Different molecular arrangements of the tetrameric annexin 2 modulate the size and dynamics of membrane aggregation

Annexin 2, a member of the annexin family of Ca²⁺-dependent membrane binding proteins is found in monomeric and heterotetrameric forms and has been involved in different membrane related functions. The heterotetrameric annexin 2 is composed of a dimer of S100A10, a member of the S100 family of Ca²⁺ binding proteins and two annexin 2 molecules ((Anx2-S100A10)₂). Different molecular models including tetramers and octamers in which S100A10 is localized in the centre of the complex with the annexin 2 molecules positioned around S100A10 had been proposed. Herein, the organization of the (Anx2-S100A10)₂ complex in conditions in which membranes are able to bridge was studied. We performed Cryo-electron microscopy observations of the tetrameric annexin 2 on the membrane surface, and study the S100A10 accessibility to antibodies by flow “cytometry”. We also studied the kinetics and size evolution of vesicle aggregates by dynamic light scattering. The results show that the protein is able to organize in three different arrangements depending on the presence of Ca²⁺ and pH and that the aggregation is faster in the presence of Ca²⁺ compared with the aggregation in its absence. In one arrangement the S100A10 molecule is exposed to the solvent allowing its interaction with other proteins. The presented results will serve as a molecular basis to explain some of the functions of the tetrameric annexin 2.     

Expression of a releasable form of annexin II by human keratinocytes

Annexin II is a multifunctional calcium-dependent phospholipid binding protein whose presence in epidermis has previously been reported. However, like other members of annexin family, annexin II has been regarded as either an intracellular protein or associated with the cellular membrane. Here, we report the presence of a releasable annexin II and p11, two monomers of annexin II tetramer, in keratinocyte-conditioned medium (KCM). Proteins present in KCM were fractionated on a gel filtration column and following further evaluation, a releasable protein with apparent MW of 36 kDa was identified. Further characterization identified this protein as the p36 monomer of annexin II tetramer. The phospho-tyrosine antibody did not visualize this protein as the phosphorylated form of p36. Several experiments were conducted to examine whether this protein is soluble or associated with keratinocyte cell membranes in the conditioned medium. A centrifugation of conditioned medium was not able to bring this protein down into the pellet. Surprisingly, the results of Western analysis identified p36 and p11, two monomers of the annexin II tetramer, in conditioned medium derived from either keratinocytes cultured alone or keratinocytes co-cultured with fibroblasts. In contrast to the keratinocyte-conditioned medium in which annexin II was easily detectable, both monomers were barely detectable in conditioned medium collected from dermal fibroblasts. This finding was in contrast to the cell lysates in which p36 was detectable in both keratinocytes and fibroblasts. However, the amount of this protein was markedly higher in keratinocyte lysate relative to that of dermal fibroblasts. Conditioned medium derived from keratinocyte established from adult showed a higher level of annexin II compared to that of keratinocytes established from newborn babies. The expression of p11 seems to increase with differentiation of keratinocytes derived from either adult or newborn skin samples. When the site of annexin synthesis in human skin was examined by immunohistochemical staining, the antibody for p36 localized the annexin to the keratinocyte cell members in the basal and suprabasal keratinocytes. In conclusion, Western blot detection of both p36 and p11 in conditioned medium from skin cells revealed that human keratinocytes, but not fibroblasts, express a releasable monomer form of annexin II which is regulated by differentiation status of keratinocytes. This finding is consistent with the localization of annexin II detected by immunohistochemical staining.     

人膜联蛋白A5:从生物标志物到药物候选物

人膜联蛋白A5 (human annexin A5,hAnxA5)是人体中一种重要的功能蛋白质分子,广泛存在于人体的细胞和体液中。hAnxA5作为具有特定结构、隶属于一个复杂成员膜联蛋白家族的一个成员分子,其生化特性也是非常独特的。它以钙离子依赖方式,可逆、特异性、高效地结合磷脂酰丝氨酸分子;并基于此特性发挥重要生物学功能,在诸多人体生理、病理现象中发挥重要作用。本文就hAnxA5的结构特点、生化特性、作用机制、影响效应以及在生物医学领域中的重要应用进行了较为系统的归纳与总结。游离态hAnxA5以单体形式存在,发挥生物学活性时通常形成聚合体形式。hAnxA5影响了人体的血管血栓疾病、自身免疫性疾病、肿瘤疾病、肺纤维化及肺损伤、非酒精性脂肪性肝炎等病理现象的发生与发展;hAnxA5作为疾病生物标志物,也应用在肿瘤、神经退行性疾病、心力衰竭、急性肾损伤和哮喘等疾病的研究中;作为新型药物候选物,hAnxA5及其衍生物被多次设计、运用于多类疾病的治疗探索,尤其在血管血栓类疾病的治疗探索上。对于hAnxA5的研究,仍存在某些空白与不足。对其研究的深入,不仅将拓展对于hAnxA5结构与功能关系的认...     

Changes in annexin (lipocortin) content in human amnion and chorion at parturition.

Arachidonic acid is mobilized from fetal membrane phospholipids at parturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometrial contractions. Phospholipid mobilization requires activation of phospholipase A2 or C, both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids and thereby may alter phospholipase activity through two mechanisms: modulation of intracellular free Ca2+ concentrations or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I-VI were identified in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pools: a pool which only associated with the membrane in the presence of calcium (calcium-dependent pool) and a calcium-independent pool that remained membrane bound in the presence of calcium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium-dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium-dependent pool of annexin V increased in chorion; and calcium-independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in total annexin I concentration with labor in amnion reflects a substantial decrease (80-90%) in the pool tightly bound to the membrane in a calcium-independent manner. This striking change distinguishes annexin I as a potential candidate inhibitor which is specifically downregulated at parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostaglandin production at labor.     

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

Annexins are calcium‐dependent phospholipid‐binding proteins involved in calcium signaling and intracellular membrane trafficking among other functions. Vesicle aggregation is a crucial event to make possible the membrane remodeling but this process is energetically unfavorable, and phospholipid membranes do not aggregate and fuse spontaneously. This issue can be circumvented by the presence of different agents such as divalent cations and/or proteins, among them some annexins. Although human annexin A5 lacks the ability to aggregate vesicles, here we demonstrate that its highly similar chicken ortholog induces aggregation of vesicles containing acidic phospholipids even at low protein and/or calcium concentration by establishment of protein dimers. Our experiments show that the ability to aggregate vesicles mainly resides in the N‐terminus as truncation of the N‐terminus of chicken annexin A5 significantly decreases this process and replacement of the N‐terminus of human annexin A5 by that of chicken switches on aggregation; in both cases, there are no changes in the overall protein structure and only minor changes in phospholipid binding. Electrostatic repulsions between negatively charged residues in the concave face of the molecule, mainly in the N‐terminus, seem to be responsible for the impairment of dimer formation in human annexin A5. Taking into account that chicken annexin A5 presents a high sequence and structural similarity with mammalian annexins absent in birds, as annexins A3 and A4, some of the physiological functions exerted by these proteins may be carried out by chicken annexin A5, even those that could require calcium‐dependent membrane aggregation.     

S100A10‐Mediated Translocation of Annexin‐A2 to SNARE Proteins in Adrenergic Chromaffin Cells Undergoing Exocytosis

In neuroendocrine cells, annexin‐A2 is implicated as a promoter of monosialotetrahexosylganglioside (GM1)‐containing lipid microdomains that are required for calcium‐regulated exocytosis. As soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) require a specific lipid environment to mediate granule docking and fusion, we investigated whether annexin‐A2‐induced lipid microdomains might be linked to the SNAREs present at the plasma membrane. Stimulation of adrenergic chromaffin cells induces the translocation of cytosolic annexin‐A2 to the plasma membrane, where it colocalizes with SNAP‐25 and S100A10. Cross‐linking experiments performed in stimulated chromaffin cells indicate that annexin‐A2 directly interacts with S100A10 to form a tetramer at the plasma membrane. Here, we demonstrate that S100A10 can interact with vesicle‐associated membrane protein 2 (VAMP2) and show that VAMP2 is present at the plasma membrane in resting adrenergic chromaffin cells. Tetanus toxin that cleaves VAMP2 solubilizes S100A10 from the plasma membrane and inhibits the translocation of annexin‐A2 to the plasma membrane. Immunogold labelling of plasma membrane sheets combined with spatial point pattern analysis confirmed that S100A10 is present in VAMP2 microdomains at the plasma membrane and that annexin‐A2 is observed close to S100A10 and to syntaxin in stimulated chromaffin cells. In addition, these results showed that the formation of phosphatidylinositol (4,5)‐bisphosphate (PIP₂) microdomains colocalized with S100A10 in the vicinity of docked granules, suggesting a functional interplay between annexin‐A2‐mediated lipid microdomains and SNAREs during exocytosis.     

Annexin A2 is required for the early steps of cytokinesis

Cytokinesis requires the formation of an actomyosin contractile ring between the two sets of sister chromatids. Annexin A2 is a calcium- and phospholipid-binding protein implicated in cortical actin remodeling. We report that annexin A2 accumulates at the equatorial cortex at the onset of cytokinesis and depletion of annexin A2 results in cytokinetic failure, due to a defective cleavage furrow assembly. In the absence of annexin A2, the small GTPase RhoA—which regulates cortical cytoskeletal rearrangement—fails to form a compact ring at the equatorial plane. Furthermore, annexin A2 is required for cortical localization of the RhoGEF Ect2 and to maintain the association between the equatorial cortex and the central spindle. Our results demonstrate that annexin A2 is necessary in the early phase of cytokinesis. We propose that annexin A2 participates in central spindle–equatorial plasma membrane communication.     

Characterization of annexin A1 glycan binding reveals binding to highly sulfated glycans with preference for highly sulfated heparan sulfate and heparin

Annexin A1 is a multifunctional, calcium-dependent phospholipid binding protein involved in a host of processes including inflammation, regulation of neuroendocrine signaling, apoptosis, and membrane trafficking. Binding of annexin A1 to glycans has been implicated in cell attachment and modulation of annexin A1 function. A detailed characterization of the glycan binding preferences of annexin A1 using carbohydrate microarrays and surface plasmon resonance served as a starting point to understand the role of glycan binding in annexin A1 function. Glycan array analysis identified annexin A1 binding to a series of sulfated oligosaccharides and revealed for the first time that annexin A1 binds to sulfated non-glycosaminoglycan carbohydrates. Using heparin/heparan sulfate microarrays, highly sulfated heparan sulfate/heparin were identified as preferred ligands of annexin A1. Binding of annexin A1 to heparin/heparan sulfate is calcium- but not magnesium-dependent. An in-depth structure-activity relationship of annexin A1-heparan sulfate interactions was established using chemically defined sugars. For the first time, a calcium-dependent heparin binding protein was characterized with such an approach. N-Sulfation and 2-O-sulfation were identified as particularly important for binding.     

Identification of annexin A1 as a novel substrate for E6AP‐mediated ubiquitylation

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high‐risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull‐down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP‐binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi‐mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca²⁺‐dependent manner. GST pull‐down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP‐mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin‐proteasome pathway. J. Cell. Biochem. 106: 1123–1135, 2009. © 2009 Wiley‐Liss, Inc.     

Identification and predominant expression of annexin A2 in epithelial-type cells of the rice field eel

Annexin is the largest family of genes encoding eukaryotic calcium-binding proteins that do not contain the EF hand motif. Annexin A2 has a common annexin core domain, consisting of four so-called annexin repeats, and each of these repeats has about 70 amino acids in length. Here we report identification of annexin A2 from rice field eel by degenerate PCR and RACE techniques. Three-dimensional structure prediction shows that it has similar annexin repeat architecture. Phylogenetic analysis shows that this gene fits with the annexin A2 clade of vertebrates. Subcellular co-localization and co-immunoprecipitation indicated annexin A2 interacted with its ligand S100A10, confirming characteristics of the rice field eel annexin A2. RT-PCR and Western blot results indicate annexin A2 expressed ubiquitously in adult tissues. Immunofluorescence analysis shows obvious immunoreactivity in the nuclear membrane of developing oocytes and base membrane of mature oocytes in ovary and ovotestis. After the gonad differentiates into testis, annexin A2 protein expressed in the site of seminal vesicles epithelium in testis. The results provided a clue to the potential role of annexin A2 in the gonadal differentiation from ovary, via ovotestis to testis of the rice field eel.     

Crystal structure of metastasis-associated protein S100A4 in the active calcium-bound form

S100A4 (metastasin) is a member of the S100 family of calcium-binding proteins that is directly involved in tumorigenesis. Until recently, the only structural information available was the solution NMR structure of the inactive calcium-free form of the protein. Here we report the crystal structure of human S100A4 in the active calcium-bound state at 2.03 Å resolution that was solved by molecular replacement in the space group P6₅ with two molecules in the asymmetric unit from perfectly merohedrally twinned crystals. The Ca^2 +-bound S100A4 structure reveals a large conformational change in the three-dimensional structure of the dimeric S100A4 protein upon calcium binding. This calcium-dependent conformational change opens up a hydrophobic binding pocket that is capable of binding to target proteins such as annexin A2, the tumor-suppressor protein p53 and myosin IIA. The structure of the active form of S100A4 provides insight into its interactions with its binding partners and a better understanding of its role in metastasis.     

A calcium-driven conformational switch of the N-terminal and core domains of annexin A1

In 1993, Huber and co-workers published the structure of an N-terminally truncated version of human annexin A1 lacking the first 32 amino acid residues (PDB code: 1AIN). In 2001, we reported the structure of full-length porcine annexin A1 including the N-terminal domain in the absence of calcium ions (PDB code: 1HM6). The latter structure did not reflect a typical annexin core fold, but rather a surprising interaction of the N-terminal domain and the core domain. Comparing these two structures revealed that in the full-length structure the first 12 residues of the N-terminal domain insert into the core of the protein, thereby replacing and unwinding one of the alpha-helices (helix D in repeat 3) that is involved in calcium binding. We hypothesized that this structure in the absence of calcium ions represents the inactive form of the protein. Furthermore, we proposed that upon calcium binding, the N-terminal domain would be expelled from the core domain and that the core D-helix would reform in the proper conformation for calcium coordination. Herein, we report the X-ray structure of full-length porcine annexin A1 in the presence of calcium. This new structure shows a typical annexin core structure as we hypothesized, with the D-helix back in place for calcium coordination while parts of the now exposed N-terminal domain are disordered. We could locate eight calcium ions in this structure, two of which are octa-coordinated and two of which were not observed in the structure of the N-terminally truncated annexin A1. Possible implications of this calcium-induced conformational switch for the membrane aggregation properties of annexin A1 will be discussed.     

A 42-kilodalton annexin-like protein is associated with plant vacuoles.

A 42-kD, calcium-dependent, membrane-binding protein (VCaB42) was associated with partially purified vacuole membrane. Membrane-dissociation assays indicated that VCaB42 binding to vacuole membranes was selective for calcium over other cations and that 50% of VCaB42 remained membrane bound at 61 +/- 11 nM free calcium. A 13-amino acid sequence obtained from VCaB42 showed 85% similarity with the endonexin fold, a sequence found in the annexin family of proteins that is thought to be essential for calcium and lipid binding. The greatest similarity in amino acid sequence was observed with annexin VIII (VAC-beta). The calcium-binding properties and sequence similarities suggest that VCaB42 is a member of the annexin family of calcium-dependent, membrane-binding proteins. Functional assays for VCaB42 on vacuole membrane transport processes indicated that it did not significantly affect the initial rate of calcium uptake into vacuole membrane vesicles. Because VCaB42 is vacuole localized (likely on the cytosolic surface of the vacuole) and is 50% dissociated within the physiological range of cytosolic free calcium, we hypothesize that this protein is a sensor that monitors cytosolic calcium levels and transmits that information to the vacuole.     

LOCALIZATION OF ANNEXIN VI IN THE ADULT AND NEONATAL HEART

Annexins are a major family of intracellular Ca²⁺-binding proteins which have been implicated in a variety of cellular functions. In this paper the authors have used confocal microscopy to compare the distribution of annexin VI in vibratome sections of the rat adult left ventricle and striated muscle of the rat oesophagus. It is shown that in rat cardiac myocytes annexin VI is associated with only the sarcolemma and intercalated discs. In contrast, it is demonstrated that in rat skeletal muscle annexin VI is associated with the sarcoplasmic reticulum, in addition to the plasma membrane, suggesting that annexin VI is regulating different processes in these tissues. Also shown is that in vibratome sections of the neonatal rat left ventricle, annexin VI has a different subcellular location to that observed in the terminally differentiated adult myocyte. In these differentiating neonatal cells annexins VI is also associated with specific subcellular structures. Furthermore, using confocal microscopy of isolated myocytes the authors demonstrate that the association of annexin VI with the sarcolemma is stable even after cells are treated with the intracellular calcium chelator BAPTA-AM, to greatly deplete cytosolic calcium levels. This demonstrates that annexin VI associates tightly with the sarcolemma, and suggests that components in addition to phospholipid are involved in binding annexin VI to the membrane. These results demonstrate that the subcellular location of annexin VI is differentially regulated, and suggest that annexin VI is required for a process or processes characteristic of the sarcolemma, and of the sarcoplasmic reticulum of skeletal but not of heart muscle.     

Structure of membrane-bound annexin A5 trimers: a hybrid cryo-EM - X-ray crystallography study

Annexins constitute a family of phospholipid- and Ca(2+)-binding proteins involved in a variety of membrane-related processes. The property of several annexins, including annexin A5, to self-organize at the surface of lipid membranes into 2D ordered arrays has been proposed to be functionally relevant in cellular contexts. To further address this question, we investigated the high-resolution structure of annexin A5 trimers in membrane-bound 2D crystals by cryo-electron microscopy (Cryo-EM). A new 2D crystal form was discovered, with p32(1) symmetry, which is significantly better ordered than the 2D crystals reported before. A 2D projection map was obtained at 6.5 A resolution, revealing protein densities within each of the four domains characteristic of annexins. A quantitative comparison was performed between this structure and models generated from the structure of the soluble form of annexin A5 in pseudo-R3 3D crystals. This analysis indicated that both structures are essentially identical, except for small local changes attributed to membrane binding. As a consequence, and contrary to the common view, annexin A5 molecules maintain their bent shape and do not flatten upon membrane binding, which implies either that the four putative Ca(2+) and membrane-binding loops present different types of interaction with the membrane surface, or that the membrane surface is locally perturbed. We propose that the trimerization of annexin A5 molecules is the relevant structural change occurring upon membrane binding. The evidence that 2D arrays of annexin A5 trimers are responsible for its in vitro property of blood coagulation inhibition supports this conclusion.     

Ligand Binding and Polymerization Characteristics of Human Milk Folate Binding Protein Depend on Concentration of Purified Protein and Presence of Amphiphatic Substances

Two folate binding proteins are present in human milk; one of 27 kDa is a cleavage product of the other one (100 kDa) which possesses a hydrophobic membrane anchor. A drastic change of radioligand binding characteristics and appearance of aggregated weak-radioligand affinity forms on gel filtration occurred at low concentrations of both proteins in the absence of Triton X-100 or other amphiphatic substances, e.g. cetyltrimethylammonium and phospholipids. These findings are consistent with a model predicting association between unliganded and liganded monomers resulting in weak-ligand affinity dimers. Amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers become hydrophilic in the liganded state) thereby preventing association between these monomeric forms prevailing at low concentrations of the protein. Bio-Gel P-300 chromatography of the 27 kDa protein revealed a pronounced polymerization tendency, which diminished with decreasing protein concentrations, however, not in the presence of cetyltrimethylammonium. The data could have some bearings on observations indicating that naturally occurring amphiphatic substances, cholesterol and phospholipids, are necessary for the important clustering of membrane folate receptors.     

Membrane-induced folding and structure of membrane-bound annexin A1 N-terminal peptides: implications for annexin-induced membrane aggregation

Annexins constitute a family of calcium-dependent membrane-binding proteins and can be classified into two groups, depending on the length of the N-terminal domain unique for each individual annexin. The N-terminal domain of annexin A1 can adopt an α-helical conformation and has been implicated in mediating the membrane aggregation behavior of this protein. Although the calcium-independent interaction of the annexin A1 N-terminal domain has been known for some time, there was no structural information about the membrane interaction of this secondary membrane-binding site of annexin A1. This study used circular dichroism spectroscopy to show that a rat annexin A1 N-terminal peptide possesses random coil structure in aqueous buffer but an α-helical structure in the presence of small unilamellar vesicles. The binding of peptides to membranes was confirmed by surface pressure (Langmuir film balance) measurements using phosphatidylcholine/phosphatidylserine monolayers, which show a significant increase after injection of rat annexin A1 N-terminal peptides. Lamellar neutron diffraction with human and rat annexin A1 N-terminal peptides reveals an intercalation of the helical peptides with the phospholipid bilayer, with the helix axis lying parallel to the surface of membrane. Our findings confirm that phospholipid membranes assist the folding of the N-terminal peptides into α-helical structures and that this conformation enables favorable direct interactions with the membrane. The results are consistent with the hypothesis that the N-terminal domain of annexin A1 can serve as a secondary membrane binding site in the process of membrane aggregation by providing a peripheral membrane anchor.     

Mechanism of annexin I-mediated membrane aggregation

It has been proposed that annexin I has two separate interaction sites that are involved in membrane binding and aggregation, respectively. To better understand the mechanism of annexin I-mediated membrane aggregation, we investigated the properties of the inducible secondary interaction site implicated in membrane aggregation. X-ray specular reflectivity measurements showed that the thickness of annexin I layer bound to the phospholipid monolayer was 31 +/- 2 A, indicating that annexin I binds membranes as a protein monomer or monolayer. Surface plasmon resonance measurements of annexin I, V, and mutants, which allowed evaluation of membrane aggregation activity of annexin I separately from its membrane binding, revealed direct correlation between the relative membrane aggregation activity and the relative affinity of the secondary interaction site for the secondary membrane. The secondary binding was driven primarily by hydrophobic interactions, unlike calcium-mediated electrostatic primary membrane binding. Chemical cross-linking of membrane-bound annexin I showed that a significant degree of lateral association of annexin I molecules precedes its membrane aggregation. Taken together, these results support a hypothetical model of annexin I-mediated membrane aggregation, in which a laterally aggregated monolayer of membrane-bound annexin I directly interacts with a secondary membrane via its induced hydrophobic interaction site.     

Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).