Phosphatidylserine Binding Sites Research Articles

Microparticle Phosphatidylserine Mediates Coagulation: Involvement in Tumor Progression and Metastasis

Tumor progression and cancer metastasis has been linked to the release of microparticles (MPs), which are shed upon cell activation or apoptosis and display parental cell antigens, phospholipids such as phosphatidylserine (PS), and nucleic acids on their external surfaces. In this review, we highlight the biogenesis of MPs as well as the pathophysiological processes of PS externalization and its involvement in coagulation activation. We review the available evidence, suggesting that coagulation factors (mainly tissue factor, thrombin, and fibrin) assist in multiple steps of tumor dissemination, including epithelial-mesenchymal transition, extracellular matrix remodeling, immune escape, and tumor angiogenesis to support the formation of the pre-metastatic niche. Platelets are not just bystander cells in circulation but are functional players in primary tumor growth and metastasis. Tumor-induced platelet aggregation protects circulating tumor cells (CTCs) from the blood flow shear forces and immune cell attack while also promoting the binding of CTCs to endothelial cells and extravasation, which activates tumor invasion and sustains metastasis. Finally, in terms of therapy, lactadherin can inhibit coagulation by competing effectively with coagulation factors for PS binding sites and may similarly delay tumor progression. Furthermore, we also investigate the therapeutic potential of coagulation factor inhibitors within the context of cancer treatment. The development of multiple therapies targeting platelet activation and platelet-tumor cell interactions may not only reduce the lethal consequences of thrombosis but also impede tumor growth and spread.

Stoichiometric analysis reveals a unique phosphatidylserine binding site in coagulation factor X

BackgroundCellular trauma or activation exposes phosphatidylserine (PS) and the substantially more abundant phospholipid, phosphatidylethanolamine (PE), on the outer layer of the plasma membrane, thereby allowing binding of many blood clotting proteins. We previously proposed the Anything But Choline (ABC) hypothesis to explain how PS and PE synergize to support binding of clotting proteins with gamma‐carboxyglutamate (Gla)‐rich domains, which posited that each Gla domain binds to a limited number of PS molecules and multiple PE molecules. However, the minimal number of PS molecules required to stably bind a Gla‐domain–containing blood clotting protein in the presence of excess PE was unknown. ObjectiveTo test the ABC hypothesis for factor X by determining the threshold binding requirement of PS molecules under conditions of PS–PE synergy. MethodsWe used surface plasmon resonance to investigate the stoichiometry of factor X binding to nanoscale membrane bilayers (Nanodiscs) of varying phospholipid composition. Results and ConclusionsWe quantified 1.05 ± 0.2 PS molecules per bound factor X molecule in Nanodiscs containing a mixture of 10% PS, 60% PE, and 30% phosphatidylcholine. Hence, there appears to be one truly PS‐specific binding site per Gla domain, while the remaining membrane binding interactions can be satisfied by PE.

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced cell binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

Abstract 2753: The molecular basis of blocking the TIM-3 checkpoint with the LY3321367 mAb in cancer immunotherapy

Abstract The anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody LY3321367 is a promising immune-checkpoint inhibitor that is being clinically tested for the treatment of multiple solid tumors (NCT03099109). LY3321367 was phenotypically selected from a large set of phage library Fabs that are TIM-3 binders. Tim3 has multiple protein and non-protein binding partners; hence, we attempt in this study to deconvolute the molecular mechanism of action of LY3321367. Surprisingly, LY3321367 in ELISA assays partially blocks the TIM-3/GAL-9 complex but does not block the TIM-3/CEACAM-1 complex. We also utilized X-ray crystallography to solve the structure the LY3321367-Fab complex with the IgV domain of TIM-3 at 2.0Å resolution. After solving the unbound structure of the LY3321367-Fab, we demonstrated that its CDRs do not undergo any conformational changes upon binding human TIM-3. The structural alignment of the human TIM-3/LY3321367-Fab complex with the available murine TIM-3 structure unexpectedly suggests that the 6.0Å epitope of LY3321367 overlaps the phosphatidylserine (PS) binding site on TIM-3. Consequently, we developed a cell-based assay to demonstrate that LY3321367 completely blocks the binding of soluble human TIM-3 to PS displayed on the surface of Campothecin-treated DO11.10 cells. Our structural results suggest an important role for PS in TIM-3 biology which is blocked by LY3321367. When combined with the partial blocking of GAL-9, the structural data of LY3321367 suggests that the binding sites of PS and GAL-9 may be proximal to each other on TIM-3. Citation Format: Jaafar N. Haidar, Stephen Antonysamy, Sneha Mathew, Lan Wu, Yi Zhang, Margaret C. Kearins, Leyi Shen, J. Michael Sauder, David Schaer, Kyla E. Driscoll, Michael Kalos. The molecular basis of blocking the TIM-3 checkpoint with the LY3321367 mAb in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2753.

Coated platelets introduce significant delay in onset of peak thrombin production: Theoretical predictions

Platelets play a crucial role in the initiation, progress, termination as well as regulation of blood coagulation. Recent studies have confirmed that not all but only a small percentage of thrombin-activated platelets (“coated” platelets) exhibit procoagulant properties (namely the expression of phosphatidylserine binding sites) required for the acceleration and progress of coagulation. A mechanistic model is developed for in vitro coagulation whose key features are distinct equations for coated platelets, thrombin dose-dependence for coated platelets, and competitive binding of coagulation factors to platelet membrane. Model predictions show significant delay in the onset of peak Va production, and peak thrombin production when dose-dependence is incorporated instead of a fixed theoretical maximum percentage of coated platelets. Further, peak thrombin concentration is significantly overestimated when either fractional presence of coated platelets is ignored (by 299.4%) or when dose-dependence on thrombin is ignored (by 24.7%).

The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles

The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.

Effect of GABRA1 Cytoplasmic Peptide on a Regulatory Protein

The pentameric neurotransmitter-gated chloride channel, GABA (type A) receptor, is well-recognized for phasic and tonic neuronal inhibition. The phasic response at the synapse is due to receptors typically containing two alpha, two beta, and one gamma subunit. Half of all GABA (type A) receptors in adult human brain have the alpha (type 1), or alpha-1, subunit (gene ID: GABRA1). Each consists of an extracellular ligand-binding domain and four transmembrane helices (TM1-4), both of which are highly conserved, as well as a large intracellular loop domain (ILD), whose structure is unknown. The human receptor is crystallized without the ILD, which connects TM3 and TM4, that is engineered out. The topology and function of the 86-residue alpha-1 ILD remains to be fully elucidated. GAPDH (d-glyceraldehyde-3-phosphate dehydrogenase) phosphorylates specific ILD residues, regulating receptor function. In this study, we examined the effect of a synthesized ILD peptide (residues 335 to 364 from GABRA1) on the oxidoreductase activity of GAPDH. The custom peptide, containing the first 30 cytosolic residues, was pre-incubated with GAPDH, and enzyme activity measured by NADH absorbance (340nm) upon oxidation of the substrate d-glyceraldehyde. When pre-incubated for 1hr at 20degC, the ILD peptide inhibited enzyme activity in a concentration-dependent manner showing an exponential non-linear function (y=a∗e ˆ (b/x+c)). The half-maximum inhibition occurred at a [peptide] = 8.7micromolar. When pre-incubated for 48hr at 4degC, the ILD peptide further enhanced inhibition of oxidoreductase activity, suggesting that hydrophobic forces play a role in the interaction between the ILD peptide and GAPDH. Molecular modeling experiments indicate that the ILD binding site on GAPDH is in close proximity to the phosphatidylserine-binding site on GAPDH. The findings may contribute to our understanding of receptor function and point to possible treatments in conditions, such as Alzheimer's dementia, where GABA receptor pharmacology is shown to play a role.

Phosphatidylserine on blood cells and endothelial cells contributes to the hypercoagulable state in cirrhosis.

The mechanism of thrombogenicity in cirrhosis is largely unknown. Our objective was to study the relationship between phosphatidylserine on blood cells and endothelial cells and the hypercoagulable state in cirrhotic patients. Patients with cirrhosis and healthy controls were studied. Lactadherin was used to quantify phosphatidylserine exposure on blood cells and endothelial cells. Procoagulant activity of cells was evaluated using clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. Phosphatidylserine exposure, fibrin strands and FVa/Xa binding on cells were observed using confocal microscopy. Our study showed that phosphatidylserine exposure on erythrocytes, platelets and leucocytes in cirrhotic patients increased progressively with Child-Pugh categories. In addition, we found that endothelial cells treated with cirrhotic serum in vitro exposed more phosphatidylserine than those exposed to healthy serum. The exposed phosphatidylserine supported a shorter coagulation time and increased FXa, thrombin and fibrin formation. Notably, phosphatidylserine+ erythrocytes also promoted shorter coagulation times and more fibrin generation in cirrhotic microparticle-depleted plasma, regardless of Child-Pugh categories. Confocal microscopy data showed that the FVa/FXa complex and fibrin fibrils colocalized with phosphatidylserine on endothelial cells. Lactadherin significantly inhibited FXa and thrombin generation and consequently decreased fibrin production in normal or cirrhotic plasma. These results lead us to believe that exposed phosphatidylserine on activated or injured erythrocytes, platelets, leucocytes and endothelial cells plays an important role in the hypercoagulable state in cirrhotic patients. Thus, blocking phosphatidylserine binding sites might be a new therapeutic target for preventing thrombosis.

The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation.

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.

Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release.

The mechanisms contributing to an increased risk of thrombosis in uremia are complex and require clarification. There is scant morphological evidence of membrane-dependent binding of factor Xa (FXa) and factor Va (FVa) on endothelial cells (EC) in vitro. Our objectives were to confirm that exposed phosphatidylserine (PS) on microparticle (MP), EC, and peripheral blood cell (PBC) has a prothrombotic role in uremic patients and to provide visible and morphological evidence of PS-dependent prothrombinase assembly in vitro. We found that uremic patients had more circulating MP (derived from PBC and EC) than controls. Additionally, patients had more exposed PS on their MPs and PBCs, especially in the hemodialysis group. In vitro, EC exposed more PS in uremic toxins or serum. Moreover, reconstitution experiments showed that at the early stages, PS exposure was partially reversible. Using confocal microscopy, we observed that PS-rich membranes of EC and MP provided binding sites for FVa and FXa. Further, exposure of PS in uremia resulted in increased generation of FXa, thrombin, and fibrin and significantly shortened coagulation time. Lactadherin, a protein that blocks PS, reduced 80% of procoagulant activity on PBC, EC, and MP. Our results suggest that PBC and EC in uremic milieu are easily injured or activated, which exposes PS and causes a release of MP, providing abundant procoagulant membrane surfaces and thus facilitating thrombus formation. Blocking PS binding sites could become a new therapeutic target for preventing thrombosis.

Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins.

Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, 31P-NMR, electron paramagnetic resonance (EPR), proton NMR (1H-NMR), deuterium NMR (2H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Neutral Phospholipids Stimulate Na,K-ATPase Activity: A SPECIFIC LIPID-PROTEIN INTERACTION

Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of αTM8-10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human α1β1 or α1β1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of α1β1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments αTM8-10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid.

Apolipoprotein E3 (ApoE3) but Not ApoE4 Protects against Synaptic Loss through Increased Expression of Protein Kinase Cϵ

Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid β oligomers (amylospheroids). Specific activators of PKCε, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCε inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCε mRNA and expression of the PKCε protein. Amyloid β specifically blocked the expression of PKCε but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCε pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD.

Snake Cytotoxins Bind to Membranes via Interactions with Phosphatidylserine Head Groups of Lipids

The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of cells.

Engineered Annexin A5 Variants Have Impaired Cell Entry for Molecular Imaging of Apoptosis Using Pretargeting Strategies

Phosphatidylserine (PS) on apoptotic cells is a target for diagnosis and therapy using annexin A5 (anxA5). Pretargeting is a strategy developed to improve signal to background ratio for molecular imaging and to minimize undesired side effects of pharmacological and radiotherapy. Pretargeting relies on accessibility of the target finder on the surface of the target cell. anxA5 binds PS and crystallizes in a two-dimensional network covering the PS-expressing cell surface. Two-dimensional crystallization is the driving force for anxA5 internalization by PS-expressing cells. Here, we report structure/function analysis of anxA5 internalization. Guided by structural bioinformatics including protein-protein docking, we revealed that the amino acids Arg(63), Lys(70), Lys(101), Glu(138), Asp(139), and Asn(160) engage in intermolecular salt bridges within the anxA5 trimer, which is the basic building block of the two-dimensional network. Disruption of the salt bridges by site-directed mutagenesis does not affect PS binding but inhibits trimer formation and cell entry of surface-bound anxA5. The anxA5 variants with impaired internalization are superior molecular imaging agents in pretargeting strategies as compared with wild-type anxA5.

Phosphatidylserine binding is important for plasma membrane targeting of 3′‐phosphoinositide‐dependent kinase‐1 pleckstrin homology domain

3′‐Phosphoinositide dependent kinase (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide‐3‐kinase activation. An increase in phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol‐3,4‐bisphosphate (PtdIns(3,4)P2) is thought to recruit PDK1 and its substrates to the 3′‐phosphoinositide‐rich regions of the plasma membrane for subsequent phosphorylation. PDK1 contains a C‐terminal catalytic domain and an N‐terminal pleckstrin homology (PH) domain that was reported to have high affinity for PtdIns(3,4,5)P3 and PtdIns(3,4)P2. To better understand how lipids regulate the subcellular localization and activation of PDK1, we rigorously investigated the membrane binding properties of PDK1 PH domain by surface plasmon resonance analysis. The results show that the PDK1 PH domain has high affinity for not only PtdIns(3,4,5)P3, PtdIns(3,4)P2 and phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P2) but also for phosphatidylserine (PS). We identified the separate binding sites for phosphoinositides and PS through molecular modeling and mutagenesis and analyzed the differential effects of phosphoinositides and PS in membrane recruitment of PDK1.Supported by a NIH grant GM68849.

The PT1−Ca2+ Gla Domain Binds to a Membrane through Two Dipalmitoylphosphatidylserines. A Computational Study

Binding of vitamin K-dependent proteins to cell membranes containing phosphatidylserine (PS) via gamma-carboxyglutamic acid (Gla) domains is one of the essential steps in the blood coagulation pathway. During activation of the coagulation cascade, prothrombin is converted to thrombin by prothrombinase, a complex consisting of serine protease FXa and cofactor FVa, anchored to anionic phospholipids on the surface of activated platelets in the presence of calcium ions. To investigate the binding of the Gla domain of prothrombin fragment 1 (PT1) to anionic lipids in the presence of Ca2+, we have conducted MD simulations of the protein with one and two dipalmitoylphosphatidylserines (DPPS) in a dipalmitoylphosphatidylcholine (DPPC) bilayer membrane. The results show a well-defined phosphatidylserine binding site, which agrees generally with crystallographic studies [Huang, M., et al. (2003) Nat. Struct. Biol. 10, 751-756]. However, in the presence of the lipid membrane, some of the interactions observed in the crystal structure adjust during the simulations possibly because in our system the PT1-Ca2+ complex is embedded in a DPPC lipid membrane. Our simulations confirm the existence of a second phospholipid headgroup binding site on the opposite face of the PT1-Ca2+ complex as suggested by MacDonald et al. [(1997) Biochemistry 36, 5120-5127]. The serine headgroup in the second site binds through a Gla domain-bound calcium ion Ca1, Gla30, and Lys11. On the basis of free energy simulations, we estimate the energy of binding of the PT1-Ca2+ complex to a single DPPS to be around -11.5 kcal/mol. The estimated free energy of binding of a DPPS lipid to the second binding site is around -8.8 kcal/mol and is in part caused by the nature of the second site and in part by entropic effects.

A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex

AbstractTightly associated factor Va (FVa) and factor Xa (FXa) serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)–containing platelet membranes during blood coagulation. We have previously shown that (1) a soluble form of PS (C6PS) triggers assembly of a fully active FVa-FXa complex in solution and (2) that 2 molecules of C6PS bind to FVa light chain with one occupying a site in the C2 domain. We expressed human factor Va (rFVa) with mutations in either the C1 domain (Y1956,L1957)A, the C2 domain (W2063,W2064)A, or both C domains (Y1956,L1957,W2063,W2064)A. Mutations in the C1 and C1-C2 domains of rFVa reduced the rate of activation of prothrombin to thrombin by FXa in the presence of 400 μM C6PS by 14 000- to 15 000-fold relative to either wild-type or C2 mutant factor rFVa. The Kd's of FXa binding with rFVa (wild-type, C2 mutant, C1 mutant, and C1-C2 mutant) were 3, 4, 564, and 624 nM, respectively. Equilibrium dialysis experiments detected binding of 4, 3, and 2 molecules of C6PS to wild-type rFVa, C1-mutated, and C1,C2-mutated rFVa, respectively. Because FVa heavy chain binds 2 molecules of C6PS, we conclude that both C2 and C1 domains bind one C6PS, with binding to the C1 domain regulating prothrombinase complex assembly.

Localization of Stereo-Specific Phosphatidylserine Binding Sites within the Factor V C2 Domain.

Binding of activated factor V (Fva) to phosphatidylserine (PS) is required for formation of the prothrombinase complex on the surface of activated platelets. The interaction between Fva and PS has been shown to be stereo-specific with bovine Fva favoring L-PS over D-PS. Our previous low resolution alanine scan data localized PS binding sites to the FV-C2 domain and identified two tryptophan residues, W2063 and W2064, which are required for high affinity binding. Two putative PS binding sites were also predicted based on modeling studies using the FV-C2 structure. In this study, the stereo-specific interaction of Fva with PS was evaluated with plasma derived human Fva (pd-HFVa), recombinant human Fva (rHFVa), rHFVa-C2 domain and rHFVa mutants. Quantitative fluorescence binding assays and functional prothrombinase assays were conducted using phospholipid vesicles containing 20 mol% dioleoyl phosphatidyl-L-serine (L-PS) or dioleoyl phosphatidyl-D-serine (D-PS). Direct binding experiments demonstrated that pd-HFVa and rHFVa bound to membranes containing L-PS with 6 to 9 fold higher affinity than to membranes containing D-PS. Analysis of prothrombinase activity on these membranes demonstrated the difference in Vmax/K1/2Va between L-PS and D-PS was 15-fold and 23-fold higher for pd-HFVa and rHFVa, respectively. These data confirm that HFVa, similar to bovine FVa, prefers L-PS over D-PS. The rHFV-C2 domain bound to L-PS membranes with an affinity of 5.3 ± 1.7 nM. In contrast, the binding of C2 domain to D-PS membranes was undetectable. This indicates that the rHFV-C2 domain contains a stereo-specific binding site for PS. We next analyzed the membrane binding of four rHFVa mutants containing alanine substitutions within the FV-C2 domain. rHFVa mutants W2063A and W2064A both bound to membranes containing 20% L-PS with high affinity similar to native rHFVa with Kd's of 0.97 ± 0.30 nM and 0.38 ± 0.18 nM, respectively. In contrast no detectable binding of W2063A or W2064A could be detected on membranes containing 20% D-PS. Mutants were also prepared containing alanine substitutions for amino acid side chains predicted to participate in two stereospecific PS binding sites based on modeling studies using the rHFV-C2 structure. The binding of (K2060, Q2085, S2115)A and (Q2078, N2082, R2187)A to L-PS and D-PS were characterized as described above. These mutants bound to both L-PS and D-PS membranes with relatively high affinity. The Kd values for binding of (K2060, Q2085, S2115)A and (Q2078, N2082, R2187)A to 20% D-PS membranes were 1.19 ± 0.31 nM and 1.11 ± 0.32 nM, respectively. These results suggest that these two sites play little or no role in the binding of FVa to PS membranes. In conclusion, the HFV-C2 domain contains stereo-specific binding sites for L-PS which require the indole side chains of W2063 and W2064. These findings provide new insights into the regulation of prothrombinase activity by PS.