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

Bouter A, Gounou C, Bérat R, Tan S, Gallois B, Granier T, d'Estaintot BL, Pöschl E, Brachvogel B, Brisson AR - Nat Commun (2011)

Bottom Line: Compared with wild-type mouse perivascular cells, AnxA5- cells exhibit a severe membrane repair defect.In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair.We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca(2+), AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging and NanoBioTechnology, IECB, UMR-5248 CBMN CNRS-University Bordeaux1-ENITAB, Talence F-33402, France.

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Structure and packing of AnxA5 and mtT-AnxA5 in three-dimensional crystals.(a, b) Structure of trimers of recombinant rat AnxA5 (a) and monomer–monomer interface of a trimer (b) in R3 crystals (created from PDB code 1a8a)3960. (a) The intramolecular pseudo twofold axes, which relate (I+IV) and (III+II) modules (indicated by crosses in each monomer of the bottom trimer) lie almost parallel (12°) to the threefold symmetry axes (black triangles) normal to the (a, b) plane. (b) Detailed structure of the monomer–monomer interface of an AnxA5 trimer, viewed along the arrow orientation in (a). A–E refer to AnxA5 α-helices, according to annexin nomenclature19. Several pairs of charged amino acids located at distances close enough to form salt bridges are indicated in ball-and-stick representation, with positively and negatively charged residues in blue and red, respectively. (c) Packing of mtT-AnxA5 molecules in high Ca2+ content 3D crystals, along the b monoclinic axis. MtT-AnxA5 molecules crystallize in the P21 space group, with lattice parameters a=51.108 Å, b=67.184 Å, c=112.313 Å, β=94.79°. The asymmetric unit contains two protein molecules related by a non-crystallographic 2ɛ screw axis parallel to the a axis, with a non-fractional pitch of ɛ=4.7 Å. The positions of two 2ɛ screw axes are indicated. MtT-AnxA5 molecules are oriented in the a, c plane with the intramolecular pseudo twofold axis (indicated by a cross in the AnxA5 monomer with I–IV numbering) lying almost normal (12°) to the a, c plane. This orientation is thus similar to that of native AnxA5 molecules shown in Figure 5a. Side chains of residues involved in salt-bridge interactions in AnxA5 trimers are shown as blue sticks. None of the residues participates in intermolecular contacts, with the exception of residue E16, which interacts through a Ca2+-mediated salt bridge with a neighbouring molecule along the b axis.
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f5: Structure and packing of AnxA5 and mtT-AnxA5 in three-dimensional crystals.(a, b) Structure of trimers of recombinant rat AnxA5 (a) and monomer–monomer interface of a trimer (b) in R3 crystals (created from PDB code 1a8a)3960. (a) The intramolecular pseudo twofold axes, which relate (I+IV) and (III+II) modules (indicated by crosses in each monomer of the bottom trimer) lie almost parallel (12°) to the threefold symmetry axes (black triangles) normal to the (a, b) plane. (b) Detailed structure of the monomer–monomer interface of an AnxA5 trimer, viewed along the arrow orientation in (a). A–E refer to AnxA5 α-helices, according to annexin nomenclature19. Several pairs of charged amino acids located at distances close enough to form salt bridges are indicated in ball-and-stick representation, with positively and negatively charged residues in blue and red, respectively. (c) Packing of mtT-AnxA5 molecules in high Ca2+ content 3D crystals, along the b monoclinic axis. MtT-AnxA5 molecules crystallize in the P21 space group, with lattice parameters a=51.108 Å, b=67.184 Å, c=112.313 Å, β=94.79°. The asymmetric unit contains two protein molecules related by a non-crystallographic 2ɛ screw axis parallel to the a axis, with a non-fractional pitch of ɛ=4.7 Å. The positions of two 2ɛ screw axes are indicated. MtT-AnxA5 molecules are oriented in the a, c plane with the intramolecular pseudo twofold axis (indicated by a cross in the AnxA5 monomer with I–IV numbering) lying almost normal (12°) to the a, c plane. This orientation is thus similar to that of native AnxA5 molecules shown in Figure 5a. Side chains of residues involved in salt-bridge interactions in AnxA5 trimers are shown as blue sticks. None of the residues participates in intermolecular contacts, with the exception of residue E16, which interacts through a Ca2+-mediated salt bridge with a neighbouring molecule along the b axis.

Mentions: By which mechanism does AnxA5 promote cell membrane repair? The most remarkable in vitro property of AnxA5 is to self-assemble into trimers and 2D ordered arrays of trimers on PS-exposing membranes2935 (Fig. 1b). To investigate whether AnxA5 2D self-assembly had an influence on membrane repair, we produced AnxA5 mutants that lack the ability to form 2D arrays. The choice of the mutations was based on the fact that the monomer–monomer interface of AnxA5 trimers (Fig. 5a) presents several pairs of amino acids of opposite charges situated at distances close enough to form salt bridges39 (Fig. 5b). In addition, Mo et al.39 have shown that mutations of single amino acids were not sufficient to abolish the ability of AnxA5 to form trimers. Consequently, we produced multiple mutants in which several positively charged residues involved in salt bridging were replaced by negatively charged ones. The ability of AnxA5 mutants to form trimers was first assessed by transmission electron microscopy (TEM), according to established procedures4041 (Supplementary Fig. S5). We found that all mutants with one, two or three mutations were still able to form 2D crystals, and thus trimers. Contrarily, all quadruple and quintuple mutants have lost the property of trimer and 2D crystal formation. The quintuple mutant mtT-AnxA5 (R16E, R23E, K27E, K56E, K191E) was selected for further analyses. To further ascertain that mtT-AnxA5 molecules are unable to form trimers, three-dimensional crystals of mtT-AnxA5 were grown in high Ca2+ concentration and their structure was determined by X-ray crystallography. Indeed, all AnxA5 structures hitherto reported from three-dimensional crystals grown at high Ca2+ concentration contain AnxA5 trimers, which are structurally homologous to membrane-bound trimers394243. MtT-AnxA5 molecules crystallize in the P21 space group (Supplementary Table S1; Supplementary Methods) in which they pack as dimers with upside-down orientations (Fig. 5c). This result confirms that mtT-AnxA5 is unable to form trimers.


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

Bouter A, Gounou C, Bérat R, Tan S, Gallois B, Granier T, d'Estaintot BL, Pöschl E, Brachvogel B, Brisson AR - Nat Commun (2011)

Structure and packing of AnxA5 and mtT-AnxA5 in three-dimensional crystals.(a, b) Structure of trimers of recombinant rat AnxA5 (a) and monomer–monomer interface of a trimer (b) in R3 crystals (created from PDB code 1a8a)3960. (a) The intramolecular pseudo twofold axes, which relate (I+IV) and (III+II) modules (indicated by crosses in each monomer of the bottom trimer) lie almost parallel (12°) to the threefold symmetry axes (black triangles) normal to the (a, b) plane. (b) Detailed structure of the monomer–monomer interface of an AnxA5 trimer, viewed along the arrow orientation in (a). A–E refer to AnxA5 α-helices, according to annexin nomenclature19. Several pairs of charged amino acids located at distances close enough to form salt bridges are indicated in ball-and-stick representation, with positively and negatively charged residues in blue and red, respectively. (c) Packing of mtT-AnxA5 molecules in high Ca2+ content 3D crystals, along the b monoclinic axis. MtT-AnxA5 molecules crystallize in the P21 space group, with lattice parameters a=51.108 Å, b=67.184 Å, c=112.313 Å, β=94.79°. The asymmetric unit contains two protein molecules related by a non-crystallographic 2ɛ screw axis parallel to the a axis, with a non-fractional pitch of ɛ=4.7 Å. The positions of two 2ɛ screw axes are indicated. MtT-AnxA5 molecules are oriented in the a, c plane with the intramolecular pseudo twofold axis (indicated by a cross in the AnxA5 monomer with I–IV numbering) lying almost normal (12°) to the a, c plane. This orientation is thus similar to that of native AnxA5 molecules shown in Figure 5a. Side chains of residues involved in salt-bridge interactions in AnxA5 trimers are shown as blue sticks. None of the residues participates in intermolecular contacts, with the exception of residue E16, which interacts through a Ca2+-mediated salt bridge with a neighbouring molecule along the b axis.
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f5: Structure and packing of AnxA5 and mtT-AnxA5 in three-dimensional crystals.(a, b) Structure of trimers of recombinant rat AnxA5 (a) and monomer–monomer interface of a trimer (b) in R3 crystals (created from PDB code 1a8a)3960. (a) The intramolecular pseudo twofold axes, which relate (I+IV) and (III+II) modules (indicated by crosses in each monomer of the bottom trimer) lie almost parallel (12°) to the threefold symmetry axes (black triangles) normal to the (a, b) plane. (b) Detailed structure of the monomer–monomer interface of an AnxA5 trimer, viewed along the arrow orientation in (a). A–E refer to AnxA5 α-helices, according to annexin nomenclature19. Several pairs of charged amino acids located at distances close enough to form salt bridges are indicated in ball-and-stick representation, with positively and negatively charged residues in blue and red, respectively. (c) Packing of mtT-AnxA5 molecules in high Ca2+ content 3D crystals, along the b monoclinic axis. MtT-AnxA5 molecules crystallize in the P21 space group, with lattice parameters a=51.108 Å, b=67.184 Å, c=112.313 Å, β=94.79°. The asymmetric unit contains two protein molecules related by a non-crystallographic 2ɛ screw axis parallel to the a axis, with a non-fractional pitch of ɛ=4.7 Å. The positions of two 2ɛ screw axes are indicated. MtT-AnxA5 molecules are oriented in the a, c plane with the intramolecular pseudo twofold axis (indicated by a cross in the AnxA5 monomer with I–IV numbering) lying almost normal (12°) to the a, c plane. This orientation is thus similar to that of native AnxA5 molecules shown in Figure 5a. Side chains of residues involved in salt-bridge interactions in AnxA5 trimers are shown as blue sticks. None of the residues participates in intermolecular contacts, with the exception of residue E16, which interacts through a Ca2+-mediated salt bridge with a neighbouring molecule along the b axis.
Mentions: By which mechanism does AnxA5 promote cell membrane repair? The most remarkable in vitro property of AnxA5 is to self-assemble into trimers and 2D ordered arrays of trimers on PS-exposing membranes2935 (Fig. 1b). To investigate whether AnxA5 2D self-assembly had an influence on membrane repair, we produced AnxA5 mutants that lack the ability to form 2D arrays. The choice of the mutations was based on the fact that the monomer–monomer interface of AnxA5 trimers (Fig. 5a) presents several pairs of amino acids of opposite charges situated at distances close enough to form salt bridges39 (Fig. 5b). In addition, Mo et al.39 have shown that mutations of single amino acids were not sufficient to abolish the ability of AnxA5 to form trimers. Consequently, we produced multiple mutants in which several positively charged residues involved in salt bridging were replaced by negatively charged ones. The ability of AnxA5 mutants to form trimers was first assessed by transmission electron microscopy (TEM), according to established procedures4041 (Supplementary Fig. S5). We found that all mutants with one, two or three mutations were still able to form 2D crystals, and thus trimers. Contrarily, all quadruple and quintuple mutants have lost the property of trimer and 2D crystal formation. The quintuple mutant mtT-AnxA5 (R16E, R23E, K27E, K56E, K191E) was selected for further analyses. To further ascertain that mtT-AnxA5 molecules are unable to form trimers, three-dimensional crystals of mtT-AnxA5 were grown in high Ca2+ concentration and their structure was determined by X-ray crystallography. Indeed, all AnxA5 structures hitherto reported from three-dimensional crystals grown at high Ca2+ concentration contain AnxA5 trimers, which are structurally homologous to membrane-bound trimers394243. MtT-AnxA5 molecules crystallize in the P21 space group (Supplementary Table S1; Supplementary Methods) in which they pack as dimers with upside-down orientations (Fig. 5c). This result confirms that mtT-AnxA5 is unable to form trimers.

Bottom Line: Compared with wild-type mouse perivascular cells, AnxA5- cells exhibit a severe membrane repair defect.In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair.We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca(2+), AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging and NanoBioTechnology, IECB, UMR-5248 CBMN CNRS-University Bordeaux1-ENITAB, Talence F-33402, France.

Show MeSH
Related in: MedlinePlus