Ca2+-bridging mechanism and phospholipid head group recognition in the membrane-binding protein annexin V

Swairjo, Manal A.; Concha, N.O.; Kaetzel, Marcia A.; Dedman, John R.; Seaton, Barbara A.

doi:10.1038/nsb1195-968

Cited by 321 publications

(276 citation statements)

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“…The X-ray structure shows that one phosphoserine head group is coordinated to two bridging Ca 2? ions that are in turn coordinated to one of the four canonical binding sites at the protein surface (Swairjo et al 1995). The requirement on the participation of calcium ions in the assay produces complications in its practical use.…”

Section: The Unlimited Choice Of Dyes Exists For Its Labelingmentioning

confidence: 99%

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

2012

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Dramatic changes in the structure of cell membranes on apoptosis allow easy, sensitive and non-destructive analysis of this process with the application of fluorescence methods. The strong plasma membrane asymmetry is present in living cells, and its loss on apoptosis is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the application of annexin V labeled with fluorescent dye is presently the most popular tool for its detection. Several methods have been suggested recently that offer important advantages over annexin V assay with the ability to study apoptosis by spectroscopy of cell suspensions, flow cytometry and confocal or twophoton microscopy. The PS exposure marks the integrated changes in the outer leaflet of cell membrane that involve electrostatic potential and hydration, and the attempts are being made to provide direct probing of these changes. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses, in comparison with the annexin V-binding assay, the novel fluorescence techniques of detecting apoptosis on cellular membrane level. In more detail we describe the detection method based on smart fluorescent dye F2N12S incorporated into outer leaflet of cell membrane and reporting on apoptotic cell transformation by easily detectable change of the spectral distribution of fluorescent emission. It can be adapted to any assay format.

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Section: The Unlimited Choice Of Dyes Exists For Its Labelingmentioning

confidence: 99%

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

2012

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“…The annexins show a marked preference for binding to interfaces, bilayers or vesicles containing anionic lipids, whereas for annexin V a specific receptor site for phosphatidylserine has been proposed [6]. No significant binding of annexins to pure phosphatidylcholine can be observed except possibly at very high Abbreviations used : CaLB, Ca 2 + -dependent phospholipid-binding domain ; (c)PLA 2 , (cytosolic) phospholipase A 2 ; DOPG, dioleoyl phosphatidylglycerol ; SAPA, 1-stearoyl 2-arachidonyl phosphatidic acid ; SAPC, 1-stearoyl 2-arachidonyl phosphatidylcholine ; SUV, small unilamellar vesicle.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of human cytosolic phospholipase A2 by human annexin V

Buckland

Wilton

1998

Biochemical Journal

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The ability of annexins, particularly annexin 1 (lipocortin 1), to inhibit phospholipase A2 (PLA2) is well known and a substrate depletion mechanism is now widely accepted as the explanation for most inhibitory studies. However, there are only a very limited number of reported studies involving annexins and the high-molecular-mass cytosolic PLA2 (cPLA2). In this study we have examined the effect of human recombinant annexin V, a potentially abundant cytosolic protein, on the ability of human recombinant cPLA2 to hydrolyse a variety of phospholipid substrates. The results show clearly that, under the conditions of our study, annexin V can inhibit cPLA2 activity by a mechanism of substrate depletion and that this inhibition is dependent on the nature of the phospholipids and the concentration of Ca2+ ions in the assay. The hydrolysis of 1-stearoyl 2-arachidonyl phosphatidylcholine by cPLA2 was not significantly affected by annexin V over a range of Ca2+ concentrations (1 μM-2.5 mM), a result that presumably reflects the zwitterionic nature of the phospholipid and the known inability of annexins to bind to such interfaces. In contrast, the hydrolysis of dioleoyl phosphatidylglycerol, which is an effective anionic phospholipid substrate for this enzyme, and more significantly that of 1-stearoyl 2-arachidonyl phosphatidic acid, were readily inhibited by annexin V, although these effects were Ca2+-dependent. The Ca2+ concentrations required for inhibition in the assay system in vitro are greater than those associated with Ca2+-stimulated events within the cell, suggesting that a role for annexin V in regulating cPLA2 activity might not involve a substrate depletion mechanism in vivo unless factors in addition to Ca2+ and phospholipids contribute to the binding of annexin V to cell membranes.

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“…There is broad agreement on the importance of calcium for the interaction of annexins with membranes at neutral pH. Calcium is thought to mediate binding via formation of calcium bridges between protein and membrane phospholipid head groups (14). Beyond this, there is little agreement on the key factors governing binding affinity, and many different theories have been suggested.…”

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confidence: 99%

“…There may be insertion of specific hydrophobic residues, such as Trp-187, into the hydrophobic region of the membrane (15,16). There may also be specific interactions between protein side chains and non-phosphate components of the phospholipid head groups (14,17). Finally, formation of trimers and higher order multimers has been postulated to promote membrane binding via formation of favorable proteinprotein interactions on the membrane surface (13, 18 -21).…”

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confidence: 99%

Entropic and Enthalpic Contributions to Annexin V-Membrane Binding

Jeppesen

Smith

Gibson

et al. 2008

Journal of Biological Chemistry

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Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quantitatively elucidated. Analysis by isothermal microcalorimetry and calcium titration under conditions of low membrane occupancy showed that there was a strongly positive entropy change upon binding. For vesicles containing 25% phosphatidylserine at 0.15 M ionic strength, the free energy of binding was ؊53 kcal/mol protein, whereas the enthalpy of binding was ؊38 kcal/mol. Addition of 4 M urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quantitatively consistent with predictions from the WimleyWhite scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more positively charged, nor decreased by making it more negatively charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Analysis by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region makes an important contribution to overall binding affinity, supplementing the role of protein-calcium-phosphate chelates.

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Ca2+-bridging mechanism and phospholipid head group recognition in the membrane-binding protein annexin V

Cited by 321 publications

References 32 publications

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

Inhibition of human cytosolic phospholipase A2 by human annexin V

Entropic and Enthalpic Contributions to Annexin V-Membrane Binding

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