Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Simmonds, A. C.; East, J. Malcolm; Jones, Owen; Rooney, E.K.; McWhirter, J M; Lee, A.G.

doi:10.1016/0005-2736(82)90447-3

Cited by 171 publications

(158 citation statements)

References 21 publications

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“…Bromine atoms quench the fluorescence of chromophores with which they are in close contact by heavy atom quenching [62,58]. To date, brominated phospholipids have been used to analyze binding of protein to vesicles [37,63], relative affinities of lipids to membrane proteins [64,65] and, through bromination at different positions in the alkyl chain, to investigate the topography of integral membrane proteins (e.g. [35,66]).…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…7 It has been found that cholesterol interacts with Ca 2ϩ -ATPase at annular sites after reconstitution of Ca 2ϩ -ATPase with cholesterol/dioleoylphosphatidylcholine mixtures, although with a somewhat lower affinity than phospholipids (41,45). However, there is also evidence from tryptophan quenching studies with a brominated cholesterol derivative that cholesterol is bound more firmly at a specific nonannular binding site (46). In shark Na ϩ ,K ϩ -ATPase (PDB code 2ZXE) (47), a cholesterol molecule has been found associated with the enzyme, although at a slightly different position between the ␣-and ␤-subunit where the pig kidney enzyme structure also shows a bound bilayer component (47).…”

Section: Interaction Of Camentioning

confidence: 99%

Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs

Winther

Liu

Sonntag

et al. 2010

Journal of Biological Chemistry

100

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Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca 2؉ -ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca 2؉ entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990 -1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.Understanding protein-membrane interaction and the activity of lipophilic drugs and specific lipids like sterols attracts increasing attention and represents a challenge to our current models of protein-solvent interactions and protein dynamics. Sarco/endoplasmic Ca 2ϩ -ATPase (SERCA) 6 offers an attractive model for the analysis of such interactions due to favorable biochemical properties with sensitive assays of function, a large resource of inhibitors, and a wealth of crystal structures related to the functional cycle. In conjunction with plasma membrane Ca 2ϩ -ATPases and Ca 2ϩ channel proteins, SERCA orchestrates spatiotemporal changes in the concentration of Ca 2ϩ inside the cell, providing the background essential for the function of Ca 2ϩ as a second messenger in the regulation of numerous cellular processes (1-3). At the same time, high cytosolic (Ͼ1 M) concentrations of Ca 2ϩ , induced by depletion of the endoplasmic Ca 2ϩ stores and uptake of extracellular Ca 2ϩ by the endoplasmic store-regulated mechanism (4), are involved as essential factors in the induction of apoptotic processes, leading to mitochondrial disruption with release of cytochrome c, activation of intracellular caspases, and cell death (5). As a consequence, interference of cellular function by inhibition of SERCA may lead to apoptotic cell death irrespective of the...

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“…When forced to interact with some membrane proteins, it has a strong inhibitory effect on their function (Warren et al, 1975). Cholesterol is normally excluded from direct contact with the Ca2+-ATPase enzyme (Bennett et al, 1975;Johannsson et al, 1981a;Simmonds et al, 1982) and excluded from other membrane enzymes as well (Houslay & …”

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confidence: 99%

Modulation of sarcoplasmic reticulum calcium pump activity by membrane fluidity

Almeida¹,

Vaz

Zachariasse

et al. 1984

Biochemistry

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Intramolecular excimerization of 1,3-di-l-pyrenylpropane [Py(3)Py] was used to assess the fluidity of sarcoplasmic reticulum membranes (SR); on the basis of the spectral data, the probe incorporates completely inside the membrane probably somewhere close to the polar head groups of phospholipid molecules, however not in the very hydrophobic core. The excimerization rate is very sensitive to lipid phase transitions, as revealed by thermal profiles of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers. Cholesterol abolishes pretransitions and broadens the thermal profiles of the main transitions which vanish completely at 50 mol % sterol. Excimer formation in liposomes of SR total lipid extracts does not show any sharp transitions, as in the case of DMPC and DPPC. However, the plots display discontinuities at about 20 OC which are broadened by cholesterol and not observed at 50 mol % sterol.Sarcoplasmic reticulum membranes (SR) have been extensively characterized in their structural and functional aspects (Weber et al., 1973;Hasselbach, 1979;Tada et al., 1978). The major protein, the Ca++-pump enzyme, is intrinsically associated with membrane lipids which greatly influence the enzyme activity (Martonosi et al., 1971;Bennett et al., 1980; Johannsson et al., 1981a). Lipids in contact with the ATPase enzyme modulate its function through physical interactions (Bennett et al., 1980; Johannsson et al., 1981a), including changing membrane fluidity. Thus, CaZ+ translocation across S R membrane and molecular mechanisms associated to energy transductions between the electroosmotic energy of Ca2+ gradients and chemical energy of ATP may be modulated by lipid-protein interactions, presumably affected by membrane fluidity. Cholesterol has a condensing effect on the acyl chains of bilayers in the fluid state (Houslay & Stanley, 1982). When forced to interact with some membrane proteins, it has a strong inhibitory effect on their function (Warren et al., 1975). Cholesterol is normally excluded from direct contact with the Ca2+-ATPase enzyme (Bennett et al., 1975; Johannsson et al., 1981a;Simmonds et al., 1982) Also cholesterol has been incorporated in native SR membranes by an exchange technique allowing progressive enrichment without changing the phospholipid/protein molar ratio. As in liposomes, discontinuities of excimer formation at 20 OC are broadened by cholesterol enrichment. The full activity of uncoupled Ca*+-ATPase is only affected by cholesterol above a molar ratio to phospholipid of 0.4. However, a significant decrease in activity (about 20%) is only noticed at a ratio of 0.6 (the highest technically achieved); at this ratio, about 28 lipid molecules per CaZ+-ATPase are expected to be relatively free from cholesterol interaction. The vesicle structure is still intact at this high ratio, as judged from the absence of basal activity (not Ca2+ stimulated). However, the sterol significantly decreases to about 60% the energetic efficiency of Ca2+ pumping (CaZ+/ATP ratio).

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Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Cited by 171 publications

References 21 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

Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs

Modulation of sarcoplasmic reticulum calcium pump activity by membrane fluidity

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Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Cited by 171 publications

References 21 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

Critical Roles of Hydrophobicity and Orientation of Side Chains for Inactivation of Sarcoplasmic Reticulum Ca2+-ATPase with Thapsigargin and Thapsigargin Analogs

Modulation of sarcoplasmic reticulum calcium pump activity by membrane fluidity

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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