Phosphorylation of annexin A1 by TRPM7 kinase: a switch regulating the induction of an α-helix

TRPM7 is an unusual bifunctional protein consisting of an α-kinase domain fused to a TRP ion channel. Previously, we have identified annexin A1 as a substrate for TRPM7 kinase and found that TRPM7 phosphorylates annexin A1 at Ser5 within the N-terminal α-helix. Annexin A1 is a Ca(2+)-dependent membrane binding protein, which has been implicated in membrane trafficking and reorganization. The N-terminal tail of annexin A1 can interact with either membranes or S100A11 protein, and it adopts the conformation of an amphipathic α-helix upon these interactions. Moreover, the existing evidence indicates that the formation of an α-helix is essential for these interactions. Here we show that phosphorylation at Ser5 prevents the N-terminal peptide of annexin A1 from adopting an α-helical conformation in the presence of membrane-mimetic micelles as well as phospholipid vesicles. We also show that phosphorylation at Ser5 dramatically weakens the binding of the peptide to S100A11. Our data suggest that phosphorylation at Ser5 regulates the interaction of annexin A1 with membranes as well as S100A11 protein.     

Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins.

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.     

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins

Annexin II is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca(2+) in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1-27) to the conserved protein core of annexin I transfers the capability of Ca(2+)-independent membrane binding to the otherwise Ca(2+)-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca(2+)-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.     

Staphylococcal superantigen-like protein 10 binds to phosphatidylserine and apoptotic cells

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins that have structural similarities to staphylococcal superantigens. Although SSLs do not have superantigenic activity, some of them have been reported to bind to host immune related molecules and they have been implicated in immune evasion by S. aureus. In this study, we showed that SSL10 is capable of binding to phospholipids. SSL10 bound to phosphatidylserine (PS) containing liposome, but not to phosphatidylcholine liposome. SSL10, but not SSL7, bound to PS containing liposome, suggesting that SSL10 specifically binds to PS. Analysis of PS binding ability among recombinant truncated SSL10 fragments revealed that the β-barrel in the N-terminal oligonucleotide/oligosaccharide-binding (OB)-fold domain contributes to PS binding capacity. Fluorescein isothiocyanate labeled OB-fold of SSL10 stained hydrogen peroxide treated Jurkat cells. Annexin V is widely utilized for detection of apoptosis. Unlike annexin V, the OB-fold domain of SSL10 also bound to apoptotic cells in the presence of EDTA, suggesting that the OB-fold of SSL10 recognizes PS and apoptotic cells in a Ca(2+) independent manner. These findings suggest SSL10 and its derived peptides may be a novel detection tool for apoptotic cells.     

Structural characterization of a dimeric complex between the short cytoplasmic domain of CEACAM1 and the pseudo tetramer of S100A10-Annexin A2 using NMR and molecular dynamics

AIIt, a heterotetramer of S100A10 (P11) and Annexin A2, plays a key role in calcium dependent, membrane associations with a variety of proteins. We previously showed that AIIt interacts with the short cytoplasmic domain (12 amino acids) of CEACAM1 (CEACAM1-SF). Since the cytoplasmic domains of CEACAM1 help regulate the formation of cis- or trans-dimers at the cell membrane, we investigated the possible role of their association with AIIt in this process. Using NMR and molecular dynamics, we show that AIIt and its pseudoheterodimer interacts with two molecules of short cytoplasmic domain isoform peptides, and that interaction depends on the binding motif 454-Phe-Gly-Lys-Thr-457 where Phe-454 binds in a hydrophobic pocket of AIIt, the null mutation Phe454Ala reduces binding by 2.5 fold, and the pseudophosphorylation mutant Thr457Glu reduces binding by three fold. Since these two residues in CEACAM1-SF were also found to play a role in the binding of calmodulin and G-actin at the membrane, we hypothesize a sequential set of three interactions are responsible for regulation of cis- to trans-dimerization of CEACAM1. The hydrophobic binding pocket in AIIt corresponds to a previously identified binding pocket for a peptide found in SMARCA3 and AHNAK, suggesting a conserved functional motif in AIIt allowing multiple proteins to reversibly interact with integral membrane proteins in a calcium dependent manner.     

Identification of a functionally relevant signal peptide of mouse ficolin A

Mouse ficolin A is a plasma protein with lectin activity, and plays a role in host defense by binding carbohydrates, especially GlcNAc, on microorganisms. The ficolin A subunit consists of an N-terminal signal peptide, a collagen-like domain, and a C-terminal fibrinogen-like domain. In this study, we show that ficolin A can be synthesized and oligomerized in a cell and secreted into culture medium. We also identify a functionally relevant signal peptide of ficolin A by using MS/MS analysis to determine the N-terminal sequence of secreted ficolin A. When the signal peptide of mouse ficolin A was fused with enhanced green fluorescent protein (EGFP), EGFP was released into HEK 293 cell medium, suggesting that the signal peptide can efficiently direct ficolin A secretion. Moreover, our results suggest that the signal peptide of ficolin A has potential application for the production of useful secretory proteins.     

The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo

Although the N-terminal BOX-I domain of the tumor suppressor protein p53 contains the primary docking site for MDM2, previous studies demonstrated that RNA stabilizes the MDM2.p53 complex using a p53 mutant lacking the BOX-I motif. In vitro assays measuring the specific activity of MDM2 in the ligand-free and RNA-bound state identified a novel MDM2 interaction site in the core domain of p53. As defined using phage-peptide display, the RNA.MDM2 isoform exhibited a notable switch in peptide binding specificity, with enhanced affinity for novel peptide sequences in either p53 or small nuclear ribonucleoprotein-U (snRNP-U) and substantially reduced affinity for the primary p53 binding site in the BOX-I domain. The consensus binding site for the RNA.MDM2 complex within p53 is SGXLLGESXF, which links the S9-S10 beta-sheets flanking the BOX-IV and BOX-V motifs in the core domain and which is a site of reversible conformational flexibility in p53. Mutation of conserved amino acids in the linker at Ser(261) and Leu(264), which bridges the S9-S10 beta-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe(270) within the S10 beta-sheet resulted in a mutant p53, which binds more stably to RNA.MDM2 complexes in vitro and which is strikingly hyper-ubiquitinated in vivo. Introducing an Ala(19) mutation into the p53(F270A) protein abolished both RNA.MDM2 complex binding and hyper-ubiquitination in vivo, thus indicating that p53(F270A) protein hyper-ubiquitination depends upon MDM2 binding to its primary site in the BOX-I domain. Together, these data identify a novel MDM2 binding interface within the S9-S10 beta-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.     

A putative DNA-binding domain in the NUCKS protein

We have studied the DNA-binding properties of a NUCKS-derived, synthetic peptide containing an extended GRP motif. This peptide binds to random-sequence DNA, but did not bind preferentially to poly(dA-dT). A synthetic peptide with the same amino acid composition but with a random sequence did not bind to DNA, suggesting that the structure of the DNA-binding domain plays a pivotal role in the interaction with DNA. NMR and graphic modeling were employed to investigate the structure of the synthetic peptide. It was shown that the DNA-binding peptide constituted an alpha helix in phosphate buffer at pH 5.5. Docking results indicated an almost perfect fit for this small, helical peptide into the major groove of DNA with the possibility of four basic residues interacting with the phosphate backbone of DNA. One consensus site for phosphorylation by Cdk1 is located in the N-terminal end of the DNA-binding peptide. Upon phosphorylation of this site, the binding to DNA was completely prohibited. Immunofluorescence experiments showed that NUCKS was located in the nuclei in proliferating cells in interphase of the cell cycle, but was distributed throughout the cytoplasm in mitotic cells.     

Structure of the Ca2+/S100B/NDR kinase peptide complex: insights into S100 target specificity and activation of the kinase

NDR, a nuclear serine/threonine kinase, belongs to the subfamily of Dbf2 kinases that is critical to the morphology and proliferation of cells. The activity of NDR kinase is modulated in a Ca(2+)/S100B-dependent manner by phosphorylation of Ser281 in the catalytic domain and Thr444 in the C-terminal regulatory domain. S100B, which is a member of the S100 subfamily of EF-hand proteins, binds to a basic/hydrophobic sequence at the junction of the N-terminal regulatory and catalytic domains (NDR(62-87)). Unlike calmodulin-dependent kinases, regulation of NDR by S100B is not associated with direct autoinhibition of the active site, but rather involves a conformational change in the catalytic domain triggered by Ca(2+)/S100B binding to the junction region. To gain further insight into the mechanism of activation of the kinase, studies have been carried out on Ca(2+)/S100B in complex with the intact N-terminal regulatory domain, NDR(1-87). Multidimensional heteronuclear NMR analysis showed that the binding mode and stoichiometry of a peptide fragment of NDR (NDR(62-87)) is the same as for the intact N-terminal regulatory domain. The solution structure of Ca(2+)/S100B and NDR(62-87) has been determined. One target molecule is found to associate with each subunit of the S100B dimer. The peptide adopts three turns of helix in the bound state, and the complex is stabilized by both hydrophobic and electrostatic interactions. These structural studies, in combination with available biochemical data, have been used to develop a model for calcium-induced activation of NDR kinase by S100B.     

NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.

In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small N-terminal domain. The solution structure of one of the N-terminal domains from Saccharomyces cerevisiae RNase HI, determined using NMR spectroscopy, is presented. The 46 residue motif comprises a three-stranded antiparallel beta-sheet and two short alpha-helices which pack onto opposite faces of the beta-sheet. Conserved residues involved in packing the alpha-helices onto the beta-sheet form the hydrophobic core of the domain. Three highly conserved and solvent exposed residues are implicated in RNA binding, W22, K38 and K39. The beta-beta-alpha-beta-alpha topology of the domain differs from the structures of known RNA binding domains such as the double-stranded RNA binding domain (dsRBD), the hnRNP K homology (KH) domain and the RNP motif. However, structural similarities exist between this domain and the N-terminal domain of ribosomal protein L9 which binds to 23 S ribosomal RNA.     

Simian virus 40 large T antigen binds a novel Bcl-2 homology domain 3-containing proapoptosis protein in the cytoplasm

A 193-kDa SV40 large T antigen (T-Ag)-binding protein, designated p193, was identified and cloned. Inspection of the deduced amino acid sequence revealed the presence of a short motif similar to the Bcl-2 homology (BH) domain 3, suggesting that p193 may be a member of a family of apoptosis promoting proteins containing only BH3 motifs. In support of this, p193 expression promoted apoptosis in NIH-3T3 cells. Deletion of the BH3 motif abolished p193 apoptosis activity. p193-induced apoptosis was antagonized by co-expression of Bcl-X(L). Immune cytologic analysis indicated that p193 is localized to the cytoplasm of transfected cells. p193-induced apoptosis was also antagonized by co-expression of T-Ag, which resulted in the cytoplasmic localization of both proteins. The p193 binding site was mapped to an N-terminal region of T-Ag previously implicated in transforming activity. These results suggest that T-Ag possesses an antiapoptosis activity, independent of p53 sequestration, which is actuated by T-Ag/p193 binding in the cytoplasm.     

Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53

In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53(1-320)) and C-terminal (p53(73-393)) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319-393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 +/- 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1(357-381) (residues 357-381 in mouse p53) and YF-O2(320-346) (residues 320-346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.     

Solution structure of the phosphotyrosine binding (PTB) domain of human tensin2 protein in complex with deleted in liver cancer 1 (DLC1) peptide reveals a novel peptide binding mode

The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.     

The N-terminal domains of cyclin-dependent kinase inhibitory proteins block the phosphorylation of cdk2/Cyclin E by the CDK-activating kinase

It has been suggested that binding of p27 and p21 kinase inhibitory proteins (KIPs) to cyclin-dependent kinases (cdks) render them inaccessible to cdk-activating kinase (CAK), presumably by steric hindrance by the C-terminal residues. However, this common mechanism of inhibition is inconsistent with the known structural divergence in the p27 and p21 C-terminal domains. Therefore, we studied the direct binding of N-terminal minimal domain of p27 (amino acids 28-81) to cdk2/cyclin E. An unlabeled p27 minimal domain, mutated in the N-terminal LFG motif, was unable to compete with a labeled minimal domain for binding to cdk2/cyclin E. The p27 and its minimal domain inhibited CAK-mediated phosphorylation of cdk2/cyclin E. This inhibitory effect was significantly diminished with p27 minimal domain mutated in the LFG motif. A synthetic peptide, ACRRLFGPVDSE, from the N-terminal residues 17-28 of p21, was also a potent inhibitor of CAK-mediated cdk2/cyclin E phosphorylation. Taken together, these results show that anchoring of p27 or p21 KIPs to cyclin E via the N-terminal LFG-containing motif can block CAK access to its cdk2/cyclin E substrate.     

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2^S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg⁹⁷/Lys⁹⁸, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.     

Two motifs within a transmembrane domain, one for homodimerization and the other for heterodimerization

Protein assembly is a critical process involved in a wide range of cellular events and occurs through extracellular and/or transmembrane domains (TMs). Previous studies demonstrated that a GXXXG motif is crucial for homodimer formation. Here we selected the TMs of ErbB1 and ErbB2 as a model since these receptors function both as homodimers and as heterodimers. Both TMs contain two GXXXG-like motifs located at the C and N termini. The C-terminal motifs were implicated previously in homodimer formation, but the role of the N-terminal motifs was not clear. We used the ToxR system and expressed the TMs of both ErbB1 and ErbB2 containing only the N-terminal GXXXG motifs. The data revealed that the ErbB2 but not the ErbB1 construct formed homodimers. Importantly, a synthetic ErbB1 TM peptide was able to form a heterodimer with ErbB2, by displacing the ErbB2 TM homodimer. The specificity of the interaction was demonstrated by using three controls: (i) Two single mutations within the GXXXG-like motif of the ErbB1 peptide reduced or preserved its activity, in agreement with similar mutations in glycophorin A. (ii) A TM peptide of the bacterial Tar receptor did not assemble with the ErbB2 construct. (iii) The ErbB1 peptide had no effect on the dimerization of a construct containing the TM-1 domain of the Tar receptor. Fluorescence microscopy demonstrated that all the peptides localized on the membrane. Furthermore, incubation with the peptides had no effect on bacterial growth and protein expression levels. Our results suggest that the N-terminal GXXXG-like motif of the ErbB1 TM plays a role in heterodimerization with the ErbB2 transmembrane domain. To our knowledge, this is the first demonstration of a transmembrane domain with two distinct recognition motifs, one for homodimerization and the other for heterodimerization.     

The S100A10-annexin A2 complex provides a novel asymmetric platform for membrane repair

Membrane repair is mediated by multiprotein complexes, such as that formed between the dimeric EF-hand protein S100A10, the calcium- and phospholipid-binding protein annexin A2, the enlargeosome protein AHNAK, and members of the transmembrane ferlin family. Although interactions between these proteins have been shown, little is known about their structural arrangement and mechanisms of formation. In this work, we used a non-covalent complex between S100A10 and the N terminus of annexin A2 (residues 1-15) and a designed hybrid protein (A10A2), where S100A10 is linked in tandem to the N-terminal region of annexin A2, to explore the binding region, stoichiometry, and affinity with a synthetic peptide from the C terminus of AHNAK. Using multiple biophysical methods, we identified a novel asymmetric arrangement between a single AHNAK peptide and the A10A2 dimer. The AHNAK peptide was shown to require the annexin A2 N terminus, indicating that the AHNAK binding site comprises regions on both S100A10 and annexin proteins. NMR spectroscopy was used to show that the AHNAK binding surface comprised residues from helix IV in S100A10 and the C-terminal portion from the annexin A2 peptide. This novel surface maps to the exposed side of helices IV and IV' of the S100 dimeric structure, a region not identified in any previous S100 target protein structures. The results provide the first structural details of the ternary S100A10 protein complex required for membrane repair.     

The molecular chaperone, ClpA, has a single high affinity peptide binding site per hexamer

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.     

Insight into the location and dynamics of the annexin A2 N-terminal domain during Ca(2+)-induced membrane bridging

Annexin A2 (AnxA2) is a Ca(2+)- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca(2+) binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth of data exists on the structure and dynamics of the core, but little is known about the N-terminal domain especially in the Ca(2+)-induced membrane-bridging process. To investigate this protein region in the monomeric AnxA2 and in the heterotetramer (AnxA2-p11)(2), the reactive Cys8 residue was specifically labelled with the fluorescent probe acrylodan and the interactions with membranes were studied by steady-state and time-resolved fluorescence. In membrane junctions formed by the (AnxA2-p11)(2) heterotetramer, the flexibility of the N-terminal domain increased as compared to the protein in solution. In "homotypic" membrane junctions formed by monomeric AnxA2, acrylodan moved to a more hydrophobic environment than in the protein in solution and the flexibility of the N-terminal domain also increased. In these junctions, this domain is probably not in close contact with the membrane surface, as suggested by the weak quenching of acrylodan observed with doxyl-PCs, but pairs of N-termini likely interact, as revealed by the excimer-forming probe pyrene-maleimide bound to Cys8. We present a model of monomeric AnxA2 N-terminal domain organization in "homotypic" bridged membranes in the presence of Ca(2+).