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Query: UNIPROT:P06889 (Mol)
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We have used electron microscopy to analyse the structure of wild-type human annexin V (recombinant and placental) and of several mutants (single and double point mutants) bound to monolayers composed of DOPS, DOPE, or brain extract (Folch fraction III). On these phospholipids and on DOPS/DOPC (3:1, w/w) protein trimers, as also found in 3-D crystals, assemble to form a hexagonal lattice with a unit vector length of about 18 nm. The resolution obtained in projection is 1.7 to 2.2 nm for wild-type and mutants. There are no significant differences between the annexin V mutants and the wild-type protein at this resolution. All proteins bind as trimers with their convex side harbouring the Ca(2+)-binding sites facing the membrane. A comparison of the 3-D reconstruction of annexin V wild-type with the high resolution crystal structure shows that the domain structure is preserved but the relative orientation of the modules (II/III) and (I/IV) is slightly changed so that the Ca(2+)-binding sites in all four domains (including the recently observed binding site in domain III) become coplanar to the membrane. The thickness of the molecule obtained in the 3-D reconstruction corresponds well with the thickness of the high resolution crystal structure indicative of peripheral binding of annexin V without substantial penetration of the membrane.
J Mol Biol 1994 Apr 29
PMID:Three-dimensional structure of membrane-bound annexin V. A correlative electron microscopy-X-ray crystallography study. 815 49

The binding of calcium ions to annexin V in the absence of phospholipids has been studied by UV-difference spectroscopy, circular dichroism, and steady-state and time-resolved fluorescence. In the absence of calcium, the unique tryptophan 187, located in domain III of annexin V, is surrounded by a strongly hydrophobic environment, as indicated by its "blue" fluorescence emission maximum (325 nm). This corresponds well with the description of the structure determined by X-ray crystallography of several crystal forms. The Trp187 time-resolved fluorescence decay shows the existence of a fast (picosecond) excited-state reaction which can involve the formation of an H-bond between the indole NH group and the proximate epsilon-OH and/or alpha-carbonyl groups of Thr224. Titration with calcium tends to stabilize the overall structure, as shown by circular dichroism, while leading to large modifications of the local structure around Trp187 making it accessible to the solvent as shown by UV-difference spectra, circular dichroism spectra, and the displacement of its fluorescence emission maximum at saturating concentrations of calcium (350 nm). A rapid (picosecond) formation of an excited-state complex, probably involving one or a few water molecules of the solvation shell, is observed. These observations correlate well with the conformational change observed in crystal structures obtained in high calcium concentrations, involving the removal of Trp187 from the buried position to the surface of the molecule [Sopkova, J., Renouard, M., & Lewit-Bentley, A. (1993) J. Mol. Biol. 234, 816-825; Concha, N. O., Head, J. F., Kaetzel, M. A., Dedman, J. R., & Seaton, B. A. (1993) Science 261, 1321-1324]. In the solvent-exposed conformation, the indole ring becomes mobile in the subnanosecond and nanosecond time range. This conformational change and the increase in local flexibility can be important for the accommodation of the protein on the surface of phospholipid membranes.
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PMID:The dynamic behavior of annexin V as a function of calcium ion binding: a circular dichroism, UV absorption, and steady-state and time-resolved fluorescence study. 816 3

Annexin V was crystallized in the presence of a high concentration of calcium and the structure refined at 1.9 A resolution. The crystals are triclinic (P1) with three molecules per asymmetric unit and pseudo-R3 symmetry, reflecting a tendency of annexin to form trimers. The overall structure of the protein is similar to that seen in other crystal forms. There are, however, significant changes in domain III, where a new calcium site is formed. The whole region surrounding this site is reorganized in our structure, rendering annexin V more symmetrical and more alike annexin I. The formation of the new calcium site causes the displacement of Trp187 from a buried to an exposed conformation, a change that has recently been demonstrated by fluorescence measurements. The affinity of the different potential calcium sites is modulated: there is no calcium bound in domains II and IV, while up to two secondary calcium ions sites (in domains I and III) can substitute, depending on the calcium concentration present. We suggest that annexin can act as a calcium buffer, binding or releasing calcium depending on its local concentration. Our results also show that annexin displays inherent mobility which, together with its capacity to modulate the calcium affinity of its sites, can be of importance for its function on the membrane surface.
J Mol Biol 1993 Dec 05
PMID:The crystal structure of a new high-calcium form of annexin V. 825 74

The annexins are a family of calcium-dependent phospholipid-binding proteins which share a high degree of primary sequence similarity. Using a model of the crystal structure of annexin V as a template, 3-dimensional models of human annexins I, II, III and VII were constructed by homology modeling (J. Greer, J. Mol. Biol. 153, 1027-1042, 1981; J.M. Chen, G. Lee, R.B. Murphy, R.P. Carty, P.W. Brant-Rauf, E. Friedman and M.R. Pincus, J. Biomolec. Str. Dyn. 6, 859-87, 1989) for the 316 amino acid portions corresponding to the annexin V structure published by Huber et al. (J. Mol. Biol. 223, 683-704, 1992). These methods were used to study structure-function correlations for calcium ion binding and calcium channel activity. Published experimental data are specifically shown to be consistent with the annexin models. Possible intramolecular disulfide bridges were identified in annexin I (between Cys297 and Cys316) and in annexins II and VII (between Cys115 and Cys243). Each of the annexin models have 3 postulated calcium binding sites, usually via a Gly-Xxx-Gly-Thr loop with an acidic Glu or Asp residue 42 positions C-terminal to the first Gly. Despite a nonconserved binding site sequence, annexins I and II are able to coordinate calcium in domain 3 since the residue in the second loop position is directed toward the solvent away from the binding pocket. This finding also suggests a mechanism for a conformational change upon binding calcium. Highly conserved Arg and acidic sidechains stabilize the channel pore structure; annexin channels probably exist in a closed state normally. Arg271 may be involved in channel opening upon activation: basic residue 254 can stabilize Glu112, which allows Arg271 to interact with residue 95 instead of Glu112. Residue 267, found on the convex surface at the pore opening, may also be important in modifying channel activity.
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PMID:Structure-function correlations of calcium binding and calcium channel activities based on 3-dimensional models of human annexins I, II, III, V and VII. 839 83

Annexin II binds in a calcium-dependent manner to acidic phospholipids and is a substrate of some protein kinases. An N-terminally shortened form of human annexin II was crystallized and its molecular structure determined. It is very similar to two previously described members of this protein family, annexin I and annexin V. The protein structure is nearly completely alpha-helical organized as four compact domains which consist of five alpha-helices each. The domains surround a hydrophilic pore. The calcium binding sites are located at the convex side of the structure as in annexin V. Recombinant and natural porcine annexin II are active as ion channel with characteristics similar to annexin V, while N-terminally shortened annexin II and the heterotetramer (annexin II-p11)2 are inactive. Two cysteine residues, Cys133 and Cys262, form a disulphide bridge connecting domains II and III, adding further weight to the notion that ion channel activity does not require major structural rearrangements.
J Mol Biol 1996 Apr 12
PMID:The crystal structure and ion channel activity of human annexin II, a peripheral membrane protein. 863 85

The ion channel properties of human annexin V, a calcium- and phospholipid-binding protein of the annexin family, have been structurally and functionally investigated by analysing the mutant Glu112 -->Gly. Glu112 forms a salt bridge with Arg271 located in the interior of the hydrophilic pore of the molecule which is conserved within the annexin family. The crystal structures of the mutant and wild-type proteins are very similar and show only marginal conformational changes around the mutation site. Electron microscopic images show a conserved four-domain structure upon membrane binding as in the wild-type annexin V. The channel properties of the mutant are drastically changed, as the mutant has lost the voltage-dependent channel gating and the selectivity for calcium ions over monovalent cations. These results strongly support the hypothesis that the central, hydrophilic pore is the ion-conducting pathway.
J Mol Biol 1996 May 17
PMID:Structural and functional characterisation of the voltage sensor in the ion channel human annexin V. 863 91

In cell culture, human osteoblasts and the osteosarcoma cell line MG-63 express annexins I, II, IV, V and VI. Small proportions of annexins IV and V are lost from MG-63 cells into the culture medium in a sedimentable form. however, the bulk of these annexins is intracellular. In non-confluent cells 3 days after passaging, annexin IV and annexin V are strongly present throughout the nucleus and are also present in the cytoplasm. On elevation of the intracellular calcium concentration with the lonophore ionomycin, the intranuclear pools of annexin IV in 38 +/- 4% of cells and annexin V in 70 +/- 5% of cells show relocation to the nuclear membrane within 40 s. Extracellular ATP, which causes a transient increase in the cytosolic free calcium concentration by acting at P2-purinoceptors, also causes relocation of the intranuclear pool of annexin IV in 22 +/- 4% of cells and of annexin V in 38 +/- 8% of cells. After stimulation no significant reversal of the relocation is observed. Elevation of intracellular calcium with ionophore and ATP also causes relocation of the cytoplasmic pools of annexins IV and V. The results support a role for annexins at cellular membranes in response to elevation of cytosolic calcium levels.
Mol Membr Biol
PMID:Calcium-induced relocation of annexins IV and V in the human osteosarcoma cell line MG-63. 874 77

The effects of lanthanide ions (Ln(3+)), La(3+), Ce(3+), Gd(3+), and Tb(3+), on the binding ability of annexin V to phospholipid vesicle were studied, and compared with that of Ca(2+). The energy transfer following excitation at 278 nm was accompanied by a small decrease (about 3%) in the fluorescence signal (310-315 nm) of the aromatic amino acid residues, followed by strong re-emission of annexin V-bound Tb(3+) at 547 nm. The results of the titration of Tb(3+)-protein interactions using the rate dialysis method suggest that there are two high affinity metal binding sites on annexin V, to which Tb(3+) binds with an average dissociation constant, Kd, of about 2.11 x 10(-7) M. The Ln(3+) seemed to assist the anticoagulant action of annexin V because it binds more strongly to phospholipid than Ca(2+) does. Annexin V had the same ability to bind phospholipids in 10 mu M Ln(3+) as it did in 1 InM Ca(2+), showing that it binds to anionic phospholipids with Ln(3+) more strongly than it does with Ca(2+).
Biochem Mol Biol Int 1996 Apr
PMID:Effects of lanthanide ions on the binding ability of annexin V to phospholipid vesicle. 913 65

This study explored whether annexin V, a protein with established phospholipase A2 inhibiting properties, plays a role in the degradation of membrane phospholipids of adult cardiac myocytes during metabolic inhibition (20 mM 2-deoxyglucose and 1 mM iodoacetic acid). Experiments were carried out on isolated cardiac myocytes prelabeled with [14C]-arachidonic acid, which were subjected to metabolic inhibition for up to 240 min. Under control conditions, annexin V was found to be localised predominantly at the sarcolemma. After 120 min of metabolic inhibition, the release of lactate dehydrogenase (LDH) was still comparable with control cells, while morphological changes were already visible. After 240 min of metabolic inhibition, LDH release was significantly elevated compared to control cells incubated for the same period of time (35% v 20% of total cellular activity). All myocytes had lost their typical elongated shape and sarcolemmal "blebs" had been formed. In metabolically inhibited cells, annexin V localisation seemed to be more pronounced at the level of the sarcolemma compared to controls, whereas membrane phospholipid hydrolysis occurred at a significantly elevated rate, as evidenced by a significantly enhanced accumulation of labeled arachidonic acid within the cells. The present findings are not in favor of the hypothesis that the increase in net degradation of phospholipids in energy-deprived cardiac myocytes is caused by a loss of annexin V from the sarcolemma, which would increase the vulnerability of the sarcolemma to phospholipase A2 activity.
J Mol Cell Cardiol 1997 May
PMID:Phospholipid degradation in energy-deprived cardiac myocytes: does annexin V play a role? 920 25

Annexin V belongs to a family of phospholipid binding proteins, the Annexins. It binds in the presence of Ca(2+)-ions with high affinity to negatively charged phospholipids like phosphatidylserine (PS). On the basis of its protein structure and biological activity Annexin V is considered as a protein exhibiting its hitherto unknown function within the intracellular environment. One argument comes from the understanding that PS is predominantly located in membrane leaflets, which face the cytosol. However, recent findings show that each cell type has the molecular machinery to expose PS at its cell surface. This machinery is activated during the execution of apoptosis. Once PS is exposed at the cell surface it exhibits procoagulant and proinflammatory activities. Annexin V will bind to the PS-exposing apoptotic cell and can inhibit thereby the procoagulant and pro-inflammatory activities of the dying cell. These findings together with the presence of Annexin V in the extracellular space depict a novel (patho)Physiological significance for Annexin V in vivo.
Cell Mol Life Sci 1997 Jun
PMID:Annexin V, the regulator of phosphatidylserine-catalyzed inflammation and coagulation during apoptosis. 923 Sep 31


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