Annexin V: the membrane-binding protein with diverse functions

https://doi.org/10.1098/rsob.210318

2

The role of procoagulant phospholipids on the surface of circulating blood cells in thrombosis and haemostasis

M. Protty, P. Jenkins, P. Collins, V. O'Donnell

Open Biology. 2022, 12, None

https://doi.org/10.1186/s12967-016-0843-7

]

. Among those membrane-binding proteins annexins form a large family which share structural properties and demonstrate calcium-dependent membrane binding.

Annexins are eukaryotic proteins and more than 100 annexins were found in different species including vertebrates, invertebrates, unicellular eukaryotes and plants

[

3,

Annexins family: insights into their functions and potential role in pathogenesis of sarcoidosis

M. Mirsaeidi, S. Gidfar, A. Vu, D. Schraufnagel

Journal of Translational Medicine. 2016, 14, None

4,

Two novel annexins from Drosophila melanogaster. Cloning, characterization, and differential expression in developmen

Johnston PA, Perin MS, Reynolds GA, Wasserman SA, Südhof TC.

J Biol Chem. 1990, 265, 11382–8

https://doi.org/10.1042/bj2700157

5

Purification and partial sequence analysis of plant annexins

M. Smallwood, J. Keen, D. Bowles

Biochemical Journal. 1990, 270, 157-161

https://doi.org/10.1186/s12967-016-0843-7

]

. The descriptor ‘A’ denotes that annexin is from vertebrates; ‘B’ denotes that annexin is from invertebrates; ‘C’ is for fungi and unicellular eukaryotes; ‘D’ is for plants; and ‘E’ is for protists. In human 12 different annexins were identified: annexins A1 – 11 and A13

[

3

Annexins family: insights into their functions and potential role in pathogenesis of sarcoidosis

M. Mirsaeidi, S. Gidfar, A. Vu, D. Schraufnagel

Journal of Translational Medicine. 2016, 14, None

https://doi.org/10.1016/s0962-8924(98)01360-9

]

.

Most of annexins are cytosolic proteins. Their messenger RNA lack a 5’-leader sequence which directs synthesized proteins to the endoplasmic reticulum as occurs in the conventional pathway of the protein secretion

[

6

Signal sequences: more than just greasy peptides

B. Martoglio, B. Dobberstein

Trends in Cell Biology. 1998, 8, 410-415

https://doi.org/10.1016/j.bbamcr.2008.01.030

]

. Inside the cell annexins play role in many processes including the cell signaling, exocytosis, membrane repair and the vesicle trafficking. However, annexins A1, A2, A4 and A5 might be found on the cell surface or in circulation and the mechanism of their secretion is largely unknown

[

7,

Extracellular annexin A5: Functions of phosphatidylserine-binding and two-dimensional crystallization

H. van Genderen, H. Kenis, L. Hofstra, J. Narula, C. Reutelingsperger

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2008, 1783, 953-963

https://doi.org/10.1016/j.bbamcr.2008.01.030

8

Extracellular annexin A5: Functions of phosphatidylserine-binding and two-dimensional crystallization

H. van Genderen, H. Kenis, L. Hofstra, J. Narula, C. Reutelingsperger

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2008, 1783, 953-963

https://doi.org/10.1007/BF02103286

]

.

Annexin A5 (or annexin V) was firstly isolated in 1979

[

9

Isolierung und Charakterisierung eines neuen plazentaspezifischen Proteins (PP10)

H. Bohn, W. Kraus

Archives of Gynecology. 1979, 227, 125-134

https://doi.org/10.1111/j.1432-1033.1985.tb09150.x

]

from human placenta and later and independently in 1985 from human arteries

[

10

Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord

C. REUTELINGSPERGER, G. HORNSTRA, H. HEMKER

European Journal of Biochemistry. 1985, 151, 625-629

]

. It is a 35.7 kDa protein which might be found both in the cellular cytosol and in the blood plasma of healthy individuals in concentration between 0.77 and 6.7 ng/ml according to different studies

[

]

. It is suggested that annexin A5 takes part in such important physiological processes as cellular membrane repair or inhibition of blood coagulation, however the mechanism and significance of its functioning are still unclear.

Annexin V has a complex mechanism of the membrane binding. It forms trimers on the phospholipid membrane and subsequently protein trimers form a two-dimensional lattice which covers the membrane surface

[

15

Two-Dimensional Crystallization of Annexin A5 on Phospholipid Bilayers and Monolayers: A Solid−Solid Phase Transition between Crystal Forms

I. Reviakine, W. Bergsma-Schutter, A. Morozov, A. Brisson

Langmuir. 2001, 17, 1680-1686

https://doi.org/10.1038/ncomms1270

]

. According to some studies this multiphase binding mechanism is important for the annexin V functioning

[

16,

Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair

A. Bouter, C. Gounou, R. Bérat, S. Tan, B. Gallois, T. Granier, B. d'Estaintot, E. Pöschl, B. Brachvogel, A. Brisson

Nature Communications. 2011, 2, None

17

Clustering of lipid-bound annexin V may explain its anticoagulant effect

Andree HA, Stuart MC, Hermens WT, Reutelingsperger CP, Hemker HC, Frederik PM, et al.

J Biol Chem. 1992, 267, 17907–12

https://doi.org/10.1016/j.bbamcr.2008.01.030

]

.

Due to its ability of binding to phospholipid membranes containing the negatively-charged phospholipid, phosphatidylserine (PS), annexin V is used in in vitro and in vivo studies as a marker of PS-positive cells

[

7,

Extracellular annexin A5: Functions of phosphatidylserine-binding and two-dimensional crystallization

H. van Genderen, H. Kenis, L. Hofstra, J. Narula, C. Reutelingsperger

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2008, 1783, 953-963

]

. In patients it is used in clinical imaging procedure of the apoptotic cells detection which includes the injection of the recombinant human annexin V into circulation

[

22,

Past, present, and future of annexin A5: from protein discovery to clinical applications.

Boersma HH, Kietselaer BLJH, Stolk LML, Bennaghmouch A, Hofstra L, Narula J, et al

J Nucl Med . 2005, 46, 2035–50

https://doi.org/10.1007/s00259-021-05641-4

23

Radionuclide imaging of apoptosis for clinical application

X. Qin, H. Jiang, Y. Liu, H. Zhang, M. Tian

European Journal of Nuclear Medicine and Molecular Imaging. 2022, 49, 1345-1359

]

.

Because of the annexin V participation in important physiological processes and the usage of annexin as a biomarker for PS-positive membranes the understanding of its precise physiological roles and mechanisms of its functioning is of great importance.

The current review is focused on the structure of annexin V and the mechanism and kinetics of the membrane binding. Lipid specificity and the multimerization process will be described. Finally, we will discuss some of the proposed annexin V functions including inhibition of the blood coagulation and the Ca²⁺ transport activity.

1. Structure of annexin A5 monomer

The crystal structure of human annexin V was firstly resolved in 1990 by Huber et al

[

24

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.

R. Huber, J. Römisch, E. Paques

The EMBO Journal. 1990, 9, 3867-3874

https://doi.org/10.1529/biophysj.106.093807

]

. Structures of rat

[

25

Protein Crystallography under Xenon and Nitrous Oxide Pressure: Comparison with In Vivo Pharmacology Studies and Implications for the Mechanism of Inhaled Anesthetic Action

N. Colloc’h, J. Sopkova-de Oliveira Santos, P. Retailleau, D. Vivarès, F. Bonneté, B. Langlois d’Estainto, B. Gallois, A. Brisson, J. Risso, M. Lemaire, T. Prangé, J. Abraini

Biophysical Journal. 2007, 92, 217-224

]

and chicken

[

26

Crystal structure of chicken annexin A5 in complex with functional modifiers Ca2+ and Zn2+ reveal Zn2+-induced formation of non-planar assemblies

Ortlund E, Chai G, Genge B, Wu LNY, Wuthier R, Lebioda L.

Annexins. 2004, 1, 183–90

https://doi.org/10.1038/s41586-021-03819-2

]

annexin V are available, and structures of bovine, mouse and chimpanzee annexin V are predicted by AlphaFold

[

27

Highly accurate protein structure prediction with AlphaFold

J. Jumper, R. Evans, A. Pritzel, T. Green, M. Figurnov, O. Ronneberger, K. Tunyasuvunakool, R. Bates, A. Žídek, A. Potapenko, A. Bridgland, C. Meyer, S. Kohl, A. Ballard, A. Cowie, B. Romera-Paredes, S. Nikolov, R. Jain, J. Adler, T. Back, S. Petersen, D. Reiman, E. Clancy, M. Zielinski, M. Steinegger, M. Pacholska, T. Berghammer, S. Bodenstein, D. Silver, O. Vinyals, A. Senior, K. Kavukcuoglu, P. Kohli, D. Hassabis

Nature. 2021, 596, 583-589

]

.

The protein consists of 320 amino acid residues and is almost entirely α-helical. The amino acid sequence contains a short N-terminal tail and four C-terminal repeats of approximately 70 residues each. Those four repeats are folded into four compact domains, numbered I to IV, which share the similar structure. Each domain consists of five α-helixes, named from A to E (for example, the helix A in domain II is referred to as helix IIA), which are connected by loops and the expression “loop IAB” refer to the loop between helixes A and B in domain I. All domains have hydrophobic cores in their centers

[

24,

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.

R. Huber, J. Römisch, E. Paques

The EMBO Journal. 1990, 9, 3867-3874

28,

Crystal and molecular structure of human annexin V after refinement

R. Huber, R. Berendes, A. Burger, M. Schneider, A. Karshikov, H. Luecke, J. Römisch, E. Paques

Journal of Molecular Biology. 1992, 223, 683-704

29

The Crystal Structure of a New High-calcium Form of Annexin V

J. Sopkova, M. Renouard, A. Lewit-Bentley

Journal of Molecular Biology. 1993, 234, 816-825

]

. Domains are bounded as follows: N-terminal tail, residues 5 – 16; domain I, residues 17 – 88; domain II, 89 – 159; linker, 160 – 167; domain III, 168 – 246; domain IV, 247 – 317

[

24

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.

R. Huber, J. Römisch, E. Paques

The EMBO Journal. 1990, 9, 3867-3874

]

(Figure 1A).

Four annexin V domains are arranged in a cyclic array. In the center of this structure there is a hydrophilic pore which is coated by charged residues

[

24,

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.

R. Huber, J. Römisch, E. Paques

The EMBO Journal. 1990, 9, 3867-3874

28,

Crystal and molecular structure of human annexin V after refinement

R. Huber, R. Berendes, A. Burger, M. Schneider, A. Karshikov, H. Luecke, J. Römisch, E. Paques

Journal of Molecular Biology. 1992, 223, 683-704

29

The Crystal Structure of a New High-calcium Form of Annexin V

J. Sopkova, M. Renouard, A. Lewit-Bentley

Journal of Molecular Biology. 1993, 234, 816-825

]

and inside it salt bridges Asp280-Arg276, Asp92–Arg117 and Glu112 -Arg271 are formed (Figure 1A). Those residues are invariant in different annexins

[

24

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.

R. Huber, J. Römisch, E. Paques

The EMBO Journal. 1990, 9, 3867-3874

]

.

The annexin V molecule has a slightly curved shape with a convex and a concave sides (Figure 1B). The N-terminal tail resides on the concave side and binds domains I and IV non-covalently

[

28

Crystal and molecular structure of human annexin V after refinement

R. Huber, R. Berendes, A. Burger, M. Schneider, A. Karshikov, H. Luecke, J. Römisch, E. Paques

Journal of Molecular Biology. 1992, 223, 683-704

]

whereas the membrane binding sites locate on the opposite, convex surface

[

29

The Crystal Structure of a New High-calcium Form of Annexin V

J. Sopkova, M. Renouard, A. Lewit-Bentley

Journal of Molecular Biology. 1993, 234, 816-825

]

.

Annexin V is a Ca²⁺-binding protein. Binding sites for Ca²⁺ ions are located on the convex side of the molecule

[

29

The Crystal Structure of a New High-calcium Form of Annexin V

J. Sopkova, M. Renouard, A. Lewit-Bentley

Journal of Molecular Biology. 1993, 234, 816-825

https://doi.org/10.1038/ncomms1270

]

(Figure 1B) and site-forming residues, revealed in different studies of human annexin V, are listed in Table 1. Briefly, seven different calcium sites were distinguished: 3 in domain I, 1 in domain II, 2 in domain III and 1 in domain IV.

In annexin V from Rattus norvegicus up to 11 Ca²⁺ binding sites were identified (2H0K in PDB Data Bank)

[

16

Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair

A. Bouter, C. Gounou, R. Bérat, S. Tan, B. Gallois, T. Granier, B. d'Estaintot, E. Pöschl, B. Brachvogel, A. Brisson

Nature Communications. 2011, 2, None

https://doi.org/10.1126/science.8362244

]

. Interestingly, human and rat annexin V are more than 90% identical (the sequences alignment is shown on Figure 1C) and amino acid residues which were found to form the Ca²⁺-binding sites are conserved in both proteins. Thus it might be speculated that the same 11 Ca²⁺-binding sites might exist in human annexin V.

The binding of Ca²⁺ ions influences the annexin V conformation. The comparison of the Ca²⁺-bound and the Ca²⁺-unbound structures revealed that significant changes occur in domain III. In the Ca²⁺ unbound structure the carbonyl oxygen of Gly183 forms a hydrogen bond with the backbone amide group of Lys186. In Ca²⁺-bound rat annexin V this bond between the corresponding residues Gly181 and Lys184 is broken and both residues bind Ca²⁺ via carbonyl oxygen

[

30

Rat Annexin V Crystal Structure: Ca ²⁺ -Induced Conformational Changes

N. Concha, J. Head, M. Kaetzel, J. Dedman, B. Seaton

Science. 1993, 261, 1321-1324

]

. As a result, in the Ca²⁺-bound form the Trp187 residue (Trp185 in rat annexin V) is exposed on the protein surface (outward-facing Trp187) whereas this residue is buried in the hydrophobic core of the protein in the Ca²⁺-unbound form (inward-facing Trp187). The protein region including residues 220 – 246 becomes more ordered in the Ca²⁺-bound form of annexin V

[

29,

The Crystal Structure of a New High-calcium Form of Annexin V

J. Sopkova, M. Renouard, A. Lewit-Bentley

Journal of Molecular Biology. 1993, 234, 816-825

31

High-resolution structures of annexin A5 in a two-dimensional array

S. Hong, S. Na, O. Kim, S. Jeong, B. Oh, N. Ha

Journal of Structural Biology. 2020, 209, 107401

]

. The molecular dynamics simulation revealed that Ca²⁺-bound annexin V has an extremely stable secondary structure with almost no fluctuations, while the Ca²⁺-unbound form demonstrates large fluctuations of the secondary structure and lower content of α-helical structures (71%) when compared to the Ca²⁺-bound form (80%)

[

32

Effect of Mg2+ versus Ca2+ on the behavior of Annexin A5 in a membrane-bound state

Z. Fezoua-Boubegtiten, B. Desbat, A. Brisson, C. Gounou, M. Laguerre, S. Lecomte

European Biophysics Journal. 2011, 40, 641-649

]

. The Ca²⁺-unbound structure is more flexible in solution

[

32

Effect of Mg2+ versus Ca2+ on the behavior of Annexin A5 in a membrane-bound state

Z. Fezoua-Boubegtiten, B. Desbat, A. Brisson, C. Gounou, M. Laguerre, S. Lecomte

European Biophysics Journal. 2011, 40, 641-649

]

.

The annexin V structure. A. The view from the convex side. Magenta, N-terminal tail; blue, domain I; yellow, domain II; green, domain III; red, domain IV; orange, Ca2+ ions. In the center of annexin V the charged residues Asp280, Arg276, Asp92, Arg117, Glu112, Arg271 are represented. B. The view from the domain II. The convex and the concave sides are marked by black arrows. Ca2+-binding sites are located on the convex surface, N-terminal tail is on the concave side. Figures were created in VMD for the current review using the structure 1ANX [29] from PDB Data Bank. C. Annexin V from human (ANXA5_HUMAN) and from rat (ANXA5_RAT) sequences alignment. Residues that form the Ca2+-binding sites are highlighted in green and yellow for human and rat annexin V respectively. The alignment was done using the UniProt Align tool.

Figure 1. The annexin V structure. A. The view from the convex side. Magenta, N-terminal tail; blue, domain I; yellow, domain II; green, domain III; red, domain IV; orange, Ca2+ ions. In the center of annexin V the charged residues Asp280, Arg276, Asp92, Arg117, Glu112, Arg271 are represented. B. The view from the domain II. The convex and the concave sides are marked by black arrows. Ca2+-binding sites are located on the convex surface, N-terminal tail is on the concave side. Figures were created in VMD for the current review using the structure 1ANX [29] from PDB Data Bank. C. Annexin V from human (ANXA5_HUMAN) and from rat (ANXA5_RAT) sequences alignment. Residues that form the Ca2+-binding sites are highlighted in green and yellow for human and rat annexin V respectively. The alignment was done using the UniProt Align tool.

2. Annexin V binding to phospholipid membrane

Annexin V is able to reversibly bind to PS-containing membranes

[

34

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM

J Biol Chem. 1990, 265, 4923–8

https://doi.org/10.1021/bi9703960

]

. The binding efficacy strongly depends on the Ca²⁺ concentration and PS content and it was studied extensively in different conditions.

Dependency on Ca²⁺ and phospholipids

At low pH (< 5) annexin V binds to PS-containing membranes even in absence of Ca²⁺. At neutral pH (= 7.4) Ca²⁺ is absolutely required for the annexin V membrane binding and the threshold concentration which initiates binding was found to be about 16 µM

[

35

Annexin V Interaction with Phosphatidylserine-Containing Vesicles at Low and Neutral pH

G. Köhler, U. Hering, O. Zschörnig, K. Arnold

Biochemistry. 1997, 36, 8189-8194

]

.

In presence of phospholipid vesicles, lipid bilayers on glass beads or planar lipid bilayers of constant composition the concentration of the bound protein rises with the increase of Ca²⁺ concentration in solution from 0.01 to 10 mM

[

34,

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM

J Biol Chem. 1990, 265, 4923–8

36,

Phospholipid binding of annexin V: Effects of calcium and membrane phosphatidylserine content

J. Tait, D. Gibson

Archives of Biochemistry and Biophysics. 1992, 298, 187-191

37

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

Stuart MC, Reutelingsperger CP, Frederik PM.

Cytometry . 1998, 33, 414–9

]

. The dependence is non-linear: for the smallest Ca²⁺ concentrations the binding is zero, it follows with the fast rise of the adsorbed protein quantity with Ca²⁺ concentration and reaches the constant value for the highest Ca²⁺ concentrations. For greater PS content less Ca²⁺ is needed to reach the maximal binding

[

34,

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM

J Biol Chem. 1990, 265, 4923–8

37

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

Stuart MC, Reutelingsperger CP, Frederik PM.

Cytometry . 1998, 33, 414–9

]

.

For small unilamellar vesicles (SUVs) (PS/PC = 20/80) the equilibrium dissociation constant K_d was determined: it decreases from 0.25 nM to 0.05 nM when the Ca²⁺ concentration increases from 0 to 0.8 mM and is almost constant for Ca²⁺ concentration higher than 0.8 mM

[

36

Phospholipid binding of annexin V: Effects of calcium and membrane phosphatidylserine content

J. Tait, D. Gibson

Archives of Biochemistry and Biophysics. 1992, 298, 187-191

]

.

Interestingly, the concentration of the bound protein in presence of saturating Ca²⁺ concentration (when the binding is maximal) was found to be the same on the planar phospholipid bilayers of substantially different composition (PS is varied from 1 to 100%)

[

34

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM

J Biol Chem. 1990, 265, 4923–8

]

. In contrast, for SUVs the number of phospholipid molecules per bound annexin V was found to be approximately 3 times higher for PS/PC = 10/90 when compared with PS/PC = 20/80 in presence of saturating Ca²⁺ concentration and that means that less annexin V was bound to vesicles with lower PS content even when the Ca²⁺ concentration is high enough to reach the maximal binding

[

36

Phospholipid binding of annexin V: Effects of calcium and membrane phosphatidylserine content

J. Tait, D. Gibson

Archives of Biochemistry and Biophysics. 1992, 298, 187-191

]

. This apparent discrepancy might be due to the difference in studied phospholipid systems.

In presence of constant Ca²⁺ concentration the dependence of annexin V binding on the PS content was determined

[

]

. The number of phospholipid molecules per bound annexin V decreased nonlinearly from 1100 to 84 when the phospholipid content of SUVs increases from 10 to 50%, the Ca²⁺ concentration was equal to 1.2 mM. It means that more protein is bound for higher PS contents. For all studied SUVs compositions the equilibrium dissociation constant K_d was unchanged and equal to 0.036 ± 0.011 nM. Thus the main changes in annexin V binding to vesicles of different content are associated with the changes in stoichiometry and density of binding sites but not with affinity to the binding site

[

36

Phospholipid binding of annexin V: Effects of calcium and membrane phosphatidylserine content

J. Tait, D. Gibson

Archives of Biochemistry and Biophysics. 1992, 298, 187-191

]

. In presence of lipid bilayers on glass beads or planar lipid bilayers the quantity of bound protein rises with PS content too for different Ca²⁺ concentrations added

[

37,

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

Stuart MC, Reutelingsperger CP, Frederik PM.

Cytometry . 1998, 33, 414–9

38

Defining the structural characteristics of annexin V binding to a mimetic apoptotic membrane

J. Lu, A. Le Brun, S. Chow, T. Shiota, B. Wang, T. Lin, G. Liu, H. Shen

European Biophysics Journal. 2015, 44, 697-708

]

. The dependency is nonlinear with rapid increase of binding for PS content less than 4% and almost constant value for more than 4% of PS

[

37

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

Stuart MC, Reutelingsperger CP, Frederik PM.

Cytometry . 1998, 33, 414–9

]

or even more complex behavior

[

38

Defining the structural characteristics of annexin V binding to a mimetic apoptotic membrane

J. Lu, A. Le Brun, S. Chow, T. Shiota, B. Wang, T. Lin, G. Liu, H. Shen

European Biophysics Journal. 2015, 44, 697-708

https://doi.org/10.1002/cyto.a.20345

]

. In a more recent study it was found out that annexin V binding demonstrates the threshold behavior: in presence of 1.5 mM Ca²⁺ the protein does not bind to membranes containing less than 8% of PS and it shows the maximal binding for higher PS content

[

40

Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death

J. Shi, Y. Shi, L. Waehrens, J. Rasmussen, C. Heegaard, G. Gilbert

Cytometry Part A. 2006, 69A, 1193-1201

]

.

The binding of annexin V is not specific for the PS only

[

34,

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM

J Biol Chem. 1990, 265, 4923–8

37

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

Stuart MC, Reutelingsperger CP, Frederik PM.

Cytometry . 1998, 33, 414–9

]

. Studies with different phospholipid types revealed that human annexin V is able to bind to planar bilayers containing 30% of cardiolipin or phosphatidylglycerol (PG) or phosphatidylinositol (PI) with 70% of phosphatidylcholine (PC) in presence of Ca²⁺ and the bound protein concentration is the same as for PS/PC = 30/70

[

34

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM

J Biol Chem. 1990, 265, 4923–8

]

. Rat annexin V binds to SUVs made of PC/ phosphatidic acid (PA) = 50/50

[

41

Annexin V Binding to the Outer Leaflet of Small Unilamellar Vesicles Leads to Altered Inner Leaflet Properties: 31P- and 1H-NMR Studies

M. Swairjo, M. Roberts, M. Campos, J. Dedman, B. Seaton

Biochemistry. 1994, 33, 10944-10950

https://doi.org/10.1006/jmbi.1997.1183

]

. Human annexin V is able to bind to large unilamellar vesicles (LUVs) composed of DOPG/DOPC = 20/80 as was observed using cryo-electron microscopy

[

42

Structural analysis of junctions formed between lipid membranes and several annexins by cryo-electron microscopy 1 1Edited by M. F. Moody

O. Lambert, V. Gerke, M. Bader, F. Porte, A. Brisson

Journal of Molecular Biology. 1997, 272, 42-55

]

and to bilayers containing more than 20% of phosphatidylethanolamine (PE). Addition of PE to vesicles containing PC and PS reduces the minimal PS content needed for the annexin V binding and leads to the earlier reaching of the maximal binding

[

37,

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

Stuart MC, Reutelingsperger CP, Frederik PM.

Cytometry . 1998, 33, 414–9

https://doi.org/10.1002/cyto.a.20345

40

Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death

J. Shi, Y. Shi, L. Waehrens, J. Rasmussen, C. Heegaard, G. Gilbert

Cytometry Part A. 2006, 69A, 1193-1201

]

.

The data on cholesterol effects are controversial. Addition of cholesterol to the PS/PC membranes on the glass beads attenuates the binding

[

37

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

Stuart MC, Reutelingsperger CP, Frederik PM.

Cytometry . 1998, 33, 414–9

https://doi.org/10.1016/j.chemphyslip.2010.01.005

]

. In contrast, for LUVs it was shown that cholesterol promotes binding to PC/PS membranes

[

39,

Effect of cholesterol content on affinity and stability of factor VIII and annexin V binding to a liposomal bilayer membrane

J. Jeon, S. Hwang, S. Cho, J. Choo, E. Lee

Chemistry and Physics of Lipids. 2010, 163, 335-340

https://doi.org/10.1006/bbrc.2001.4748

43

Cholesterol Enhances Phospholipid Binding and Aggregation of Annexins by Their Core Domain

J. Ayala-Sanmartin

Biochemical and Biophysical Research Communications. 2001, 283, 72-79

https://doi.org/10.1016/j.chemphyslip.2010.01.005

]

. The discrepancy might be due to the difference in phospholipid systems or PS contents used.

Cholesterol stabilizes the membrane-bound annexin V and the dissociation rate constant k_d decreases from 1.69×10⁻³ s⁻¹ for PS/PC = 20/80 to 3.24×10⁻⁴ s⁻¹ for PS/PC/cholesterol = 20/70/10

[

39

Effect of cholesterol content on affinity and stability of factor VIII and annexin V binding to a liposomal bilayer membrane

J. Jeon, S. Hwang, S. Cho, J. Choo, E. Lee

Chemistry and Physics of Lipids. 2010, 163, 335-340

https://doi.org/10.1006/bbrc.1997.7177

]

.

Annexin V do not bind to 100% PC membranes

[

44

Presence and Comparison of Ca2+Transport Activity of Annexins I, II, V, and VI in Large Unilamellar Vesicles

R. Matsuda, N. Kaneko, Y. Horikawa

Biochemical and Biophysical Research Communications. 1997, 237, 499-503

https://doi.org/10.1016/j.chemphyslip.2010.01.005

]

or membranes composed of PC and cholesterol

[

39

Effect of cholesterol content on affinity and stability of factor VIII and annexin V binding to a liposomal bilayer membrane

J. Jeon, S. Hwang, S. Cho, J. Choo, E. Lee

Chemistry and Physics of Lipids. 2010, 163, 335-340

]

. However, existing studies with phospholipids of different types are quite limited and they utilize annexin V from different species. Thus additional extensive studies are required to fully elucidate the effect of different phospholipid head groups and fatty acid chains types on annexin V membrane interaction.

The annexin V binding is specific for Ca²⁺ ions. In presence of other divalent cations including Zn²⁺, Mn²⁺ and Mg²⁺ substantially less or almost no protein binds to the membrane

[

32,

Effect of Mg2+ versus Ca2+ on the behavior of Annexin A5 in a membrane-bound state

Z. Fezoua-Boubegtiten, B. Desbat, A. Brisson, C. Gounou, M. Laguerre, S. Lecomte

European Biophysics Journal. 2011, 40, 641-649

34

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM

J Biol Chem. 1990, 265, 4923–8

]

. In a molecular dynamics simulation annexin V structures with 4 Mg²⁺ or 4 Ca²⁺ bound ions were compared and it was shown that Mg²⁺ ions are significantly embedded inside the protein structure and are less accessible to solvent molecules than Ca²⁺ ions are

[

32

Effect of Mg2+ versus Ca2+ on the behavior of Annexin A5 in a membrane-bound state

Z. Fezoua-Boubegtiten, B. Desbat, A. Brisson, C. Gounou, M. Laguerre, S. Lecomte

European Biophysics Journal. 2011, 40, 641-649

]

. From those data it was proposed that Mg²⁺ is less accessible for the phospholipid phosphate group which participates in the Ca²⁺-mediated membrane binding of annexin V and thus the binding is impaired.

Addition of the Zn²⁺ ion in small concentration (50 µM) to solution which contains Ca²⁺ ions improves the annexin V binding to PS-containing membranes and less Ca²⁺ is needed to reach the maximal binding

[

34

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM

J Biol Chem. 1990, 265, 4923–8

]

. Thus Ca²⁺ and Zn²⁺ were shown to have the synergistic effect and the precise reason for it is currently unknown.

The binding of annexin V depends on the membrane curvature: the protein binds better to planar phospholipid bilayers than to small phospholipid vesicles with high curvature and more Ca²⁺ is needed to reach the maximal binding to vesicles than to planar bilayers

[

17

Clustering of lipid-bound annexin V may explain its anticoagulant effect

Andree HA, Stuart MC, Hermens WT, Reutelingsperger CP, Hemker HC, Frederik PM, et al.

J Biol Chem. 1992, 267, 17907–12

]

. This effect might be due to the annexin V ability to form multimeric arrays on the membrane, and formation of such arrays might be counteracted by steric constraints on small vesicles surfaces where neighboring annexin V monomers may make an angle with each other.

Structural considerations concerning the membrane binding

Studies of the annexin V binding to membranes at a submolecular level revealed that all four domains of the protein are involved in interactions with phospholipids

[

45

Characterizing the binding of annexin V to a lipid bilayer using molecular dynamics simulations

Z. Chen, Y. Mao, J. Yang, T. Zhang, L. Zhao, K. Yu, M. Zheng, H. Jiang, H. Yang

Proteins: Structure, Function, and Bioinformatics. 2014, 82, 312-322

]

. There are three types of protein-membrane interactions observed.

The first type is so-called “Ca²⁺-bridges”, when the Ca²⁺ ion interacts simultaneously with the protein binding site and phospholipid charged groups. Both phosphate and PS carboxyl groups might be involved in this type of binding

[

45

Characterizing the binding of annexin V to a lipid bilayer using molecular dynamics simulations

Z. Chen, Y. Mao, J. Yang, T. Zhang, L. Zhao, K. Yu, M. Zheng, H. Jiang, H. Yang

Proteins: Structure, Function, and Bioinformatics. 2014, 82, 312-322

]

. One Ca²⁺ ion might be chelated by more than one lipid molecule

[

46

High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

A. Miyagi, C. Chipot, M. Rangl, S. Scheuring

Nature Nanotechnology. 2016, 11, 783-790

https://doi.org/10.1074/jbc.M210286200

]

. This type of interactions was also proposed for the rat annexin V in the X-ray diffraction study

[

47

Interfacial Basic Cluster in Annexin V Couples Phospholipid Binding and Trimer Formation on Membrane Surfaces

Y. Mo, B. Campos, T. Mealy, L. Commodore, J. Head, J. Dedman, B. Seaton

Journal of Biological Chemistry. 2003, 278, 2437-2443

]

.

The second type of interactions involves the hydrogen bonds formation between carboxyl groups of PS or phosphate oxygen and amino acid residues of the protein, which include Lys76, Lys101, Lys108, Thr74 and Ser305

[

45

Characterizing the binding of annexin V to a lipid bilayer using molecular dynamics simulations

Z. Chen, Y. Mao, J. Yang, T. Zhang, L. Zhao, K. Yu, M. Zheng, H. Jiang, H. Yang

Proteins: Structure, Function, and Bioinformatics. 2014, 82, 312-322

]

.

The third type of contacts is formed because of the hydrophobic effect. In this type Trp187 is involved

[

45

Characterizing the binding of annexin V to a lipid bilayer using molecular dynamics simulations

Z. Chen, Y. Mao, J. Yang, T. Zhang, L. Zhao, K. Yu, M. Zheng, H. Jiang, H. Yang

Proteins: Structure, Function, and Bioinformatics. 2014, 82, 312-322

]

.

Annexin V multimerization

Annexin V is supposed to be monomeric in solution but it is able to form two-dimensional lattices on the membrane surface. Such lattices are formed when monomers of annexin V bind each other and organize into large highly structurized arrays of bound molecules. The main structural subunit of those arrays is trimer of annexin V which was visualized using X-ray crystallography

[

29,

The Crystal Structure of a New High-calcium Form of Annexin V

J. Sopkova, M. Renouard, A. Lewit-Bentley

Journal of Molecular Biology. 1993, 234, 816-825

31

High-resolution structures of annexin A5 in a two-dimensional array

S. Hong, S. Na, O. Kim, S. Jeong, B. Oh, N. Ha

Journal of Structural Biology. 2020, 209, 107401

]

, atomic force microscopy

[

15,

Two-Dimensional Crystallization of Annexin A5 on Phospholipid Bilayers and Monolayers: A Solid−Solid Phase Transition between Crystal Forms

I. Reviakine, W. Bergsma-Schutter, A. Morozov, A. Brisson

Langmuir. 2001, 17, 1680-1686

46

High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

A. Miyagi, C. Chipot, M. Rangl, S. Scheuring

Nature Nanotechnology. 2016, 11, 783-790

]

and electron microscopy

[

15,

Two-Dimensional Crystallization of Annexin A5 on Phospholipid Bilayers and Monolayers: A Solid−Solid Phase Transition between Crystal Forms

I. Reviakine, W. Bergsma-Schutter, A. Morozov, A. Brisson

Langmuir. 2001, 17, 1680-1686

33,

Structural and Electrophysiological Analysis of Annexin V Mutants

A. Burger, D. Voges, P. Demange, C. Perez, R. Huber, R. Berendes

Journal of Molecular Biology. 1994, 237, 479-499

https://doi.org/10.1126/science.7692599

48,

Structure-Function Analysis of the Ion Channel Selectivity Filter in Human Annexin V

R. Berendes, D. Voges, P. Demange, R. Huber, A. Burger

Science. 1993, 262, 427-430

https://doi.org/10.1006/jsbi.2000.4337

49

Trimers, Dimers of Trimers, and Trimers of Trimers Are Common Building Blocks of Annexin A5 Two-Dimensional Crystals

F. Oling, W. Bergsma-Schutter, A. Brisson

Journal of Structural Biology. 2001, 133, 55-63

]

(for schematic representation see Figure 2A).

Two different types of annexin V 2D arrays were observed on the membranes of different composition: so-called p6 and p3 lattices (Figure 2B, C). Lattice p3 is approximately 1.2 times denser than p6. The p6 lattice was the only one presented on the membranes containing less than 40% of PS, and p3 was observed on membranes containing 40% of PS or more. P3 lattice might transform into p6 when Ca²⁺ in the system is depleted or when the lipid surface available for the array formation increases via addition of phospholipids to the membrane covered by annexin V. P6 lattice may transform into p3 when an excess of annexin V is added

[

15

Two-Dimensional Crystallization of Annexin A5 on Phospholipid Bilayers and Monolayers: A Solid−Solid Phase Transition between Crystal Forms

I. Reviakine, W. Bergsma-Schutter, A. Morozov, A. Brisson

Langmuir. 2001, 17, 1680-1686

]

.

The structure of human annexin V lattices and contacts between individual annexin V trimers were extensively studied using X-ray crystallography and combination of atomic force microscopy and structure modeling

[

31,

High-resolution structures of annexin A5 in a two-dimensional array

S. Hong, S. Na, O. Kim, S. Jeong, B. Oh, N. Ha

Journal of Structural Biology. 2020, 209, 107401

46

High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

A. Miyagi, C. Chipot, M. Rangl, S. Scheuring

Nature Nanotechnology. 2016, 11, 783-790

https://doi.org/10.1074/jbc.M210286200

]

.

In p6 lattice two types of annexin V trimers were distinguished: “p6 trimers” (green on Figure 2B) and central “non-p6 trimers” (orange on Figure 2B), which are structurally similar. Both types are supposed to have annexin V domain II in their center (Figure 2A) and this kind of trimer was previously visualized using X-ray crystallography for rat annexin V

[

47

Interfacial Basic Cluster in Annexin V Couples Phospholipid Binding and Trimer Formation on Membrane Surfaces

Y. Mo, B. Campos, T. Mealy, L. Commodore, J. Head, J. Dedman, B. Seaton

Journal of Biological Chemistry. 2003, 278, 2437-2443

]

.

The contacts between two p6 trimers (marked by yellow triangle on Figure 2B) involve domains III of two neighboring monomers. Amino acid residues which might participate in binding are Gln174, Gln177, Phe180, Thr215, Ile216, Ser217 and the side chains of Phe180 are supposed to form π stacking. The distance between backbones of proteins is approximately 3 A

[

46

High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

A. Miyagi, C. Chipot, M. Rangl, S. Scheuring

Nature Nanotechnology. 2016, 11, 783-790

https://doi.org/10.1006/jsbi.2000.4337

]

. The same contact was previously observed between trimers of rat annexin V in p6 lattice

[

49

Trimers, Dimers of Trimers, and Trimers of Trimers Are Common Building Blocks of Annexin A5 Two-Dimensional Crystals

F. Oling, W. Bergsma-Schutter, A. Brisson

Journal of Structural Biology. 2001, 133, 55-63

]

.

Contacts between p6 and non-p6 trimers involve domain IV (amino acid residues Ser295, Tyr297, Ser298) from p6 and domain III (amino acid residues Thr215, Ile216, Ser217, Gly218) from non-p6 monomers (marked by magenta triangle on Figure 2B). The distance between backbones of proteins is much higher than for two p6 trimers and is approximately 6 – 8 A. The non-p6 trimers possess the high degree of rotational freedom and exhibit rotational diffusion

[

46

High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

A. Miyagi, C. Chipot, M. Rangl, S. Scheuring

Nature Nanotechnology. 2016, 11, 783-790

]

. It is supposed that non-p6 trimers possess lower affinity for the Ca²⁺ ions as those trimers dissociate from the membrane earlier than p6 trimers in experiments with Ca²⁺ depletion

[

46

High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

A. Miyagi, C. Chipot, M. Rangl, S. Scheuring

Nature Nanotechnology. 2016, 11, 783-790

]

.

For p3 lattice three types of contacts between human annexin V monomers were observed

[

31

High-resolution structures of annexin A5 in a two-dimensional array

S. Hong, S. Na, O. Kim, S. Jeong, B. Oh, N. Ha

Journal of Structural Biology. 2020, 209, 107401

]

. The first type (marked by black triangle on Figure 2C) forms the annexin V trimer with domain II in the center, as was for p6 lattice. Salt bridges are formed between acidic amino acid residues from domains II and III (Asp162, Glu192, Glu138) of one monomer and basic amino acid residues from domain I (Arg18, Lys29, Arg25, Lys58) of another

[

31

High-resolution structures of annexin A5 in a two-dimensional array

S. Hong, S. Na, O. Kim, S. Jeong, B. Oh, N. Ha

Journal of Structural Biology. 2020, 209, 107401

https://doi.org/10.1074/jbc.M210286200

]

. For the same type of trimer of rat annexin V residues Arg23, Lys27 and Glu190 were found to form contacts between monomers and those residues correspond to Arg25, Lys29 and Glu192 of human annexin V

[

47

Interfacial Basic Cluster in Annexin V Couples Phospholipid Binding and Trimer Formation on Membrane Surfaces

Y. Mo, B. Campos, T. Mealy, L. Commodore, J. Head, J. Dedman, B. Seaton

Journal of Biological Chemistry. 2003, 278, 2437-2443

]

.

The second type of contacts (marked by red triangle on Figure 2C) forms annexin V trimers with domain III in their center. Amino acid residues Gln177 and Gln181 form a circular network of hydrogen bonds with water molecules in the center of trimer. The residues Trp187 from three monomers in their outward-facing conformation are grouped together near the center of trimer

[

31

High-resolution structures of annexin A5 in a two-dimensional array

S. Hong, S. Na, O. Kim, S. Jeong, B. Oh, N. Ha

Journal of Structural Biology. 2020, 209, 107401

]

.

The third type of contacts (marked by blue triangle on Figure 2C) forms annexin V trimers with domain IV in their center. The same type of trimer was observed earlier using X-ray crystallography

[

29

The Crystal Structure of a New High-calcium Form of Annexin V

J. Sopkova, M. Renouard, A. Lewit-Bentley

Journal of Molecular Biology. 1993, 234, 816-825

]

(Figure 2D). Amino acid residues Lys26 and Tyr297 from one monomer form bonds with corresponding residues Glu223 and Ala293 from another

[

31

High-resolution structures of annexin A5 in a two-dimensional array

S. Hong, S. Na, O. Kim, S. Jeong, B. Oh, N. Ha

Journal of Structural Biology. 2020, 209, 107401

https://doi.org/10.1038/ncomms1270

]

.

From those data it might be concluded that different domains of one annexin V monomer might form bonds with varied domains of another monomer (domain III of one monomer with domains III or IV or I of another; domain I of one monomer with domains II or III of another and so on) and protein multimerization might involve formation of multiple contacts between different amino acids. In agreement with this conclusion it was found out that 4 or 5 different mutations are needed to construct the annexin V mutant that lack the ability of trimer formation

[

16

Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair

A. Bouter, C. Gounou, R. Bérat, S. Tan, B. Gallois, T. Granier, B. d'Estaintot, E. Pöschl, B. Brachvogel, A. Brisson

Nature Communications. 2011, 2, None

]

. Rat annexin V with mutations Arg16Glu, Arg23Glu, Lys27Glu, Lys56Glu, Lys191Gly (those residues correspond to Arg18, Arg25, Lys29, Lys58 and Lys193 of human annexin V, the majority of which were shown to participate in trimerization

[

31

High-resolution structures of annexin A5 in a two-dimensional array

S. Hong, S. Na, O. Kim, S. Jeong, B. Oh, N. Ha

Journal of Structural Biology. 2020, 209, 107401

https://doi.org/10.1006/jsbi.2000.4337

]

) was not able to form 2D arrays that additionally emphasize the role of domains I and III in multimerization process.

In addition to the p6 and p3 lattices some other crystal forms of annexin V were observed on PS-containing membranes including the dimers of annexin V trimers or trimers of trimers. Those forms coexists with p6 lattice

[

49

Trimers, Dimers of Trimers, and Trimers of Trimers Are Common Building Blocks of Annexin A5 Two-Dimensional Crystals

F. Oling, W. Bergsma-Schutter, A. Brisson

Journal of Structural Biology. 2001, 133, 55-63

]

.

The annexin V 2D lattice formation is a reversible process as was shown via sequential addition and depletion of Ca²⁺ in solution

[

46

High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

A. Miyagi, C. Chipot, M. Rangl, S. Scheuring

Nature Nanotechnology. 2016, 11, 783-790

]

. Addition of Ca²⁺ leads to the rapid (within 5 s) array formation, but lattice might be completely dissolved when Ca²⁺ is depleted using EDTA. This process might be repeated several times. The boundaries of the formed lattices are highly dynamic and annexin V molecules constantly associate to and dissociate from the lattice with rate constants k_a = 2.3 s^-1 and k_d = 2.0 s^-1 respectively

[

50

Annexin-V stabilizes membrane defects by inducing lipid phase transition

Y. Lin, C. Chipot, S. Scheuring

Nature Communications. 2020, 11, None

]

. The height of the annexin lattice on the membrane was equal to 2.5 nm above the membrane surface

[

38,

Defining the structural characteristics of annexin V binding to a mimetic apoptotic membrane

J. Lu, A. Le Brun, S. Chow, T. Shiota, B. Wang, T. Lin, G. Liu, H. Shen

European Biophysics Journal. 2015, 44, 697-708

50,

Annexin-V stabilizes membrane defects by inducing lipid phase transition

Y. Lin, C. Chipot, S. Scheuring

Nature Communications. 2020, 11, None

https://doi.org/10.1016/S0002-9440(10)63479-7

51

Human Monoclonal Antiphospholipid Antibodies Disrupt the Annexin A5 Anticoagulant Crystal Shield on Phospholipid Bilayers

J. Rand, X. Wu, A. Quinn, P. Chen, K. McCrae, E. Bovill, D. Taatjes

The American Journal of Pathology. 2003, 163, 1193-1200

]

and was found to decrease to approximately 2.2 nm on the membranes with higher PS content

[

38

Defining the structural characteristics of annexin V binding to a mimetic apoptotic membrane

J. Lu, A. Le Brun, S. Chow, T. Shiota, B. Wang, T. Lin, G. Liu, H. Shen

European Biophysics Journal. 2015, 44, 697-708

]

.

The annexin V lattice formation on the phospholipid membrane of vesicles leads to the decrease of the membrane curvature and to formation of planar phospholipid surfaces

[

17,

Clustering of lipid-bound annexin V may explain its anticoagulant effect

Andree HA, Stuart MC, Hermens WT, Reutelingsperger CP, Hemker HC, Frederik PM, et al.

J Biol Chem. 1992, 267, 17907–12

41

Annexin V Binding to the Outer Leaflet of Small Unilamellar Vesicles Leads to Altered Inner Leaflet Properties: 31P- and 1H-NMR Studies

M. Swairjo, M. Roberts, M. Campos, J. Dedman, B. Seaton

Biochemistry. 1994, 33, 10944-10950

]

. The vesicle changes its shape from round to polyhedral that was observed using cryoelectron microscopy

[

17

Clustering of lipid-bound annexin V may explain its anticoagulant effect

Andree HA, Stuart MC, Hermens WT, Reutelingsperger CP, Hemker HC, Frederik PM, et al.

J Biol Chem. 1992, 267, 17907–12

https://doi.org/10.1016/j.jcis.2021.05.067

]

.

The annexin V lattice formation might be interrupted by presence of different membrane-binding proteins. Simultaneous addition of annexin V and another member of the annexin family, annexin A4, in a 1:1 mixture to the PS-containing membrane results in the irregular packing of individual annexin V and A4 trimers without the lattice formation. Interestingly, the addition of annexin A4 even in small concentrations to preformed annexin V lattice disrupt its regular structure, but the alternative situation when annexin V is added to preformed A4 lattice does not alter the A4 lattice structure significantly

[

52

Simultaneous membrane binding of Annexin A4 and A5 suppresses 2D lattice formation while maintaining curvature induction

A. Mularski, S. Sønder, A. Heitmann, J. Nylandsted, A. Simonsen

Journal of Colloid and Interface Science. 2021, 600, 854-864

https://doi.org/10.1016/S0002-9440(10)63479-7

]

.

The 2D annexin V lattice might be disrupted by antiphospholipid antibodies

[

51

Human Monoclonal Antiphospholipid Antibodies Disrupt the Annexin A5 Anticoagulant Crystal Shield on Phospholipid Bilayers

J. Rand, X. Wu, A. Quinn, P. Chen, K. McCrae, E. Bovill, D. Taatjes

The American Journal of Pathology. 2003, 163, 1193-1200

https://doi.org/10.1006/jmbi.2000.4183

]

.

The annexin V 2D lattice formation. A. Schematic representation of the annexin V trimer. Domains are marked with roman numbers. B. Scheme of the p6 annexin V lattice. Green, p6 trimer; orange, non-p6 trimer. Contacts between two p6 or p6 and non-p6 trimers are marked by yellow and magenta triangles respectively. C. Scheme of the p3 annexin V lattice. Centers of different types of trimers are marked by red, blue and black triangles. D. The structure of the annexin V trimer. Magenta, N-terminal tail; blue, domain I; yellow, domain II; green, domain III; red, domain IV; orange, Ca2+ ions. The figure was created in VMD using the structure 1ANX [29] from PDB Data Bank.

Figure 2. The annexin V 2D lattice formation. A. Schematic representation of the annexin V trimer. Domains are marked with roman numbers. B. Scheme of the p6 annexin V lattice. Green, p6 trimer; orange, non-p6 trimer. Contacts between two p6 or p6 and non-p6 trimers are marked by yellow and magenta triangles respectively. C. Scheme of the p3 annexin V lattice. Centers of different types of trimers are marked by red, blue and black triangles. D. The structure of the annexin V trimer. Magenta, N-terminal tail; blue, domain I; yellow, domain II; green, domain III; red, domain IV; orange, Ca2+ ions. The figure was created in VMD using the structure 1ANX [29] from PDB Data Bank.

Structural alterations of annexin V and membrane during binding

Both experimental

[

53

Structure of Membrane-bound Annexin A5 Trimers: A Hybrid Cryo-EM - X-ray Crystallography Study

F. Oling, J. Santos, N. Govorukhina, C. Mazères-Dubut, W. Bergsma-Schutter, G. Oostergetel, W. Keegstra, O. Lambert, A. Lewit-Bentley, A. Brisson

Journal of Molecular Biology. 2000, 304, 561-573

]

and theoretical

[

45

Characterizing the binding of annexin V to a lipid bilayer using molecular dynamics simulations

Z. Chen, Y. Mao, J. Yang, T. Zhang, L. Zhao, K. Yu, M. Zheng, H. Jiang, H. Yang

Proteins: Structure, Function, and Bioinformatics. 2014, 82, 312-322

https://doi.org/10.1006/jmbi.2000.4183

]

evidences exist that annexin V does not change its structure significantly in the process of the membrane binding. The molecule maintains its bent shape with a convex and a concave sides. Only small local differences between membrane-bound and unbound structures were observed

[

53

Structure of Membrane-bound Annexin A5 Trimers: A Hybrid Cryo-EM - X-ray Crystallography Study

F. Oling, J. Santos, N. Govorukhina, C. Mazères-Dubut, W. Bergsma-Schutter, G. Oostergetel, W. Keegstra, O. Lambert, A. Lewit-Bentley, A. Brisson

Journal of Molecular Biology. 2000, 304, 561-573

]

. Differences in the secondary structure of two forms are negligible too

[

41

Annexin V Binding to the Outer Leaflet of Small Unilamellar Vesicles Leads to Altered Inner Leaflet Properties: 31P- and 1H-NMR Studies

M. Swairjo, M. Roberts, M. Campos, J. Dedman, B. Seaton

Biochemistry. 1994, 33, 10944-10950

]

. The membrane-bound annexin V demonstrates smaller fluctuations in a molecular dynamics simulation

[

45

Characterizing the binding of annexin V to a lipid bilayer using molecular dynamics simulations

Z. Chen, Y. Mao, J. Yang, T. Zhang, L. Zhao, K. Yu, M. Zheng, H. Jiang, H. Yang

Proteins: Structure, Function, and Bioinformatics. 2014, 82, 312-322

https://doi.org/10.1021/bi9819677

]

that is consistent with stabilization of its structure due to the membrane binding

[

54

Stability of Annexin V in Ternary Complexes with Ca²⁺ and Anionic Phospholipids: IR Studies of Monolayer and Bulk Phases

F. Wu, C. Flach, B. Seaton, T. Mealy, R. Mendelsohn

Biochemistry. 1999, 38, 792-799

]

.

However, in the process of annexin V adsorption major changes of the phospholipid membrane were observed.

In early studies it was found out that the annexin V binding leads to the increase of the phospholipid monolayer surface pressure, to its rigidification

[

55,

Annexin V membrane interaction: an electrostatic potential study

A. Karshikov, R. Berendes, A. Burger, A. Cavali�, H. Lux, R. Huber

European Biophysics Journal. 1992, 20, None

https://doi.org/10.1016/S0006-3495(98)78034-8

56

Domain Structure and Molecular Conformation in Annexin V/1,2-Dimyristoyl-sn-Glycero-3-Phosphate/Ca2+ Aqueous Monolayers: A Brewster Angle Microscopy/Infrared Reflection-Absorption Spectroscopy Study

F. Wu, A. Gericke, C. Flach, T. Mealy, B. Seaton, R. Mendelsohn

Biophysical Journal. 1998, 74, 3273-3281

https://doi.org/10.1016/S0006-3495(98)78034-8

]

and to significant ordering of the phospholipid acyl chains

[

56

Domain Structure and Molecular Conformation in Annexin V/1,2-Dimyristoyl-sn-Glycero-3-Phosphate/Ca2+ Aqueous Monolayers: A Brewster Angle Microscopy/Infrared Reflection-Absorption Spectroscopy Study

F. Wu, A. Gericke, C. Flach, T. Mealy, B. Seaton, R. Mendelsohn

Biophysical Journal. 1998, 74, 3273-3281

https://doi.org/10.1021/bi971484n

]

. The PS and PC diffusivity is significantly reduced too

[

57

Influence of Annexin V on the Structure and Dynamics of Phosphatidylcholine/Phosphatidylserine Bilayers: A Fluorescence and NMR Study

O. Saurel, L. Cézanne, A. Milon, J. Tocanne, P. Demange

Biochemistry. 1998, 37, 1403-1410

]

.

Later the detailed studies using atomic force microscopy and fluorescence techniques were conducted. It was found out that the bilayer composed of DOPC/DOPS = 50/50 undergoes shrinkage due to the annexin V adsorption

[

50

Annexin-V stabilizes membrane defects by inducing lipid phase transition

Y. Lin, C. Chipot, S. Scheuring

Nature Communications. 2020, 11, None

]

. Additionally, the decrease of the diameter was observed for unilamellar vesicles. The membrane thickness increases after the annexin V binding from 3.15 to 3.45 nm and it corresponds to the more ordered gel rather than the liquid crystal state. The phospholipid diffusivity decreases to only 15% of the original rate

[

50

Annexin-V stabilizes membrane defects by inducing lipid phase transition

Y. Lin, C. Chipot, S. Scheuring

Nature Communications. 2020, 11, None

]

. Comparable results (shrinkage of the membrane and reduction of diffusivity) were obtained in a molecular dynamics simulation of the membrane with bound and fixed Ca²⁺ ions in positions corresponding to positions in a membrane-bound annexin V trimer

[

50

Annexin-V stabilizes membrane defects by inducing lipid phase transition

Y. Lin, C. Chipot, S. Scheuring

Nature Communications. 2020, 11, None

]

. It was proposed that the binding of annexin V leads to the increase of the local Ca²⁺ concentration on the membrane surface up to 606 mM and such high ion concentration might alter the bilayer properties

[

50

Annexin-V stabilizes membrane defects by inducing lipid phase transition

Y. Lin, C. Chipot, S. Scheuring

Nature Communications. 2020, 11, None

]

.

The binding of annexin V induces the local membrane curvature due to the bent shape of the molecule

[

45

Characterizing the binding of annexin V to a lipid bilayer using molecular dynamics simulations

Z. Chen, Y. Mao, J. Yang, T. Zhang, L. Zhao, K. Yu, M. Zheng, H. Jiang, H. Yang

Proteins: Structure, Function, and Bioinformatics. 2014, 82, 312-322

https://doi.org/10.1038/s41598-018-28481-z

]

. In a large-scale process on the membrane with free edges this curvature induction leads to the membrane rolling

[

58

Annexins induce curvature on free-edge membranes displaying distinct morphologies

T. Boye, J. Jeppesen, K. Maeda, W. Pezeshkian, V. Solovyeva, J. Nylandsted, A. Simonsen

Scientific Reports. 2018, 8, None

https://doi.org/10.1038/s41598-018-28481-z

]

. The rolling of the membrane starts from multiple points on the free edge and results in formation of one major roll parallel to the membrane edge thus the process was suggested to be cooperative

[

58

Annexins induce curvature on free-edge membranes displaying distinct morphologies

T. Boye, J. Jeppesen, K. Maeda, W. Pezeshkian, V. Solovyeva, J. Nylandsted, A. Simonsen

Scientific Reports. 2018, 8, None

https://doi.org/10.1016/j.jcis.2021.05.067

]

. In contrast to the 2D lattice formation, the annexin V induced membrane rolling is not disturbed by the presence of annexin A4

[

52

Simultaneous membrane binding of Annexin A4 and A5 suppresses 2D lattice formation while maintaining curvature induction

A. Mularski, S. Sønder, A. Heitmann, J. Nylandsted, A. Simonsen

Journal of Colloid and Interface Science. 2021, 600, 854-864

https://doi.org/10.1016/j.jcis.2021.05.067

]

. However, it should be noted that both annexin V and annexin A4 are able to induce membrane rolling.

Annexin V properties of the membrane stabilization and rolling were suggested to be important for the membrane repair process

[

52

Simultaneous membrane binding of Annexin A4 and A5 suppresses 2D lattice formation while maintaining curvature induction

A. Mularski, S. Sønder, A. Heitmann, J. Nylandsted, A. Simonsen

Journal of Colloid and Interface Science. 2021, 600, 854-864

https://doi.org/10.1042/bj3080591

]

.

The binding of annexin V to various proteins

In early studies it was discovered that annexin V is able to bind to the membrane of thrombin or А23187-stimulated platelets in two distinct ways

[

59

Ca2+ concentration during binding determines the manner in which annexin V binds to membranes

P. Trotter, M. Orchard, J. Walker

Biochemical Journal. 1995, 308, 591-598

https://doi.org/10.1042/bj3080591

]

. The first way occurs in presence of high Ca²⁺ concentrations and correspond to the membrane binding discussed earlier. The second way occurs in presence of low Ca²⁺ concentrations and the bound protein is resistant to EGTA treatment. From those data it was suggested that annexin V is able to form complex with a membrane protein

[

59

Ca2+ concentration during binding determines the manner in which annexin V binds to membranes

P. Trotter, M. Orchard, J. Walker

Biochemical Journal. 1995, 308, 591-598

https://doi.org/10.1006/excr.1999.4553

]

. Later it was found out that annexin V colocalizes with actin in stimulated platelets and the 85 kDa complex of actin and annexin V was identified

[

60

Annexin V Binds to the Actin-Based Cytoskeleton at the Plasma Membrane of Activated Platelets

E. Tzima, P. Trotter, M. Orchard, J. Walker

Experimental Cell Research. 1999, 251, 185-193

https://doi.org/10.1046/j.1432-1327.2000.01525.x

]

. The association is Ca²⁺-dependent and it was shown that annexin V binds to the γ-actin isoform

[

61

Annexin V relocates to the platelet cytoskeleton upon activation and binds to a specific isoform of actin

E. Tzima, P. Trotter, M. Orchard, J. Walker

European Journal of Biochemistry. 2000, 267, 4720-4730

https://doi.org/10.1042/BJ20020997

]

.

Annexin V is able to bind annexin A2. The binding is independent of phospholipid and occurs in solution but requires presence of the millimolar Ca²⁺ concentration. Both monomeric and trimeric forms of annexin A2 bind annexin V and this interaction might be inhibited by addition of the low-molecular-mass heparin

[

62

Ca2+-dependent and phospholipid-independent binding of annexin 2 and annexin 5

N. BROOKS, J. GRUNDY, N. LAVIGNE, M. DERRY, C. RESTALL, R. MacKENZIE, D. WAISMAN, E. PRYZDIAL

Biochemical Journal. 2002, 367, 895-900

https://doi.org/10.1111/j.1432-1033.1985.tb09150.x

]

.

3. Annexin V functions

Inhibition of the blood coagulation

In 1985 it was revealed that annexin V is able to inhibit the tissue factor (TF) or factor Xa initiated blood plasma clotting but does not affect the fibrinogen cleavage by thrombin

[

10

Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord

C. REUTELINGSPERGER, G. HORNSTRA, H. HEMKER

European Journal of Biochemistry. 1985, 151, 625-629

]

. Its proposed inhibitory action is based on the ability of high affinity binding to phospholipid membranes. Reactions of the blood clotting require the presence of negatively-charged phospholipid membranes for the maximal reaction efficiency

[

63,

Lipid-protein interactions in blood coagulation

Zwaal RF, Comfurius P, Bevers EM

Biochim Biophys Acta. 1998, 1376, 433–53

https://doi.org/10.1055/s-0035-1564044

64

Structure–Function Relationship of the Interaction between Tissue Factor and Factor VIIa

J. Gajsiewicz, J. Morrissey

Seminars in Thrombosis and Hemostasis. 2015, 41, 682-690

https://doi.org/10.1023/A:1013985911759

]

and the mechanism of those membrane-dependent reactions includes the stage of proteins association with the membrane, two-dimensional diffusion on the membrane surface and the membrane-associated active complexes formation

[

65,

Blood coagulation

Butenas S, Mann KG, Butenas

Biochemistry. 2002, 67, 3-12

https://doi.org/10.1016/j.jtbi.2017.09.015

66,

Substrate delivery mechanism and the role of membrane curvature in factor X activation by extrinsic tenase

T. Kovalenko, M. Panteleev, A. Sveshnikova

Journal of Theoretical Biology. 2017, 435, 125-133

https://doi.org/10.1182/blood-2008-01-077909

67

Advances in understanding pathogenic mechanisms of thrombophilic disorders

B. Dahlbäck

Blood. 2008, 112, 19-27

https://doi.org/10.1111/j.1432-1033.1985.tb09150.x

]

. The annexin V membrane binding might interrupt those processes.

The anticoagulant effect of annexin V was extensively studied.

Annexin V inhibits the prothrombin activation by the prothrombinase complex (formed by FVa and FXa) in a dose-dependent manner. Higher annexin V concentrations lead to the higher degree of inhibition

[

10,

Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord

C. REUTELINGSPERGER, G. HORNSTRA, H. HEMKER

European Journal of Biochemistry. 1985, 151, 625-629

https://doi.org/10.1111/j.1538-7836.2006.01985.x

68,

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells

R. FERNANDES, C. KIRSZBERG, V. RUMJANEK, R. MONTEIRO

Journal of Thrombosis and Haemostasis. 2006, 4, 1546-1552

https://doi.org/10.1097/CMR.0b013e32832e40fe

69

Simultaneous tissue factor expression and phosphatidylserine exposure account for the highly procoagulant pattern of melanoma cell lines

C. Kirszberg, L. Lima, A. Da Silva de Oliveira, W. Pickering, E. Gray, T. Barrowcliffe, V. Rumjanek, R. Monteiro

Melanoma Research. 2009, 19, 301-308

]

. It was shown that the binding of annexin V cannot fully displace coagulation factors (factor Xa, Va or thrombin) from the vesicle surface and enough factors are kept on the membrane for the prothrombinase functioning, so it was concluded that inhibition of the reaction is associated with the annexin V 2D lattice formation

[

17

Clustering of lipid-bound annexin V may explain its anticoagulant effect

Andree HA, Stuart MC, Hermens WT, Reutelingsperger CP, Hemker HC, Frederik PM, et al.

J Biol Chem. 1992, 267, 17907–12

]

. Such a lattice might slow down the factors diffusion on the membrane surface and thus their collisions and binding. The extent of inhibition depends on the membrane curvature with more prominent inhibition on the surface of planar bilayers than on SUVs

[

17

Clustering of lipid-bound annexin V may explain its anticoagulant effect

Andree HA, Stuart MC, Hermens WT, Reutelingsperger CP, Hemker HC, Frederik PM, et al.

J Biol Chem. 1992, 267, 17907–12

https://doi.org/10.1160/TH02-12-0291

]

. Annexin V inhibits prothrombinase activity on the surface of apoptotic smooth muscle cells

[

70

Shedding of active tissue factor by aortic smooth muscle cells (SMCs) undergoing apoptosis

A. Brisset, A. Terrisse, D. Dupouy, L. Tellier, S. Pech, C. Navarro, P. Sié

Thrombosis and Haemostasis. 2003, 90, 511-518

https://doi.org/10.1111/j.1538-7836.2006.01985.x

]

and on the malignant cell lines

[

68,

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells

R. FERNANDES, C. KIRSZBERG, V. RUMJANEK, R. MONTEIRO

Journal of Thrombosis and Haemostasis. 2006, 4, 1546-1552

https://doi.org/10.1097/CMR.0b013e32832e40fe

69

Simultaneous tissue factor expression and phosphatidylserine exposure account for the highly procoagulant pattern of melanoma cell lines

C. Kirszberg, L. Lima, A. Da Silva de Oliveira, W. Pickering, E. Gray, T. Barrowcliffe, V. Rumjanek, R. Monteiro

Melanoma Research. 2009, 19, 301-308

https://doi.org/10.1111/j.1538-7836.2006.01985.x

]

.

The FX activation by the intrinsic tenase complex (formed by FVIIIa and FIXa) is inhibited by annexin V on the surface of different cell lines

[

68,

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells

R. FERNANDES, C. KIRSZBERG, V. RUMJANEK, R. MONTEIRO

Journal of Thrombosis and Haemostasis. 2006, 4, 1546-1552

https://doi.org/10.1097/CMR.0b013e32832e40fe

69,

Simultaneous tissue factor expression and phosphatidylserine exposure account for the highly procoagulant pattern of melanoma cell lines

C. Kirszberg, L. Lima, A. Da Silva de Oliveira, W. Pickering, E. Gray, T. Barrowcliffe, V. Rumjanek, R. Monteiro

Melanoma Research. 2009, 19, 301-308

https://doi.org/10.1111/j.1538-7836.2004.00607.x

71

Characterization of the cell-surface procoagulant activity of T-lymphoblastoid cell lines

W. Pickering, E. Gray, A. Goodall, S. Ran, P. Thorpe, T. Barrowcliffe

Journal of Thrombosis and Haemostasis. 2004, 2, 459-467

https://doi.org/10.1021/jp2048579

]

. It was demonstrated that the addition of annexin V reduces the factor VIIIa binding to LUVs containing 10% of POPS with the rest of POPC. The effect depends on the annexin V concentration and is more prominent for higher concentrations

[

72

FVIII Binding to PS Membranes Differs in the Activated and Non-Activated Form and Can Be Shielded by Annexin A5

H. Engelke, S. Lippok, I. Dorn, R. Netz, J. Rädler

The Journal of Physical Chemistry B. 2011, 115, 12963-12970

https://doi.org/10.1021/jp2048579

]

. For vesicles with 15% of PS the dependence of FVIIIa binding on annexin V concentration is more complex. The presence of low annexin concentrations leads to the moderate increase of FVIIIa binding, but higher concentrations inhibit binding. The dependences of the inhibitory effect on annexin V concentration cannot be explained by a simple model of competition for the binding sites and require a more complex model of “phospholipid shielding” by annexin V

[

72

FVIII Binding to PS Membranes Differs in the Activated and Non-Activated Form and Can Be Shielded by Annexin A5

H. Engelke, S. Lippok, I. Dorn, R. Netz, J. Rädler

The Journal of Physical Chemistry B. 2011, 115, 12963-12970

https://doi.org/10.1160/TH02-12-0291

]

.

The extrinsic tenase (formed by FVIIa and tissue factor TF) activity on the surface of apoptotic smooth muscle cells is inhibited by annexin V

[

70

Shedding of active tissue factor by aortic smooth muscle cells (SMCs) undergoing apoptosis

A. Brisset, A. Terrisse, D. Dupouy, L. Tellier, S. Pech, C. Navarro, P. Sié

Thrombosis and Haemostasis. 2003, 90, 511-518

https://doi.org/10.1074/jbc.M411710200

]

.

For the extrinsic tenase one more, alternative mechanism of inhibition was proposed. In etoposide stimulated macrophages (THP-1 cell line) it was observed that annexin V might be internalized and it induces the TF internalization

[

73

Annexin A5 Down-regulates Surface Expression of Tissue Factor

S. Ravassa, A. Bennaghmouch, H. Kenis, T. Lindhout, T. Hackeng, J. Narula, L. Hofstra, C. Reutelingsperger

Journal of Biological Chemistry. 2005, 280, 6028-6035

https://doi.org/10.1074/jbc.M411710200

]

. Internalized TF and annexin V co-localize in endocytic vesicles. The TF internalization leads to the decrease of the cell-surface TF procoagulant activity. In vivo in mouse model it was shown that exogenously added annexin V significantly reduces the TF expression on the surface of smooth muscle cells after mechanical injury when compared to situation without added annexin V

[

73

Annexin A5 Down-regulates Surface Expression of Tissue Factor

S. Ravassa, A. Bennaghmouch, H. Kenis, T. Lindhout, T. Hackeng, J. Narula, L. Hofstra, C. Reutelingsperger

Journal of Biological Chemistry. 2005, 280, 6028-6035

https://doi.org/10.1056/NEJM199707173370303

]

.

It was suggested that annexin V anticoagulant activity plays role in preventing thrombotic processes in placenta

[

74

Pregnancy Loss in the Antiphospholipid-Antibody Syndrome — A Possible Thrombogenic Mechanism

J. Rand, X. Wu, H. Andree, C. Lockwood, S. Guller, J. Scher, P. Harpel

New England Journal of Medicine. 1997, 337, 154-160

https://doi.org/10.1016/S0002-9440(10)63479-7

]

. Antiphospholipid antibodies inhibit the anticoagulant function of annexin V because of the two-dimensional lattice disruption

[

51

Human Monoclonal Antiphospholipid Antibodies Disrupt the Annexin A5 Anticoagulant Crystal Shield on Phospholipid Bilayers

J. Rand, X. Wu, A. Quinn, P. Chen, K. McCrae, E. Bovill, D. Taatjes

The American Journal of Pathology. 2003, 163, 1193-1200

]

.

Annexin V as a Ca²⁺ channel

In early studies it was found out that annexin V is able to transport Ca²⁺ ions across the membrane of phospholipid vesicles and it was suggested that annexin V forms Ca²⁺ channels

[

33,

Structural and Electrophysiological Analysis of Annexin V Mutants

A. Burger, D. Voges, P. Demange, C. Perez, R. Huber, R. Berendes

Journal of Molecular Biology. 1994, 237, 479-499

https://doi.org/10.1006/bbrc.1997.7177

44,

Presence and Comparison of Ca2+Transport Activity of Annexins I, II, V, and VI in Large Unilamellar Vesicles

R. Matsuda, N. Kaneko, Y. Horikawa

Biochemical and Biophysical Research Communications. 1997, 237, 499-503

https://doi.org/10.1016/0014-5793(93)81507-v

75

Calcium influx through annexin V ion channels into large unilamellar vesicles measured with fura-2

R. Berendes, A. Burger, D. Voges, P. Demange, R. Huber

FEBS Letters. 1993, 317, 131-134

]

. Those channels are voltage-gated and demonstrate selectivity for Ca²⁺ ions

[

33,

Structural and Electrophysiological Analysis of Annexin V Mutants

A. Burger, D. Voges, P. Demange, C. Perez, R. Huber, R. Berendes

Journal of Molecular Biology. 1994, 237, 479-499

55

Annexin V membrane interaction: an electrostatic potential study

A. Karshikov, R. Berendes, A. Burger, A. Cavali�, H. Lux, R. Huber

European Biophysics Journal. 1992, 20, None

]

.

The role of individual amino acid residues in the process of ion transport was investigated using point mutations of the protein and structural studies. In the X-ray diffraction study residues T33, E35 and K70, L73, E78 (Table 1) were suggested to form intermediate binding sites for Ca²⁺ on the protein surface and thus play role in the ion transport

[

28

Crystal and molecular structure of human annexin V after refinement

R. Huber, R. Berendes, A. Burger, M. Schneider, A. Karshikov, H. Luecke, J. Römisch, E. Paques

Journal of Molecular Biology. 1992, 223, 683-704

]

. The residue Glu95 is responsible for the ion selectivity, as the mutant Glu95Ser demonstrates the higher permeability for Na⁺ and K⁺ ions. Furthermore, it demonstrates the slightly reduced Ca²⁺ flux through the channel and different current-voltage dependence when compared to wild-type annexin V

[

33,

Structural and Electrophysiological Analysis of Annexin V Mutants

A. Burger, D. Voges, P. Demange, C. Perez, R. Huber, R. Berendes

Journal of Molecular Biology. 1994, 237, 479-499

https://doi.org/10.1126/science.7692599

48

Structure-Function Analysis of the Ion Channel Selectivity Filter in Human Annexin V

R. Berendes, D. Voges, P. Demange, R. Huber, A. Burger

Science. 1993, 262, 427-430

https://doi.org/10.1006/jmbi.1996.0268

]

. The mutation Glu112Gly leads to similar changes in the channel activity as were for Glu95Ser (the loss of selectivity for Ca²⁺ and different current-voltage dependences). In addition, Glu112Gly mutant demonstrates two levels of the ions transport: high, which corresponds to the level of wild-type annexin V, and low, and this property was not demonstrated by Glu95Ser or wild type annexin V, which have only one level of the transport

[

76

Structural and Functional Characterisation of the Voltage Sensor in the Ion Channel Human Annexin V

S. Liemann, J. Benz, A. Burger, D. Voges, A. Hofmann, R. Huber, P. Göttig

Journal of Molecular Biology. 1996, 258, 555-561

]

. Both residues Glu95 and Glu112 locate inside the hydrophilic pore in the annexin V center.

Glu78 and Glu17 play role in the ion transport as their mutations into Gln and Gly alter the transport kinetics significantly

[

33

Structural and Electrophysiological Analysis of Annexin V Mutants

A. Burger, D. Voges, P. Demange, C. Perez, R. Huber, R. Berendes

Journal of Molecular Biology. 1994, 237, 479-499

https://doi.org/10.1016/0014-5793(93)81507-v

]

. Both mutants have two levels of the ion transport (high and low) and the double mutant Glu78Gln/Glu17Gly exhibits only the smaller level. The Glu17Gly mutant provides the significantly slower Ca²⁺ influx into the phospholipid vesicle

[

75

Calcium influx through annexin V ion channels into large unilamellar vesicles measured with fura-2

R. Berendes, A. Burger, D. Voges, P. Demange, R. Huber

FEBS Letters. 1993, 317, 131-134

]

.

Interestingly, obtained structures of p6 lattices made of wild-type or mutant (Glu95Ser, Glu112Gly, Glu17Gly and Glu78Gln) annexin V are the same and the main difference between them is located around the mutated residue

[

33,

Structural and Electrophysiological Analysis of Annexin V Mutants

A. Burger, D. Voges, P. Demange, C. Perez, R. Huber, R. Berendes

Journal of Molecular Biology. 1994, 237, 479-499

https://doi.org/10.1126/science.7692599

48,

Structure-Function Analysis of the Ion Channel Selectivity Filter in Human Annexin V

R. Berendes, D. Voges, P. Demange, R. Huber, A. Burger

Science. 1993, 262, 427-430

https://doi.org/10.1006/jmbi.1996.0268

76

Structural and Functional Characterisation of the Voltage Sensor in the Ion Channel Human Annexin V

S. Liemann, J. Benz, A. Burger, D. Voges, A. Hofmann, R. Huber, P. Göttig

Journal of Molecular Biology. 1996, 258, 555-561

https://doi.org/10.1016/0014-5793(93)81507-v

]

.

The N-terminal domain of annexin V is required for the ion channel activity as the mutated annexin V without 14 N-terminal amino acids does not support the Ca²⁺ flux across the membrane

[

75

Calcium influx through annexin V ion channels into large unilamellar vesicles measured with fura-2

R. Berendes, A. Burger, D. Voges, P. Demange, R. Huber

FEBS Letters. 1993, 317, 131-134

]

.

The hydrophilic pore in the center of the annexin V monomer was suggested to form the ion transport pathway through the molecule. Salt bridges inside the pore form the “gate”

[

55

Annexin V membrane interaction: an electrostatic potential study

A. Karshikov, R. Berendes, A. Burger, A. Cavali�, H. Lux, R. Huber

European Biophysics Journal. 1992, 20, None

]

. Hypotheses concerning the ways of the ion path opening include the hinge movement of the molecule that leads to the change of the inter module angle which opens the pore. The alternative salt bridge formation was also suggested

[

28,

Crystal and molecular structure of human annexin V after refinement

R. Huber, R. Berendes, A. Burger, M. Schneider, A. Karshikov, H. Luecke, J. Römisch, E. Paques

Journal of Molecular Biology. 1992, 223, 683-704

55,

Annexin V membrane interaction: an electrostatic potential study

A. Karshikov, R. Berendes, A. Burger, A. Cavali�, H. Lux, R. Huber

European Biophysics Journal. 1992, 20, None

https://doi.org/10.1093/protein/11.10.891

77

Hinge-bending motions in annexins: molecular dynamics and essential dynamics of apo-annexin V and of calcium bound annexin V and I

D. Cregut, G. Drin, J. Liautard, L. Chiche

Protein Engineering Design and Selection. 1998, 11, 891-900

https://doi.org/10.1093/protein/11.10.891

]

. The hinge movement of annexin V domains was observed in a molecular dynamics simulation. The angle between two parts of the molecule was bigger in a Ca²⁺-bound form of annexin V than in a Ca²⁺-unbound one and thus the Ca²⁺-bound form might represent a more open conformation of the channel. However the simulation time was relatively small

[

77

Hinge-bending motions in annexins: molecular dynamics and essential dynamics of apo-annexin V and of calcium bound annexin V and I

D. Cregut, G. Drin, J. Liautard, L. Chiche

Protein Engineering Design and Selection. 1998, 11, 891-900

https://doi.org/10.1093/protein/11.10.891

]

. Free diffusion through the pore is supposed to be impossible. The channel width was found to be 0.8 A in the most opened conformation and those dimensions are still not sufficient for the ion passage so it was proposed that even larger annexin V hinge motions than those observed in simulation might be expected

[

77

Hinge-bending motions in annexins: molecular dynamics and essential dynamics of apo-annexin V and of calcium bound annexin V and I

D. Cregut, G. Drin, J. Liautard, L. Chiche

Protein Engineering Design and Selection. 1998, 11, 891-900

https://doi.org/10.1016/S0006-3495(92)81764-2

]

.

The precise mechanism of the ion transport by annexin V is currently unknown but two hypotheses exist. The first one includes the structural changes of the protein and its deep penetration into the membrane surface

[

78

Calcium channel and membrane fusion activity of synexin and other members of the Annexin gene family

H. Pollard, H. Guy, N. Arispe, M. de la Fuente, G. Lee, E. Rojas, J. Pollard, M. Srivastava, Z. Zhang-Keck, N. Merezhinskaya

Biophysical Journal. 1992, 62, 15-18

https://doi.org/10.1021/bi9703960

]

. It is often emphasized that annexin V is a soluble protein and it has very few hydrophobic areas on its surface at neutral pH = 7.4

[

35

Annexin V Interaction with Phosphatidylserine-Containing Vesicles at Low and Neutral pH

G. Köhler, U. Hering, O. Zschörnig, K. Arnold

Biochemistry. 1997, 36, 8189-8194

]

so its membrane insertion seems to be not favorable and might require extensive structural changes of the molecule

[

41

Annexin V Binding to the Outer Leaflet of Small Unilamellar Vesicles Leads to Altered Inner Leaflet Properties: 31P- and 1H-NMR Studies

M. Swairjo, M. Roberts, M. Campos, J. Dedman, B. Seaton

Biochemistry. 1994, 33, 10944-10950

]

. However it was shown that annexin V does not demonstrate any significant changes of the structure after the membrane binding

[

41

Annexin V Binding to the Outer Leaflet of Small Unilamellar Vesicles Leads to Altered Inner Leaflet Properties: 31P- and 1H-NMR Studies

M. Swairjo, M. Roberts, M. Campos, J. Dedman, B. Seaton

Biochemistry. 1994, 33, 10944-10950

]

(discussed in details in previous sections). Furthermore, it binds predominantly to lipid head groups and does not disturb lipid tails significantly and that contradicts the model of the membrane insertion

[

41

Annexin V Binding to the Outer Leaflet of Small Unilamellar Vesicles Leads to Altered Inner Leaflet Properties: 31P- and 1H-NMR Studies

M. Swairjo, M. Roberts, M. Campos, J. Dedman, B. Seaton

Biochemistry. 1994, 33, 10944-10950

https://doi.org/10.1021/bi9703960

]

. On the other hand, the hydrophobicity of the protein increases substantially at acidic pH = 4.0 and those conditions might facilitate the membrane insertion

[

35

Annexin V Interaction with Phosphatidylserine-Containing Vesicles at Low and Neutral pH

G. Köhler, U. Hering, O. Zschörnig, K. Arnold

Biochemistry. 1997, 36, 8189-8194

]

, so the possibility of this process in different conditions is still under question.

The second hypothesis includes changes in the membrane structure and the process of electroporation, the reversible membrane pore formation under action of the external electric field

[

55

Annexin V membrane interaction: an electrostatic potential study

A. Karshikov, R. Berendes, A. Burger, A. Cavali�, H. Lux, R. Huber

European Biophysics Journal. 1992, 20, None

https://doi.org/10.1146/annurev-biophys-052118-115451

]

(for resent review of electroporation see

[

79

Membrane Electroporation and Electropermeabilization: Mechanisms and Models

T. Kotnik, L. Rems, M. Tarek, D. Miklavčič

Annual Review of Biophysics. 2019, 48, 63-91

]

). It was shown that Ca²⁺-bound annexin V is able to exert the electric field in the region of the protein-membrane contact and this field is strong enough to induce the pore formation

[

55

Annexin V membrane interaction: an electrostatic potential study

A. Karshikov, R. Berendes, A. Burger, A. Cavali�, H. Lux, R. Huber

European Biophysics Journal. 1992, 20, None

https://doi.org/10.1021/bi9626867

]

.

The key limitation of the early studies of annexin V ion transport activity is the usage of extremely acidic phospholipid membranes (PS/PE = 90/10) or small membrane patches on the micropipettes. More recently the annexin V channel activity was studied on the phospholipid vesicles of different composition (25, 50, 75 or 90% of PS with the rest of PE)

[

80

Annexin V-Mediated Calcium Flux across Membranes Is Dependent on the Lipid Composition: Implications for Cartilage Mineralization

T. Kirsch, H. Nah, D. Demuth, G. Harrison, E. Golub, S. Adams, M. Pacifici

Biochemistry. 1997, 36, 3359-3367

https://doi.org/10.1021/bi9626867

]

. It was revealed that the significant Ca²⁺ flux across the membrane exists only for PS/PE = 90/10 vesicles but is not observed on vesicles with lower PS content. On vesicles composed of PG/PE or PI/PE annexin V does not demonstrate Ca²⁺ channel activity, and only vesicles composed of PA/PE = 90/10 support the Ca²⁺ flux across the membrane

[

80

Annexin V-Mediated Calcium Flux across Membranes Is Dependent on the Lipid Composition: Implications for Cartilage Mineralization

T. Kirsch, H. Nah, D. Demuth, G. Harrison, E. Golub, S. Adams, M. Pacifici

Biochemistry. 1997, 36, 3359-3367

https://doi.org/10.1006/jmbi.1997.1183

]

. Those findings suggest that for the Ca²⁺ transport by annexin V the acidic surface is required. However, this study was performed for chicken annexin V which has different from human annexin V properties (for example human annexin V does not promote the vesicles aggregation, while the chicken annexin V does

[

42,

Structural analysis of junctions formed between lipid membranes and several annexins by cryo-electron microscopy 1 1Edited by M. F. Moody

O. Lambert, V. Gerke, M. Bader, F. Porte, A. Brisson

Journal of Molecular Biology. 1997, 272, 42-55

https://doi.org/10.1002/pro.119

81

Key role of the N-terminus of chicken annexin A5 in vesicle aggregation

J. Turnay, A. Guzmán-Aránguez, E. Lecona, J. Barrasa, N. Olmo, M. Lizarbe

Protein Science. 2009, 18, 1095-1106

https://doi.org/10.1016/0014-5793(95)00032-5

]

), thus the human annexin V channel activity on membranes of different composition might be different.

In some studies it was noted that authors were unable to detect the annexin V channel activity

[

82

Annexin V perturbs or stabilises phospholipid membranes in a calcium-dependent manner

E. Goossens, C. Reutelingsperger, F. Jongsma, R. Kraayenhof, W. Hermens

FEBS Letters. 1995, 359, 155-158

https://doi.org/10.1016/0014-5793(95)00032-5

]

. However the leakage of fura-2 from the acidic phospholipid vesicle (PS/PE = 90/10) in presence of high (2.5 mM) Ca²⁺ concentration or annexin V without Ca²⁺ was observed

[

82

Annexin V perturbs or stabilises phospholipid membranes in a calcium-dependent manner

E. Goossens, C. Reutelingsperger, F. Jongsma, R. Kraayenhof, W. Hermens

FEBS Letters. 1995, 359, 155-158