Annexin-phospholipid interactions. Functional implications

Abstract

Annexins constitute an evolutionary conserved multigene protein superfamily characterized by their ability to interact with biological membranes in a calcium dependent manner. They are expressed by all living organisms with the exception of certain unicellular organisms. The vertebrate annexin core is composed of four (eight in annexin A6) homologous domains of around 70 amino acids, with the overall shape of a slightly bent ring surrounding a central hydrophilic pore. Calcium- and phospholipid-binding sites are located on the convex side while the N-terminus links domains I and IV on the concave side. The N-terminus region shows great variability in length and amino acid sequence and it greatly influences protein stability and specific functions of annexins. These proteins interact mainly with acidic phospholipids, such as phosphatidylserine, but differences are found regarding their affinity for lipids and calcium requirements for the interaction. Annexins are involved in a wide range of intra- and extracellular biological processes in vitro, most of them directly related with the conserved ability to bind to phospholipid bilayers: membrane trafficking, membrane-cytoskeleton anchorage, ion channel activity and regulation, as well as antiinflammatory and anticoagulant activities. However, the in vivo physiological functions of annexins are just beginning to be established.

Figure 1

Three-dimensional structure of rat annexin A5. The three-dimensional structure of rat annexin A5 with the four Type II calcium-binding sites saturated with calcium was obtained using MOLMOL [11] and is based on the protein data bank (PDB) file 1A8A [12]. A lateral view (A), a top view of the molecule (B), a detail of the interactions in Domain III with calcium and the polar head of phosphatidylserine (PS) (C), and a hydrophobicity surface representation (D) are shown. The four domains in the protein core are represented in different colors: I, blue; II, cyan; III, orange; and IV, red. The letters assigned to the α-helices are shown only in Domain III. Calcium ions are represented by yellow spheres. Panel B allows a clear view of the central hydrophilic hole; the different domains are indicated. The calcium-dependent binding of glycerophosphoserine (GPS) to Domain III of the annexin core is shown in (C) together with the coordinations that bind calcium to the protein at the “AB”, “B” and “DE” sites. The carbon atoms of the polypeptide backbone and the lateral chains of key residues for the interaction with calcium are represented in grey, whereas carbons in GPS are green (oxygen, red; nitrogen, blue; phosphorus, magenta).

Figure 2

Crystal structure of full-length pig annexin A1 in the absence and presence of calcium. The three-dimensional structure of pig annexin A1 in the absence of calcium (A) (PDB file 1HM6) [22] shows the intact N-terminus with the appearance of two consecutive α-helices (residues 2–18 and 19–28; green), the most N-terminal one being inserted into Domain III (orange) with the consequent disappearance of Helix 3D. The structure of this annexin in the presence of calcium (B) (PDB file 1MCX) [24] does not show the disordered N-terminus; as observed, Helix 3D is refolded when the N-terminal helix moves out of the protein core. Arrows show the most affected region of the protein core after the interaction with calcium. Three-dimensional structures were drawn using MOLMOL.

Figure 3

Proposed mechanisms for annexin-mediated membrane-vesicle aggregation. Several mechanisms have been proposed to explain the annexin-mediated induction of vesicle aggregation. Essentially, the mechanism depends on the molecular characteristics of the annexin involved in the process. In all cases, calcium seems to be essential to induce the process. (A) Aggregation induced by annexin-annexin interactions through the concave side of the molecules (probable involvement of the N-terminus); (B) Aggregation induced by the formation of heterotetrameric annexin bridges. Annexin A2 does not require calcium to interact with S100A10 as this small S100 molecule is permanently in the “activated” form without binding calcium. Other annexins require calcium for the formation of the complex with S100 proteins that are found in an “inactivated” form at low calcium concentrations (i.e., annexin A1 with S100A11); (C) Vesicle aggregation induced by the appearance of a second phospholipid- binding site in the N-terminus after calcium-dependent binding to one membrane and consequent annexin structural rearrangement. (Part of the figure is based on the model proposed in [1] for annexin A1).

Figure 4

Intra- and extracellular functions of annexins. Annexins can be found in different intracellular compartments, including the nucleus, in equilibrium between soluble and a membrane-bound or cytoskeleton-bound forms depending on local intracellular calcium variations and lipid composition. In addition, acidification in the close proximity of the membrane due to acidic phospholipids can induce a conformational rearrangement of specific annexins with insertion of the annexin helices into the membrane. Although they lack signal sequences for secretion, they can be localized on the outer plasma membrane or soluble as circulating proteins where they can interact with biological membranes that expose PS, such as activated platelets.

Figure 5

Proposed interactions of annexin B12 with cell membranes. Annexin B12 is quite similar to annexin A5 and it can interact with cell membranes in a superficial manner in response to an increase in calcium concentration. This interaction may induce alterations in the membrane and allow electroporation of calcium ions (A). It has also been proposed that a hexamer of annexin B12 (PDB file 1AEI; [129]) may integrate into the membrane in the presence of calcium (B), and could function as a calcium channel due to the existence of a central hydrophilic pore in the hexamer (ribbon and surface representations are shown from an upper view showing the hydrophilic pore). At low calcium concentration but in the presence of mild acidic pH in the proximity of the membrane, annexin B12 may experiment an overall structural rearrangement with formation of seven transmembrane helices that may allow the calcium channel activity (the helix distribution is based on a scheme in [1] and the work of Langen and coworkers [–60]) (C).