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Midori Shima - One of the best experts on this subject based on the ideXlab platform.
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interactions between residues 2228 2240 within factor viiia C2 Domain and factor ixa gla Domain contribute to propagation of clot formation
Thrombosis and Haemostasis, 2011Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Kenichi Ogiwara, Midori ShimaAbstract:Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 Domain (residues 2228–2240) interacts with the FIXa Gla Domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228–2240. The peptide 2228–2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 μM). This effect was significantly greater than that obtained by peptide 1804–1818 (IC50; ~180 μM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228–2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228–2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 Domain (residues 2228–2240) with the FIXa Gla Domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.
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the factor viiia C2 Domain residues 2228 2240 interacts with the factor ixa gla Domain in the factor xase complex
Journal of Biological Chemistry, 2009Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Katsumi Nishiya, Masahiro Takeyama, Kenichi Ogiwara, Yoichi Sakata, Akira Yoshioka, Midori ShimaAbstract:Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 Domain of factor VIIIa and the Gla Domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these Domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 Domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla Domain of factor IXa inhibited binding by approximately 95%, and Gla Domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 Domain interacted with the Gla Domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 Domain constitutes an interactive site for the Gla Domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.
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a factor viii neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 Domain inhibit factor viii binding to von willebrand factor and to phosphatidylserine
Thrombosis and Haemostasis, 1993Co-Authors: Midori Shima, Akira Yoshioka, Dorothea Scandella, Hiroaki Nakai, I Tanaka, Seiki Kamisue, S Terada, Hiromu FukuiAbstract:A neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 Domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 micrograms/ml for IgG and 0.2 micrograms/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 micrograms/ml by IgG and 6.3 micrograms/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 micrograms/ml.
William H Kane - One of the best experts on this subject based on the ideXlab platform.
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location of the multimerin 1 binding site in coagulation factor v an update
Thrombosis Research, 2008Co-Authors: Samira Jeimy, Mary Ann Quinnallen, William H Kane, Nola Fuller, Catherine P M HaywardAbstract:Activated coagulation factor V (FVa) is an important cofactor that accelerates thrombin production. In human blood, 25% of the factor V (FV) is stored in platelets, complexed to the polymeric, FV binding protein multimerin 1 (MMRN1). The light chain of FV is required for MMRN1 binding, and its C2 Domain contains a MMRN1 binding site that overlaps phospholipid binding residues essential for FVa procoagulant function. The homologous structures and roles of the FVa light chain C1 and C2 Domains led us to investigate if the C1 Domain also contains a MMRN1 binding site. The MMRN1 binding properties of FV constructs were tested by modified enzyme-linked immunoassays, before and after thrombin activation. The constructs tested included the combined C1 and C2 Domain deleted FV, and B-Domain deleted forms of FV containing C1 Domain point mutations or combined C1 and C2 Domain phospholipid binding site mutations. The MMRN1 binding site in FV/FVa was mapped to a large region that included the C1 Domain phospholipid binding residues Y1956 and L1957. The FV construct with combined C1 and C2 Domain phospholipid binding site mutations had no MMRN1 binding, highlighting the critical role of the FV C1 and C2 Domain phospholipid binding residues in MMRN1 binding. Our data update the information on the structural features of FV and FVa important for MMRN1 binding, and suggest that the extended MMRN1 binding site in the C1 and C2 Domains is important for the storage of FV-MMRN1 complexes in platelets.
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trp2063 and trp2064 in the factor va C2 Domain are required for high affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex
Biochemistry, 2004Co-Authors: Weimin Peng, Mary Ann Quinnallen, Kenneth A Alexander, William H KaneAbstract:Interactions between factor Va and membrane phosphatidylserine (PS) regulate activity of the prothrombinase complex. Two solvent-exposed hydrophobic residues located in the C2 Domain, Trp2063 and Trp2064, have been proposed to contribute to factor Va membrane interactions by insertion into the hydrophobic membrane bilayer. However, the prothrombinase activity of rHFVa W(2063, 2064)A was found to be significantly impaired only at low concentrations of PS (5 mol %). In this study, we find that 10-fold higher concentrations of mutant factor Va are required for half-maximal prothrombinase activity on membranes containing 25% PS. The ability of the mutant factor Va to interact with factor Xa on a membrane was also impaired since 4-fold higher concentrations of factor Xa were required for half-maximal prothrombinase activity. The interaction of factor Va with 25% PS membranes was also characterized using fluorescence energy transfer and surface plasmon resonance. We found that the affinity of mutant factor Va f...
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identification of functionally important amino acid residues within the C2 Domain of human factor v using alanine scanning mutagenesis
Biochemistry, 2000Co-Authors: Suhng Wook Kim, Mary Ann Quinnallen, Sandra Macedoribeiro, Wolfram Bode, J T Camp, Pablo Fuentesprior, William H KaneAbstract:We have previously determined that the C2-Domain of human factor V (residues 2037−2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. ...
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crystal structures of the membrane binding C2 Domain of human coagulation factor v
Nature, 1999Co-Authors: Sandra Macedoribeiro, Mary Ann Quinnallen, Thomas L Ortel, Wolfram Bode, Robert Huber, Suhng Wook Kim, Gleb Bourenkov, Hans D Bartunik, Milton T Stubbs, William H KaneAbstract:Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 Domains of both cofactors. Here we report two crystal structures of the C2 Domain of human factor Va. The conserved beta-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.
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localization of functionally important epitopes within the second c type Domain of coagulation factor v using recombinant chimeras
Journal of Biological Chemistry, 1994Co-Authors: Thomas L Ortel, Mary Ann Quinnallen, F G Keller, J A Peterson, D Larocca, William H KaneAbstract:Coagulation factor V, an integral component of the prothrombinase complex, possesses two C-type Domains at the carboxyl-terminal end of the molecule. Homologous C-type Domains are present in factor VIII as well as several non-coagulation proteins. Deletion of the second C-type Domain of factor V results in the loss of procoagulant activity and the ability to bind phosphatidylserine. We now report the effect of substitution of all or a portion of the C2 Domain of factor V with the corresponding regions of factor VIII or the human breast carcinoma protein BA46. Substitution of the entire Domain with a heterologous C2 Domain does not restore significant procoagulant activity, although smaller, exon-size substitutions do result in chimeras with partial activity (approximately 10% of factor Va). Using chimeras with partial substitutions, we determined that the amino-terminal region of the Domain is involved in binding to phosphatidylserine. In contrast, the central region of the Domain is not involved in phosphatidylserine binding, but an antibody binding at or near this site inhibits procoagulant activity, suggesting that this region is involved in a separate function. Lastly, the molecular basis for the light chain doublet, which is important in the expression of full procoagulant activity, is located within the carboxyl-terminal region of the C2 Domain.
Joseph J Falke - One of the best experts on this subject based on the ideXlab platform.
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membrane docking geometry and target lipid stoichiometry of membrane bound pkcα C2 Domain a combined molecular dynamics and experimental study
Journal of Molecular Biology, 2010Co-Authors: Kyle E Landgraf, Gregory A Voth, Joseph J FalkeAbstract:Protein kinase Cα (PKCα) possesses a conserved C2 Domain (PKCα C2 Domain) that acts as a Ca2+-regulated membrane targeting element. Upon activation by Ca2+, the PKCα C2 Domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca2+ concentrations, binding of the C2 Domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca2+ concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP2) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCα C2 Domain docked to (i) PS alone and (ii) both PS and PIP2 simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP2 headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP2-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP2:C2 stoichiometry approaching 2:1 at a high PIP2 mole density. Direct binding measurements titrating the C2 Domain with PIP2 in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP2 mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP2 stoichiometries are discussed.
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effect of pip2 binding on the membrane docking geometry of pkcα C2 Domain an epr site directed spin labeling and relaxation study
Biochemistry, 2008Co-Authors: Kyle E Landgraf, Nathan J Malmberg, Joseph J FalkeAbstract:Many diverse cell signaling processes rapidly modulate the levels of intracellular small molecule second messengers in order to spatially and temporally regulate the activity of downstream effectors. Important reactions on membrane surfaces are often stimulated by second messenger signals which recruit signaling enzymes to the appropriate membrane, thereby bringing these enzymes to their membrane-bound substrates. Such membrane recruitment is typically controlled by a membrane targeting Domain activated by the binding of a second messenger, often a signaling lipid or cytoplasmic Ca2+. One the most prevalent membrane targeting motifs is the C2 Domain, which can be activated by Ca2+ binding and is widely found in membrane-targeted signaling proteins [reviewed in refs 1–8]. In a typical cell, transient cytoplasmic Ca2+ signals recruit multiple C2 Domain-containing proteins to specific intracellular membrane surfaces, thereby modulating crucial membrane-associated signaling pathways. The conventional isoforms of protein kinase C (PKCα,1 PKCβ and PKCγ) possess Ca2+-activated C2 Domains which recruit their parent proteins specifically to the inner leaflet of the plasma membrane where they phosphorylate membrane-bound substrate proteins [reviewed in refs 7, 9–13]. Their shared topology consists of an N-terminal pseudosubstrate motif that provides kinase autoinhibition, followed by a pair of C1 Domains that bind the signaling lipid diacylgycerol (DAG), then by a single plasma membrane-targeting C2 Domain, and finally by the C-terminal kinase Domain. The present study focuses on the C2 Domain of the conventional PKCα protein, which binds two Ca2+ ions and associates with two lipids essential for its plasma membrane targeting: phosphatidylserine (PS), the most abundant anionic lipid of the plasma membrane; and phosphatidylinositol-4,5-bisphosphate (PIP2), the most abundant phosphorylated PIP lipid (14–24). The activation of conventional PKCs has been described as a sequential process in which, following a transient Ca2+ signal, the Ca2+-occupied C2 Domain first associates with plasma membrane PS and PIP2, thereby allowing the C1 Domains to search for the more rare DAG messenger (11, 24, 25). Ultimately, the simultaneous binding of the C1 and C2 Domains to their lipid targets displaces the pseudosubstrate from the kinase active site. The ensuing loss of autoinhibition, together with close proximity to membrane-bound substrates, provides dual activation of the kinase Domain. High resolution X-ray crystal structures are available for PKCα C2 Domain and several of its complexes (16, 26). As for other C2 Domains, the core of this Domain is an eight-stranded antiparallel β-sandwich. At one edge of the sandwich lie three interstrand Ca2+- and membrane-binding loops (CMBL1–3). The crystal structure of a complex between PKCα C2 Domain and the PS headgroup reveals that the two bound Ca2+ ions chelated by CMBL1–3 also receive direct and indirect coordination from the 1-phosphate of the PS headgroup (16), indicating that the Ca2+ binding site is central to PS recognition and binding. In addition, the structure of a different PS complex shows that a basic cluster of four lysine residues (K197, K199, K209, K211), all located on the β3-β4 hairpin, directly contacts a second PS head-group, indicating that this basic cluster serves as a second binding site for anionic lipics such as PS (26). Recently, it has been established that the lysine cluster binds PIP2 with higher affinity than PS, and that PIP2 binding is essential for high affinity membrane docking in vitro as well as specific plasma membrane targeting in live cells (20, 22–24). Despite advances in the structural analysis of isolated PKC C2 Domains, the structures of their membrane-docked states remain poorly described. Of particular interest for the conventional PKC C2 Domains is a structural understanding of their simultaneous docking to its two target lipids, PS and PIP2, on a bilayer surface. Currently, high resolution methods are not yet capable of analyzing the structures of peripheral proteins docked to lipid bilayers, thus medium resolution approaches must be employed to generate molecular pictures of peripheral proteins in their active, membrane-bound states. In recent years, an EPR approach involving site-directed spin labeling and spin relaxation measurements (27–29) has been shown to be an effective method for elucidating membrane docking geometries, and has been successfully applied to several C2 Domains (30–35). The docking geometry provided by EPR analysis, in turn, can serve as an experimentally defined starting point for subsequent molecular dynamics simulations designed to develop atomic resolution models of the membrane-docked protein (36). Previously, in an initial study of the PKCα C2 Domain, we used EPR and fluorescence spectroscopies to identify the membrane docking surfaces, and to develop a preliminary model for the membrane docking geometry (37). The resulting model of this C2 Domain docked to 3:1 PC:PS membranes suggested that the CMBLs penetrate the headgroup region of the bilayer, while a cluster of lysine residues lies near the surface of the headgroup region. However, the simplified lipid mixture used in the model target membranes lacked the important target lipid PIP2 as well as other lipids found in the inner leaflet of the plasma membrane. Moreover, this previous study employed a low density of spin label positions on the membrane docking surface, yielding only a preliminary, qualitative picture of the membrane docking geometry. The present study utilizes EPR site-directed spin labeling and relaxation techniques to generate the first medium resolution structural model of the PKCα C2 Domain docked to a lipid bilayer composed of a physiological mixture of lipids. Specifically, the membrane penetration depth and docking angle are determined for the C2 Domain bound to physiological membranes lacking or containing the target lipid PIP2. The results provide the first molecular view of a peripheral membrane protein simultaneously docked to two target lipids, thereby significantly extending our mechanistic understanding of the dual lipid specificity required for plasma membrane targeting by conventional PKC C2 Domains. Moreover, the findings reveal that PIP2 binding to the lysine cluster significantly alters the membrane docking geometry of the C2 Domain by tilting the Domain relative to the membrane surface. This advance demonstrates that the EPR approach can be used to analyze changes in membrane docking geometry triggered by protein switching between different functional states. Finally, the results imply that target lipid-triggered geometry changes could play an important role in modulating the bound state lifetime and function of membrane-bound, conventional PKCs.
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Self-Induced Docking Site of a Deeply Embedded Peripheral Membrane Protein
Biophysical Journal, 2006Co-Authors: Simon Jaud, Joseph J Falke, Douglas J. Tobias, Stephen H. WhiteAbstract:As a first step toward understanding the principles of the targeting of C2 Domains to membranes, we have carried out a molecular dynamics simulation of the C2 Domain of cytosolic phospholipase A2 (cPLA2-C2) in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer at constant pressure and temperature (NPT, 300 K and 1 atm). Using the high-resolution crystal structure of cPLA2-C2 as a starting point, we embedded two copies of the C2 Domain into a preequilibrated membrane at the depth and orientation previously defined by electron paramagnetic resonance (EPR). Noting that in the membrane-bound state the three calcium binding loops are complexed to two calcium ions, we initially restrained the calcium ions at the membrane depth determined by EPR. But the depth and orientation of the Domains remained within EPR experimental errors when the restraints were later removed. We find that the thermally disordered, chemically heterogeneous interfacial zones of phosphatidylcholine bilayers allow local lipid remodeling to produce a nearly perfect match to the shape and polarity of the C2 Domain, thereby enabling the C2 Domain to assemble and optimize its own lipid docking site. The result is a cuplike docking site with a hydrophobic bottom and hydrophilic rim. Contrary to expectations, we did not find direct interactions between the protein-bound calcium ions and lipid headgroups, which were sterically excluded from the calcium binding cleft. Rather, the lipid phosphate groups provided outer-sphere calcium coordination through intervening water molecules. These results show that the combined use of high-resolution protein structures, EPR measurements, and molecular dynamics simulations provides a general approach for analyzing the molecular interactions between membrane-docked proteins and lipid bilayers.
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independent folding and ligand specificity of the C2 calcium dependent lipid binding Domain of cytosolic phospholipase a2
Journal of Biological Chemistry, 1998Co-Authors: Eric A Nalefski, Joseph J Falke, Thomas Mcdonagh, William Stuart Somers, Jasbir Seehra, James D ClarkAbstract:Abstract The Ca2+-dependent lipid binding Domain of the 85-kDa cytosolic phospholipase A2 (cPLA2) is a homolog of C2 Domains present in protein kinase C, synaptotagmin, and numerous other proteins involved in signal transduction. NH2-terminal fragments of cPLA2 spanning the C2 Domain were expressed as inclusion bodies in Escherichia coli, extracted with solvent to remove phospholipids, and refolded to yield a Domain capable of binding phospholipid vesicles in a Ca2+-dependent manner. Unlike other C2 Domains characterized to date, the cPLA2 C2 Domain bound preferentially to vesicles comprised of phosphatidylcholine in response to physiological concentrations of Ca2+. Binding of the cPLA2 C2 Domain to vesicles in the presence of excess Ca2+ chelator was induced by high concentrations of salts that promote hydrophobic interactions. Despite the selective hydrolysis of arachidonyl-containing phospholipid vesicles by cPLA2, the cPLA2 C2 Domain did not discriminate among phospholipid vesicles containing saturated or unsaturated sn-2 fatty acyl chains. Moreover, the cPLA2 C2 Domain bound to phospholipid vesicles containing sn-1 and -2 ether linkages and sphingomyelin at Ca2+ concentrations that caused binding to vesicles containing ester linkages, demonstrating that the carbonyl oxygens of the sn-1 and-2 ester linkage are not critical for binding. These results suggest that the cPLA2C2 Domain interacts primarily with the headgroup of the phospholipid. The cPLA2 C2 Domain displayed selectivity among group IIA cations, preferring Ca2+ approximately 50-fold over Sr2+ and nearly 10,000-fold over Ba2+ for vesicle binding. No binding to vesicles was observed in the presence of greater than 10 mm Mg2+. Such strong selectivity for Ca2+ over Mg2+ reinforces the view that C2 Domains link second messenger Ca2+ to signal transduction events at the membrane.
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the C2 Domain calcium binding motif structural and functional diversity
Protein Science, 1996Co-Authors: Eric A Nalefski, Joseph J FalkeAbstract:The C2 Domain is a Ca(2+)-binding motif of approximately 130 residues in length originally identified in the Ca(2+)-dependent isoforms of protein kinase C. Single and multiple copies of C2 Domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 Domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 Domains are regulated by Ca2+, suggesting that some C2 Domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 Domain and provides a novel sequence alignment of 65 C2 Domain primary structures. This alignment predicts that C2 Domains form two distinct topological folds, illustrated by the recent crystal structures of C2 Domains from synaptotagmin 1 and phosphoinositide-specific phospholipase C-delta 1, respectively. The alignment highlights residues that may be critical to the C2 Domain fold or required for Ca2+ binding and regulation.
Olga Perisic - One of the best experts on this subject based on the ideXlab platform.
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mapping the phospholipid binding surface and translocation determinants of the C2 Domain from cytosolic phospholipase a2
Journal of Biological Chemistry, 1999Co-Authors: Olga Perisic, Hugh F Paterson, Georgina Mosedale, Samuel Laragonzalez, R WilliamsAbstract:Cytosolic phospholipase A2 (cPLA2) plays a key role in the generation of arachidonic acid, a precursor of potent inflammatory mediators. Intact cPLA2 is known to translocate in a calcium-dependent manner from the cytosol to the nuclear envelope and endoplasmic reticulum. We show here that the C2 Domain of cPLA2 alone is sufficient for this calcium-dependent translocation in living cells. We have identified sets of exposed hydrophobic residues in loops known as calcium-binding region (CBR) 1 and CBR3, which surround the C2 Domain calcium-binding sites, whose mutation dramatically decreased phospholipid binding in vitro without significantly affecting calcium binding. Mutation of a residue that binds calcium ions (D43N) also eliminated phospholipid binding. The same mutations that prevent phospholipid binding of the isolated C2 Domain in vitro abolished the calcium-dependent translocation of cPLA2 to internal membranes in vivo, suggesting that the membrane targeting is driven largely by direct interactions with the phospholipid bilayer. Using fluorescence quenching by spin-labeled phospholipids for a series of mutants containing a single tryptophan residue at various positions in the cPLA2 C2 Domain, we show that two of the calcium-binding loops, CBR1 and CBR3, penetrate in a calcium-dependent manner into the hydrophobic core of the phospholipid bilayer, establishing an anchor for docking the Domain onto the membrane.
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calcium dependent membrane penetration is a hallmark of the C2 Domain of cytosolic phospholipase a2 whereas the C2a Domain of synaptotagmin binds membranes electrostatically
Journal of Biological Chemistry, 1998Co-Authors: Bazbek Davletov, Olga Perisic, Roger WilliamsAbstract:C2 Domains have been identified in a wide range of intracellular proteins, including lipid modifying enzymes, protein kinases, GTPases, and proteins involved in membrane trafficking. Many C2 Domains bind membranes in a calcium-dependent manner. The first C2 Domain from synaptotagmin I (SytIC2A) and the C2 Domain from cytosolic phospholipase A2 (cPLA2C2) are among the best characterized C2 Domains in terms of their structures and calcium binding. Here we demonstrate that the protein-lipid interaction is dramatically different for these two Domains. Photolabeling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) in the presence of phospholipid vesicles indicates that cPLA2C2 penetrates into the hydrophobic region of the membrane. Hydrophobic surfaces on cPLA2C2 are exposed even in the absence of calcium, but only in its presence does the Domain penetrate into the nonpolar core of the membrane. The interaction of SytIC2A with phospholipid membranes is primarily electrostatic with binding being abolished in 500 mM NaCl. Because soluble phospholipid head group analogues do not compete with binding of either SytIC2A or cPLA2C2 to vesicles, it is likely that membrane binding by these Domains involves multiple interactions.
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crystal structure of a calcium phospholipid binding Domain from cytosolic phospholipase a2
Journal of Biological Chemistry, 1998Co-Authors: Olga Perisic, Sun Fong, Denise E Lynch, Mark Bycroft, R WilliamsAbstract:Abstract Cytosolic phospholipase A2 (cPLA2) is a calcium-sensitive 85-kDa enzyme that hydrolyzes arachidonic acid-containing membrane phospholipids to initiate the biosynthesis of eicosanoids and platelet-activating factor, potent inflammatory mediators. The calcium-dependent activation of the enzyme is mediated by an N-terminal C2 Domain, which is responsible for calcium-dependent translocation of the enzyme to membranes and that enables the intact enzyme to hydrolyze membrane-resident substrates. The 2.4-A x-ray crystal structure of this C2 Domain was solved by multiple isomorphous replacement and reveals a β-sandwich with the same topology as the C2 Domain from phosphoinositide-specific phospholipase Cδ1. Two clusters of exposed hydrophobic residues surround two adjacent calcium binding sites. This region, along with an adjoining strip of basic residues, appear to constitute the membrane binding motif. The structure provides a striking insight into the relative importance of hydrophobic and electrostatic components of membrane binding for cPLA2. Although hydrophobic interactions predominate for cPLA2, for other C2 Domains such as in “conventional” protein kinase C and synaptotagmins, electrostatic forces prevail.
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a ternary metal binding site in the C2 Domain of phosphoinositide specific phospholipase c delta1
Biochemistry, 1997Co-Authors: Larsoliver Essen, Denise E Lynch, Olga Perisic, Matilda Katan, Roger WilliamsAbstract:We have determined the crystal structures of complexes of phosphoinositide-specific phospholipase C-delta1 from rat with calcium, barium, and lanthanum at 2.5-2.6 A resolution. Binding of these metal ions is observed in the active site of the catalytic TIM barrel and in the calcium binding region (CBR) of the C2 Domain. The C2 Domain of PLC-delta1 is a circularly permuted topological variant (P-variant) of the synaptotagmin I C2A Domain (S-variant). On the basis of sequence analysis, we propose that both the S-variant and P-variant topologies are present among other C2 Domains. Multiple adjacent binding sites in the C2 Domain were observed for calcium and the other metal/enzyme complexes. The maximum number of binding sites observed was for the calcium analogue lanthanum. This complex shows an array-like binding of three lanthanum ions (sites I-III) in a crevice on one end of the C2 beta-sandwich. Residues involved in metal binding are contained in three loops, CBR1, CBR2, and CBR3. Sites I and II are maintained in the calcium and barium complexes, whereas sites II and III coincide with a binary calcium binding site in the C2A Domain of synaptotagmin I. Several conformers for CBR1 are observed. The conformation of CBR1 does not appear to be strictly dependent on metal binding; however, metal binding may stabilize certain conformers. No significant structural changes are observed for CBR2 or CBR3. The surface of this ternary binding site provides a cluster of freely accessible liganding positions for putative phospholipid ligands of the C2 Domain. It may be that the ternary metal binding site is also a feature of calcium-dependent phospholipid binding in solution. A ternary metal binding site might be a conserved feature among C2 Domains that contain the critical calcium ligands in their CBR's. The high cooperativity of calcium-mediated lipid binding by C2 Domains described previously is explained by this novel type of calcium binding site.
Tetsuhiro Soeda - One of the best experts on this subject based on the ideXlab platform.
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interactions between residues 2228 2240 within factor viiia C2 Domain and factor ixa gla Domain contribute to propagation of clot formation
Thrombosis and Haemostasis, 2011Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Kenichi Ogiwara, Midori ShimaAbstract:Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 Domain (residues 2228–2240) interacts with the FIXa Gla Domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228–2240. The peptide 2228–2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 μM). This effect was significantly greater than that obtained by peptide 1804–1818 (IC50; ~180 μM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228–2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228–2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 Domain (residues 2228–2240) with the FIXa Gla Domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.
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the factor viiia C2 Domain residues 2228 2240 interacts with the factor ixa gla Domain in the factor xase complex
Journal of Biological Chemistry, 2009Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Katsumi Nishiya, Masahiro Takeyama, Kenichi Ogiwara, Yoichi Sakata, Akira Yoshioka, Midori ShimaAbstract:Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 Domain of factor VIIIa and the Gla Domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these Domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 Domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla Domain of factor IXa inhibited binding by approximately 95%, and Gla Domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 Domain interacted with the Gla Domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 Domain constitutes an interactive site for the Gla Domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.
