the Coomassie Blue staining of the gel and of the amount present on the polyvinylidene difluoride membrane. For three peptides of the gastric H+K+-ATPase, the sequencing yield after urea treatment increased almost threefold.

Amino Acid Sequence↗