Properties and partial protein sequence of plant annexins

We have examined the characteristics of Ca²⁺-dependent phospholipid-binding proteins (annexins) in maize (Zea mays L.) coleoptiles and tip-growing pollen tubes of Lilium longiflorum. In maize, there are three such proteins, p35, p33, and p23. Partial sequence analysis reveals that peptides from p35 and p33 have identity to members of the annexin family of animal proteins and to annexins from tomato. Interestingly, multiple sequence alignments reveal that the domain responsible for Ca²⁺ binding in animal annexins is not conserved in these plant peptide sequences. Although p33 and p35 share the annexin characteristic of binding to membrane lipid, unlike annexins II and VI they do not associate with detergent-insoluble cytoskeletal proteins or with F-actin from either plants or animals. Immunoblotting with antiserum raised to p33/p35 from maize reveals that cross-reactive polypeptides of 33 to 35 kilodaltons are also present in protein extracts from pollen tubes of L. longiflorum. Immunolocalization at the light microscope level suggests that these proteins are predominantly confined to the nongranular zone at the tube tip, a region rich in secretory vesicles. Our hypothesis that plant annexins mediate exocytotic events is supported by the finding that p23, p33, and p35 bind to these secretory vesicles in a Ca²⁺-dependent manner.     

Identification of α-11 giardin as a flagellar and surface component of Giardia lamblia

Giardia lamblia is a protozoan pathogen with distinct cytoskeletal structures, including median bodies and eight flagella. In this study, we examined components comprising G. lamblia flagella. Crude flagellar extracts were prepared from G. lamblia trophozoites, and analyzed by two-dimensional (2-D) gel electrophoresis. The 19 protein spots were analyzed by MALDI–TOF mass spectrometry, identifying ten metabolic enzymes, six distinct giardins, Giardia trophozoite antigen 1, translational initiation factor eIF-4A, and an extracellular signal-regulated kinase 2. Among the identified proteins, we studied α-11 giardin which belongs to a group of cytoskeletal proteins specific to Giardia. Western blot analysis and real-time PCR indicated that expression of α-11 giardin is not significantly increased during encystation of G. lamblia. Immunofluorescence assays using anti-α-11 giardin antibodies revealed that α-11 giardin protein mainly localized to the plasma membranes and basal bodies of the anterior flagella of G. lamblia trophozoites, suggesting that α-11 giardin is a genuine component of the G. lamblia cytoskeleton.     

Crystal-structure and biochemical characterization of recombinant human calcyphosine delineates a novel EF-hand-containing protein family

Calcyphosine is an EF-hand protein involved in both Ca^2 +-phosphatidylinositol and cyclic AMP signal cascades, as well as in other cellular functions. The crystal structure of Ca^2 +-loaded calcyphosine was determined up to 2.65 Å resolution and reveals a protein containing two pairs of Ca^2 +-binding EF-hand motifs. Calcyphosine shares a highly similar overall topology with calmodulin. However, there are striking differences between EF-hand 4, both N-terminal and C-terminal regions, and interdomain linkers. The C-terminal domain of calcyphosine possesses a large hydrophobic pocket in the presence of calcium ions that might be implicated in ligand binding, while its N-terminal hydrophobic pocket is almost shielded by an additional terminal helix. Calcyphosine is largely monomeric, regardless of the presence of Ca^2 +. Differences in structure, oligomeric state in the presence and in the absence of Ca^2 +, a highly conserved sequence with low similarity to other proteins, and phylogeny define a new EF-hand-containing family of calcyphosine proteins that extends from arthropods to humans.     

Induction of annexin by heavy metals and jasmonic acid in Zea mays

Plant annexins are Ca²⁺- and phospholipid-binding proteins forming an evolutionary conserved multi-gene family. They are implicated in the regulation of plant growth, development, and stress responses. With the availability of the maize genome sequence information, we identified 12 members of the maize annexin genes. Analysis of protein sequence and gene structure of maize annexins led to their classification into five different orthologous groups. Expression analysis by RT-PCR revealed that these genes are responsive to heavy metals (Ni, Zn, and Cd). The maize annexin genes were also found to be regulated by Ustilago maydis and jasmonic acid. Additionally, the promoter of the maize annexin gene was analyzed for the presence of different stress-responsive cis-elements, such as ABRE, W-box, GCC-box, and G-box. RT-PCR and microarray data show that all 12 maize annexin genes present differential, organ-specific expression patterns in the maize developmental steps. These results indicate that maize annexin genes may play important roles in the adaptation of plants to various environmental stresses.     

Directly observed hydrogen bonds at calcium-binding-sites of calmodulin in solution relate to affinity of the calcium-binding

Molecular tuning to calcium-binding in the EF-hand motif of holo-calmodulin was studied in solution by NMR ^h3J_NC′ H-bond couplings. In the N-terminus lobe of holo-calmodulin, the glutamate crucial for Ca²⁺ coordination has network of H-bonds weaker than inferred from the X-ray crystal structure. This glutamate at position 12 appears shifted away from the Ca²⁺ preferred coordination, which can explain the lower affinity of the calcium-binding to the N-terminus with respect to C-terminus EF hands.     

Tensin2 reduces intracellular phosphatidylinositol 3,4,5-trisphosphate levels at the plasma membrane

Tensins are proposed cytoskeleton-regulating proteins. However, Tensin2 additionally inhibits Akt signalling and cell survival. Structural modelling of the Tensin2 phosphatase (PTPase) domain revealed an active site-like pocket receptive towards phosphoinositides. Tensin2-expressing HEK293 cells displayed negligible levels of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) under confocal microscopy. However, mock-transfected cells, and Tensin2 cells harbouring a putative phosphatase-inactivating mutation, exhibited significant PtdIns(3,4,5)P₃ levels, which decreased upon phosphatidylinositol 3-kinase inhibition with LY294002. In contrast, wtTensin3, mock and mutant cells were identical in membrane PtdIns(3,4,5)P₃ and Akt phosphorylation. In vitro lipid PTPase activity was however undetectable in isolated recombinant PTPase domains of both Tensins, indicating a possible loss of structural stability when expressed in isolation. In summary, we provide evidence that Tensin2, in addition to regulating cytoskeletal dynamics, influences phosphoinositide-Akt signalling through its PTPase domain.     

Spatial distribution of cytoplasmic domains of the Mg-transporter MgtE,in a solution lacking Mg,revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg²⁺ transporter that controls cellular Mg²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg²⁺-free and Mg²⁺-bound forms. The Mg²⁺-binding sites lay at the interface of the 2 domains, making the Mg²⁺-bound form compact and globular. In the Mg²⁺-free structure, however, the domains are far apart, and the Mg²⁺-binding sites are destroyed. Therefore, it is unclear how Mg²⁺-free MgtE changes its conformation to accommodate Mg²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg²⁺ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg²⁺-bound crystal structure, facilitating efficient capture of Mg²⁺ with increased intracellular Mg²⁺ concentration, which is necessary to close the gate.     

Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of Ca²⁺-bound LipL32 was determined at 2.3 Å resolution. LipL32 has a novel polyD sequence of seven aspartates that forms a continuous acidic surface patch for Ca²⁺ binding. A significant conformational change was observed for the Ca²⁺-bound form of LipL32. Calcium binding to LipL32 was determined by isothermal titration calorimetry. The binding of fibronectin to LipL32 was observed by Stains-all CD and enzyme-linked immunosorbent assay experiments. The interaction between LipL32 and fibronectin might be associated with Ca²⁺ binding. Based on the crystal structure of Ca²⁺-bound LipL32 and the Stains-all results, fibronectin probably binds near the polyD region on LipL32. Ca²⁺ binding to LipL32 might be important for Leptospira to interact with the extracellular matrix of the host cell.     

Calcium-dependent phospholipid-binding proteins in plants

There is evidence that Ca²⁺ can regulate vesicle-mediated secretion in plant cells, but the mechanism for this is not known. One possibility is that Ca²⁺ -dependent phospholipid-binding proteins (annexins) couple the Ca²⁺ stimulus to the exocytotic response. Using a protocol developed for the isolation of animal annexins we have identified proteins in maize (Zea mays L.) coleoptiles that have similar characteristics to annexins. The predominant polypeptide species run as a doublet of relative molecular mass (M_r) 33000–35000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); another less-abundant protein of M_r 23000 is also present. In the presence of Ca²⁺ these proteins bind to liposomes composed of acidic phospholipids. Calcium-sensitivity of binding differs for each protein and is also influenced by the pH of the buffer used for the liposome-binding assay. Antiserum raised to the 33 to 35-kDa doublet purified on SDS-PAGE recognises the doublet in crude extracts from maize and proteins of similar M_r in Tradescantia virginiana and tobacco Nicotiana tabacum L. The antiserum also recognises p68 (Annexin VI) from chicken gizzard extracts, indicating homology between animal annexins and the maize proteins. For the maize proteins to be involved in the regulation of exocytosis, binding to phospholipids would be expected to occur at physiological levels of Ca²⁺. The characteristics of the maize annexin-like proteins are described and attention drawn to the marked effect of pH in lowering the requirement for Ca²⁺ for phospholipid binding.Abbreviations DEAE diethylaminoethyl - EGTA ethylene glycol-bis (-aminoethyiether)-N,N,N,N-tetraacetic acid - kDa kilodalton(s) - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis This work was funded by the Agricultural and Food Research Council. Our thanks also to Professor P. Lowry and Dr R. Woods, Department of Biochemistry, University of Reading for facilities and advice for antiserum production, and C. Boustead, Department of Biochemistry, University of Leeds for advice on immunoblotting and phospholipid-binding assays.     

Millisecond time-resolved changes occurring in Ca2+-regulated myosin filaments upon relaxation

Contraction of many muscles is activated in part by the binding of Ca²⁺ to, or phosphorylation of, the myosin heads on the surface of the thick filaments. In relaxed muscle, the myosin heads are helically ordered and undergo minimal interaction with actin. On Ca²⁺ binding or phosphorylation, the head array becomes disordered, reflecting breakage of the head-head and other interactions that underlie the ordered structure. Loosening of the heads from the filament surface enables them to interact with actin filaments, bringing about contraction. On relaxation, the heads return to their ordered positions on the filament backbone. In scallop striated adductor muscle, the disordering that takes place on Ca²⁺ binding occurs on the millisecond time scale, suggesting that it is a key element of muscle activation. Here we have studied the reverse process. Using time-resolved negative staining electron microscopy, we show that the rate of reordering on removal of Ca²⁺ also occurs on the same physiological time scale. Direct observation of images together with analysis of their Fourier transforms shows that activated heads regain their axial ordering within 20 ms and become ordered in their final helical positions within 50 ms. This rapid reordering suggests that reformation of the ordered structure, and the head-head and other interactions that underlie it, is a critical element of the relaxation process.     

Kinetics and ion specificity of Na/Ca exchange mediated by the reconstituted beef heart mitochondrial Na/Ca antiporter

The Na⁺/Ca²⁺ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na⁺ or Ca²⁺. Na⁺/Ca²⁺ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca²⁺ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K_m for Ca²⁺. When present on the same side as Ca²⁺, K⁺ activated exchange by lowering the K_m for Ca²⁺ from 2  to 0.9 μM. The K_m for Na⁺ was 8 mM. In the absence of Ca²⁺, the exchanger catalyzed high rates of Na⁺/Li⁺ and Na⁺/K⁺ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na⁺/Ca²⁺ and Na⁺/K⁺ exchange with IC₅₀ values of 10 and 0.6 μM, respectively. The V_max for Na⁺/Ca²⁺ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.     

Effect of Serine Phosphorylation and Ser25 Phospho-Mimicking Mutations on Nuclear Localisation and Ligand Interactions of Annexin A2

Annexin A2 (AnxA2) interacts with numerous ligands, including calcium, lipids, mRNAs and intracellular and extracellular proteins. Different post-translational modifications participate in the discrimination of the functions of AnxA2 by modulating its ligand interactions. Here, phospho-mimicking mutants (AnxA2-S25E and AnxA2-S25D) were employed to investigate the effects of Ser25 phosphorylation on the structure and function of AnxA2 by using AnxA2-S25A as a control. The overall α-helical structure of AnxA2 is not affected by the mutations, since the thermal stabilities and aggregation tendencies of the mutants differ only slightly from the wild-type (wt) protein. Unlike wt AnxA2, all mutants bind the anxA2 3′ untranslated region and β-γ-G-actin with high affinity in a Ca^2 +-independent manner. AnxA2-S25E is not targeted to the nucleus in transfected PC12 cells. In vitro phosphorylation of AnxA2 by protein kinase C increases its affinity to mRNA and inhibits its nuclear localisation, in accordance with the data obtained with the phospho-mimicking mutants. Ca^2 +-dependent binding of wt AnxA2 to phosphatidylinositol, phosphatidylinositol-3-phosphate, phosphatidylinositol-4-phosphate and phosphatidylinositol-5-phosphate, as well as weaker but still Ca^2 +-dependent binding to phosphatidylserine and phosphatidylinositol-3,5-bisphosphate, was demonstrated by a protein–lipid overlay assay, whereas binding of AnxA2 to these lipids, as well as its binding to liposomes, is inhibited by the Ser25 mutations. Thus, introduction of a modification (mutation or phosphorylation) at Ser25 appears to induce a conformational change leading to increased accessibility of the mRNA- and G-actin-binding sites in domain IV independent of Ca^2 + levels, while the Ca^2 +-dependent binding of AnxA2 to phospholipids is attenuated.     

Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges

S100A3, a member of the EF-hand-type Ca²⁺-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn²⁺ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca²⁺/Zn²⁺ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A + C68A) abolished the Ca²⁺ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca²⁺ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A + C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15−1.40 Å resolution shows that SS1 renders the C-terminal classical Ca²⁺-binding loop flexible, which are essential for its Ca²⁺ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn²⁺ by (Cys)₃His residues that is compatible with SS2 formation in S100A3.     

Mechanism of the Ca-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA

The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca²⁺ binds to the EF2 domain of S100A4 with micromolar affinity and that the K_d value for Ca²⁺ is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in K_d results from a reduced dissociation rate constant (from 16 s^− 1 to 0.3 s^− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca²⁺]. However, for Ca²⁺ transients to be effective in further promoting dissociation, the elevated Ca²⁺ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein-myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.     

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

Annexin A2 (AnxA2) is a Ca²⁺- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca²⁺ 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 Ca²⁺-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 Ca²⁺.     

Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca²⁺ signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca²⁺-specific) and structural (Ca²⁺- or Mg²⁺-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca²⁺ binding using NMR and EF-hand mutants. Ca²⁺ titration, as monitored by [¹⁵N,¹H] heteronuclear single quantum coherence, suggests that Ca²⁺ binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg²⁺ binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca²⁺-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca²⁺ and not Mg²⁺. Ca²⁺ binding induces conformational rearrangements in the protein by reversing Mg²⁺-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg²⁺ with Ca²⁺ reduces the Ca²⁺-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca²⁺-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg²⁺-bound NCS-1 are similar, the early unfolding transitions of Ca²⁺-bound NCS-1 are partially influenced in the presence of Mg²⁺. This study demonstrates the importance of Mg²⁺ as a modulator of calcium homeostasis and active-state behavior of NCS-1.     

Acid sensing ion channels regulate neuronal excitability by inhibiting BK potassium channels

Acid sensing ion channels (ASICs), Ca²⁺ and voltage-activated potassium channels (BK) are widely present throughout the central nervous system. Previous studies have shown that when expressed together in heterologous cells, ASICs inhibit BK channels, and this inhibition is relieved by acidic extracellular pH. We hypothesized that ASIC and BK channels might interact in neurons, and that ASICs may regulate BK channel activity. We found that ASICs inhibited BK currents in cultured wild-type cortical neurons, but not in ASIC1a/2/3 triple knockout neurons. The inhibition in the wild-type was partially relieved by a drop in extracellular pH to 6. To test the consequences of ASIC-BK interaction for neuronal excitability, we compared action potential firing in cultured cortical neurons from wild-type and ASIC1a/2/3 null mice. We found that in the knockout, action potentials were narrow and exhibited increased after-hyperpolarization. Moreover, the excitability of these neurons was significantly increased. These findings are consistent with increased BK channel activity in the neurons from ASIC1a/2/3 null mice. Our data suggest that ASICs can act as endogenous pH-dependent inhibitors of BK channels, and thereby can reduce neuronal excitability.