The Cytoplasmic Loop between Putative Transmembrane Segments 6 and 7 in Sarcoplasmic Reticulum Ca2+-ATPase Binds Ca2+ and Is Functionally Important

Falson, Pierre; Menguy, Thierry; Corre, Fabienne; Bouneau, Laurence; Gracia, Adrienne Gomez de; Soulié, Stéphanie; Centeno, Francisco; Møller, Jesper; Champeil, Philippe; Maire, Marc le

doi:10.1074/jbc.272.28.17258

Cited by 53 publications

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“…These residues are both established proteinase K cleavage sites in loop 6-7 of the Ca 2ϩ -ATPase [5,11]. The C-terminal amino acid of both M7-L and M7-S was equivalent to A847 (rather than a more C-terminal residue), due to chemical considerations.…”

Section: Resultsmentioning

confidence: 99%

“…The dashed horizontal line is considered to indicate the top of the stalk. Continuous and dashed arrows respectively indicate well known cleavage sites for proteinase K [5,11] and a new proteinase K site at L833 appearing in low yield when proteolysis is performed at 37°C (Soulié , S. and le Maire, unpublished results) near the membrane/water interface. Circled P indicates the location of the phosphorylable amino acid residue (Asp351).…”

Section: Resultsmentioning

confidence: 99%

“…However, many aspects of the topology of the C-terminal domain, including the precise location of the TM boundaries and their possible variations during enzyme turnover, remain unconfirmed (see [12] for a discussion of the plasticity concept). For example, the hydropathy plots of the M6 region predict the C-terminal boundary to be at N810 [6] whereas limited proteolysis with proteinase K shows that the preceding L807-G808 peptide bond is, at moderate Ca 2ϩ concentration, readily accessible to digestion ( [5,11], see Fig. 1A).…”

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confidence: 99%

“…Furthermore, the precise part of the polypeptide chain which is embedded in the membrane is often uncertain (see e.g. [4,5]). In the case of sarcoplasmic reticulum Ca 2ϩ -ATPase most authors favor the ten-span model (designated M1ϪM10) initially proposed by Brandl et al [6].…”

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confidence: 99%

“…1 B, C). This region of the Ca 2ϩ -ATPase is of particular interest because of its controversial topology and important functional role in Ca 2ϩ transport, span M6 is known to include three amino acid residues essential for Ca 2ϩ -binding [13, 31Ϫ34] and loop 6-7 also contains three Asp residues critical for the Ca 2ϩ -transport process [5].…”

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confidence: 99%

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

Soulié

Foresta

Møller

et al. 1998

European Journal of Biochemistry

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The transmembrane sector of sarcoplasmic reticulum Ca 2ϩ -ATPase comprises ten putative transmembrane spans (M1ϪM10) 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 Ca 2ϩ binding: peptide M6 (amino acid residues 785Ϫ810), peptide M7-L (amino acid residues 808Ϫ847) corresponding to loop 6-7 and the majority of span M7, and peptide M7-S (amino acid residues 818Ϫ847) 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 A-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 Ca 2ϩ -ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n-dodecyl β- D-maltoside 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 β-D-maltoside/ 7,8-dibromododecyl β-maltoside, using tryptophan octyl ester and solubilized Ca 2ϩ -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 Ca 2ϩ -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 Ca 2ϩ -ATPase and the rather short A-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 Ca 2ϩ 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 Ca 2ϩ binding.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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

Soulié

Foresta

Møller

et al. 1998

European Journal of Biochemistry

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Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca²⁺‐ATPase

Huang

2009

J Comput Chem

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Calcium ATPase is a member of the P-type ATPase, and it pumps calcium ions from the cytoplasm into the reticulum against a concentration gradient. Several X-ray structures of different conformations have been solved in recent years, providing basis for elucidating the active transport mechanism of Ca2+ ions. In this work, molecular dynamics (MD) simulations were performed at atomic level to investigate the dynamical process of calcium ions moving from the outer mouth of the protein to their binding sites. Five initial locations of Ca2+ ions were considered, and the simulations lasted for 2 or 6 ns, respectively. Specific pathways leading to the binding sites and large structural rearrangements around binding sites caused by uptake of calcium ions were identified. A cooperative binding mechanism was observed from our simulation. Firstly, the first Ca2+ ion binds to site I, and then, the second Ca2+ ion approaches. The interactions between the second Ca2+ and the residues around site I disturb the binding state of site I and weaken its binding ability for the first bound Ca2+. Because of the electrostatic repulsion of the second Ca2+ and the electrostatic attraction of site II, the first bound Ca2+ shifts from site I to site II. Concertedly, the second Ca2+ binds to site I, forming a binding state with two Ca2+ ions, one at site I and the other at site II. Both of Glu908 and Asp800 coordinate with the two Ca2+ ions simultaneously during the concerted binding process, which is believed to be the hinge to achieve the concerted binding. In our simulations, four amino acid residues that serve as the channel to link the outer mouth and the binding sites during the binding process were recognized, namely Tyr837, Tyr763, Asn911, and Ser767. The analyses regarding the activity of the proteins via mutations of some key residues also supported our cooperative mechanism.

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Calcium binding to the transmembrane domain of the sarcoplasmic reticulum Ca²⁺‐ATPase: Insights from molecular modeling

Costa

Carloni

2002

Proteins

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Sarcoplasmic reticulum Ca(2+)- ATPase pumps Ca(2+) ions from muscle cells to the sarcoplasmic reticulum. Here we use molecular dynamics and electrostatic modeling to investigate structural and dynamical features of key intermediates in the Ca(2+) binding process of the protein. Structural models of the protein (containing either two, one, or no calcium ions in the transmembrane domain) are constructed based on the X-ray structure by Toyoshima et al. (Nature 2000;405:647-655). The protein is embedded in a water/octane bilayer, which mimics the water/membrane environment. Our calculations provide information on the hydration of the two Ca(2+) ions, not emerging from the X-ray structure. Furthermore, they indicate that uptake of the metal ions causes large structural rearrangements of the metal binding sites. In addition, they suggest that the two ions reach their binding sites via two specific pathways. Finally, they allow identification of residues in the outer mouth of the protein that might interact with the Ca(2+) ions during the binding process.

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The Cytoplasmic Loop between Putative Transmembrane Segments 6 and 7 in Sarcoplasmic Reticulum Ca2+-ATPase Binds Ca2+ and Is Functionally Important

Cited by 53 publications

References 69 publications

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

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

Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca²⁺‐ATPase

Calcium binding to the transmembrane domain of the sarcoplasmic reticulum Ca²⁺‐ATPase: Insights from molecular modeling

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The Cytoplasmic Loop between Putative Transmembrane Segments 6 and 7 in Sarcoplasmic Reticulum Ca2+-ATPase Binds Ca2+ and Is Functionally Important

Cited by 53 publications

References 69 publications

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

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

Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca2+‐ATPase

Calcium binding to the transmembrane domain of the sarcoplasmic reticulum Ca2+‐ATPase: Insights from molecular modeling

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

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

Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca²⁺‐ATPase

Calcium binding to the transmembrane domain of the sarcoplasmic reticulum Ca²⁺‐ATPase: Insights from molecular modeling