Localization of phosphatidylserine binding sites to structural domains of factor Xa.

Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor X(a) by 60-fold (Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Biochemistry 35, 7482-7491). In the present study, we locate three C6PS binding sites to different domains of factor X(a) using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor X(a). Our results demonstrate that the structural responses of human and bovine factor X(a) to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor X(a) are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the gamma-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca(2+) (k(d) approximately 1 mm), although this PS site does not influence the functional response of factor X(a). Second, a Ca(2+)-dependent binding site is in the epidermal growth factor domains (EGF(NC)) that are linked by Ca(2+) and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor X(a). Third, a Ca(2+)-requiring site seems to be in the EGF(C)-catalytic domain. This site appears not to be a lipid regulatory site but rather to share residues with the substrate recognition site. Finally, the full functional response to C6PS requires linkage of the Gla, EGF(NC), and catalytic domains in the presence of Ca(2+), meaning that PS regulation of factor X(a) involves linkage between widely separated parts of the protein.     

Interaction of vasopressin with phosphatidylserine bilayers

Vasopressin causes a decrease in electrical resistance of phosphatidylserine bilayers. The magnitude of the decrease is a function of vasopressin and salt concentrations. The conducting channel is produced probably by aggregation of 4–5 molecules of the hormone.     

Crystallization of phosphatidylserine bilayers induced by lithium

Utilizing differential scanning calorimetry and x-ray diffraction, 1,2-dimyristoyl-L-glycero-3-phospho-L-serine (DMPS) was shown to form hydrated bilayer membrane structures exhibiting a gel leads to liquid crystalline transition at 39 degrees C (delta H = 7.2 kcal/mol). Addition of up to molar concentrations of the alkali halides NaCl, KCl, Rl Cl, and CsCl produced relatively minor changes in DMPS bilayer structure or stability. For example, in the presence of 0.5 M NaCl, the transition temperature (Tc = 42 degrees C) and transition enthalpy (delta H = 7.0 kcal/mol) show only minor changes. In marked contrast, addition of LiCl results in "'crystallization" of the DMPS bilayer membrane structure. At 0.5 M LiCl, the crystalline DMPS exhibits a bilayer gel leads to liquid crystal transition at 89 degrees C accompanied by a high enthalpy change, delta H = 16.0 kcal/mol. Thus, Li+ induces an isothermal crystallization of DMPS bilayers, the hydrocarbon chains adopting a more ordered packing mode than the "hexagonal" arrangement of the gel state. In view of the widespread use of lithium in the treatment of manic-depressive illness, we also raise the possibility that interaction of Li+ with anionic membrane phospholipids could play a role in its pharmacological action.     

Energetic and structural aspects of ethidium cation intercalation into DNA minihelices

The enthalpy ΔH for the intercalation of the ethidium cation (EC) into DNA minihelices can be decomposed into (1) an energy of conformational adjustment (i.e., the energy of minihelix extension and unwinding from the B-form to the intercalated form) and (2) EC minihelix intermolecular interactions. In the present study, we have focused our attention mainly on a decomposition of the energetic factors of the EC minihelix intermolecular interactions, while the essential features of the energy of conformational adjustment have been discussed in detail elsewhere by us. The structural features of the various resulting energy-minimized EC-intercalated complexes are compared with each other and the initial x-ray model structure. ΔH is estimated to be in the range of ?12.3 to ?24.0 kcal/mol. This theoretical estimate is qualitatively and quantitatively in agreement with a variety of available experimental data. The energy of conformational adjustment is an energetically unfavorable step, while the energetically favorable contribution of the EC minihelix intermolecular interactions is responsible for the overall favorable nature of the intercalation process involving the EC. On the other hand, the observed preference for intercalation into Pyr(3′–5′)Pur DNA sequences over their isomeric Pur(3′–5′)Pyr sequences is controlled by the energy of conformational adjustment and not by the EC minihelix intermolecular interaction contribution. No base-composition effect is expected at EC concentrations normally found at cellular conditions. Moreover, the structural features of the various EC-intercalated complexes are very similar regardless of minihelix base sequence or composition. These results compare favorably with available evidence. The nature of biologically preferred sites of EC binding with the minihelices is discussed.     

Divalent cation binding to ceruloplasmin

Binding of calcium to human and sheep ceruloplasniin was investigated by metal substitution with manganese and competitive displacement of bound manganese by calcium monitored by electron paramagnetic resonance spectroscopy. The K _d for calcium was found to be 1.4mm. Magnesium also bound to ceruloplasmin, with K _d = 0.3 and 0.7 mm for the human and sheep protein, respectively. The thermal stability of ceruloplasmin, as studied by differential scanning calorimetry, was affected by calcium but not by magnesium. A considerable increase of the T _m value, from 73.8 to 83.1°C, was observed for sheep ceruloplasmin in the presence of calcium. The T _m value of the human protein was only slightly altered by calcium (from 85.1 to 87°C). The interaction of ceruloplasmin with the chromatographic material used for its isolation, Sepharose 4B derivatized with chloroethylamine, was weakened by calcium. This allowed us to set up a novel purification scheme that made it possible to efficiently isolate ceruloplasmin and prothrombin from plasma with the same single-step chromatography.     

Clustering of lecithin molecules in phosphatidylserine membranes induced by calcium ion binding to phosphatidylserine

The effects of calcium ion on phosphatidyl L-serine (PS) have been studied with PS membranes containing a lecithin spin label (L¹). The calcium ion makes the ESR spectra of the L¹ in PS membranes broadened owing to the intermolecular spin-spin exchange interactions. The results indicate that the calcium ion binds to PS molecules to form rapidly rigid calcium ion-bound PS aggregates, the lecithin molecules being thereby separated from the host PS bilayers to form clusters. The magnesium ion is ineffective for the aggregation and exerts a quite different effect only at much higher concentrations.     

Comparative structural analyses of corticosteroid binding globulin

Some physicochemical characteristics of corticosteroid binding globulin (CBG) in several species have been determined. Molecular radii were determined from Ferguson plots and were used in conjunction with sedimentation coefficients determined by sucrose density gradient centrifugation to calculate the molecular weights of the CBG. These were found to range from 44,200 (dog) to 60,000 (turtle) for most species. The squirrel monkey was found to have a molecular weight twice that of other species (119,800). Purified CBG was prepared from human, rat, and guinea pig sera. The molecular weights of the purified material, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate, were in excellent agreement with those determined by Ferguson analysis. Careful examination of the purified proteins by electrophoresis at pH 8.3 revealed that each consisted of two closely related electrophoretic variants. Tryptic peptides were prepared from the purified proteins and separated by reversed phase HPLC chromatography. The peptide patterns were identical for the three proteins with the exception of three hydrophilic peptides. Amino terminal sequence analysis of the rat and human proteins revealed no apparent homology, however. The immunologic relatedness of the three purified proteins was also examined, but no crossreactivity was observed. The results obtained suggest that while the molecular size and hydrophobicity of peptides have been conserved across species considerable surface differences must exist.     

Specific RNA binding to ordered phospholipid bilayers

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions.     

Role of structural water for prediction of cation binding sites in apoproteins

Structures of many metal-binding proteins are often obtained without structural cations in their apoprotein forms. Missing cation coordinates are usually updated from structural templates constructed from many holoprotein structures. Such templates usually do not include structural water, the important contributor to the ion binding energy. Structural templates are also inconvenient for taking into account structural modifications around the binding site at apo-/holo- transitions. An approach based upon statistical potentials readily takes into account structural modifications associated with binding as well as contribution of structural water molecules. Here, we construct a set of statistical potentials for Mg²⁺, Ca²⁺, and Zn²⁺ contacting with protein atoms of a different type or structural water oxygens. Each type of the cations tends to form tight contacts with protein atoms of specific types. Structural water contributes relatively more into the binding pseudo-energy of Mg²⁺ and Ca²⁺ than of Zn²⁺. We have developed PIONCA (Protein-Ion Calculator), a fast CUDA GPGPU-based algorithm that predicts ion-binding sites in apoproteins. Comparative tests demonstrate that PIONCA outperforms most of the tools based on structural templates or docking. Our software can be also used for locating bound cations in holoprotein structures with missing cation heteroatoms. PIONCA is equipped with an interactive web interface based upon JSmol.     

Comparative structural analysis of lipid binding START domains

Background

Steroidogenic acute regulatory (StAR) protein related lipid transfer (START) domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease.

Principal Findings

We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains.

Significance

Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity.

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The binding of organic ions to phospholipid bilayers

The binding of organic anions and cations, mainly tetraphenylboride and tetraphenylarsonium, to phospholipid membranes has been studied using an NMR method. Binding is appreciable and is affected by cholesterol in the membrane and counterions in solution. The passage of the organic anions through the membrane has also been followed. These measurements indicate that it is naive to use organic anions to measure membrane potentials in a simple manner.     

Effect of organic mercury on the electrical resistance of phosphatidylserine bilayers

Acidic phospholipids have been shown to form complexes with methyl mercury chloride, at physiological pH, in vitro. To check if this interaction had any effect on the physical properties of membranes made with these lipids, the specific resistance of phosphatidylserine bilayers was monitored, as a function of time, in the absence and in the presence of methyl mercury chloride in the bathing solution. While the resistance of the bilayer remained constant in the absence of the toxic, it dropped an average of 17% in four hours in the presence of 100 microM methyl mercury chloride. Such observations suggest that the physical integrity of these membranes is modified by the interaction with organic mercury. This result may be relevant to the observed degeneration of nerve membranes in Minamata disease.     

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.     

Calcium ion binding between lipid bilayers: the four-component system of phosphatidylserine, phosphatidylcholine, calcium chloride, and water

Ca2+ binding between lamellae of phosphatidylserine (PS) and phosphatidylcholine (PC) gives rise to a rigid phase of Ca(PS)2. When aqueous Ca2+, hydrated PS/PC, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant and is characteristic of the PS/PC ratio. This characteristic Ca2+ concentration is 0.040 microM for palmitoyloleoylphosphatidylserine without PC and increases as the inverse square of the PS mole fraction at high PS concentration (Raoult's law) and as the inverse square of the PS mole fraction multiplied by a constant at low PS concentration (Henry's law). For example, for palmitoyloleoylphosphatidylserine/palmitoyloleoylphosphatidylcholi ne = 0.6/0.4 or 0.2/0.8, this characteristic Ca2+ concentration is about 0.1 or about 6 microM, respectively. These observations at constant temperature are summarized in a quaternary phase diagram for the four-component system CaCl2/PS/PC/water.     

Structure and fluctuations of charged phosphatidylserine bilayers in the absence of salt

Using x-ray diffraction and NMR spectroscopy, we present structural and material properties of phosphatidylserine (PS) bilayers that may account for the well documented implications of PS headgroups in cell activity. At 30 degrees C, the 18-carbon monounsaturated DOPS in the fluid state has a cross-sectional area of 65.3 A(2) which is remarkably smaller than the area 72.5 A(2) of the DOPC analog, despite the extra electrostatic repulsion expected for charged PS headgroups. Similarly, at 20 degrees C, the 14-carbon disaturated DMPS in the gel phase has an area of 40.8 A(2) vs. 48.1 A(2) for DMPC. This condensation of area suggests an extra attractive interaction, perhaps hydrogen bonding, between PS headgroups. Unlike zwitterionic lipids, stacks of PS bilayers swell indefinitely as water is added. Data obtained for osmotic pressure versus interbilayer water spacing for fluid phase DOPS are well fit by electrostatic interactions calculated for the Gouy-Chapman regime. It is shown that the electrostatic interactions completely dominate the fluctuational pressure. Nevertheless, the x-ray data definitively exhibit the effects of fluctuations in fluid phase DOPS. From our measurements of fluctuations, we obtain the product of the bilayer bending modulus K(C) and the smectic compression modulus B. At the same interbilayer separation, the interbilayer fluctuations are smaller in DOPS than for DOPC, showing that B and/or K(C) are larger. Complementing the x-ray data, (31)P-chemical shift anisotropy measured by NMR suggest that the DOPS headgroups are less sensitive to osmotic pressure than DOPC headgroups, which is consistent with a larger K(C) in DOPS. Quadrupolar splittings for D(2)O decay less rapidly with increasing water content for DOPS than for DOPC, indicating greater perturbation of interlamellar water and suggesting a greater interlamellar hydration force in DOPS. Our comparisons between bilayers of PS and PC lipids with the same chains and the same temperature enable us to focus on the effects of these headgroups on bilayer properties.     

Calorimetric investigation of polymyxin binding to phosphatidic acid bilayers

The cooperative binding process between the antibiotic peptide polymyxin-B and negatively-charged phosphatidic acid bilayers was investigated by differential thermal analysis and completed by fluorescence polarization measurements. The sigmoidal binding curves were analyzed in terms of the interaction energy within a domain formed by polymyxin and phosphatidic acid molecules. The formation of such a heterogeneous domain structure was favoured by high concentration of external monovalent ions. The cooperativity of the binding increased while a charge-induced decrease in the phase transition temperature of the pure lipid phase was observed with increasing ion concentration at a given pH. The reduced lateral coupling within the lipid bilayer in the presence of salt ions, as demonstrated by an increase in the lipid phase transition enthalpy, was considered to facilitate the cooperative domain formation. Moreover, an increase in the cooperativity of the polymyxin binding could be observed if phosphatidic acids of smaller chain length and thus of a lowered phase transition temperature were used. By the use of chemically-modified polymyxin we were able to demonstrate the effect of electrostatic and hydrophobic interaction. Acetylated polymyxin with a reduced positive charge was used to demonstrate the pure hydrophobic effect of polymyxin binding leading to a decrease in the phosphatidic acid phase transition temperature by about 20°C. The cooperativity of the binding was strongly reduced. Cleavage of the hydrophobic polymyxin tail yielded a colistinnonapeptide which caused an electrostatically-induced increase in the phosphatidic acid phase transition temperature. With unmodified polymyxin we observed the combined effects of electrostatic as well as hydrophobic interaction making this model system interesting for the understanding of lipid-protein interactions. Evidence is presented that the formation of the polymyxin-phosphatidic acid complex is a lateral phase separation phenomenon.     

Calorimetric investigation of polymyxin binding to phosphatidic acid bilayers

The cooperative binding process between the antibiotic peptide polymyxin-B and negatively-charged phosphatidic acid bilayers was investigated by differential thermal analysis and completed by fluorescence polarization measurements. The sigmoidal binding curves were analyzed in terms of the interaction energy within a domain formed by polymyxin and phosphatidic acid molecules. The formation of such a heterogeneous domain structure was favoured by high concentration of external monovalent ions. The cooperativity of the binding increased while a charge-induced decrease in the phase transition temperature of the pure lipid phase was observed with increasing ion concentration at a given pH. The reduced lateral coupling within the lipid bilayer in the presence of salt ions, as demonstrated by an increase in the lipid phase transition enthalpy, was considered to facilitate the cooperative domain formation. Moreover, an increase in the cooperativity of the polymyxin binding could be observed if phosphatidic acids of smaller chain length and thus of a lowered phase transition temperature were used. By the use of chemically-modified polymyxin we were able to demonstrate the effect of electrostatic and hydrophobic interaction. Acetylated polymyxin with a reduced positive charge was used to demonstrate the pure hydrophobic effect of polymyxin binding leading to a decrease in the phosphatidic acid phase transition temperature by about 20 degrees C. The cooperativity of the binding was strongly reduced. Cleavage of the hydrophobic polymyxin tail yielded a colistinnonapeptide which caused an electrostatically-induced increase in the phosphatidic acid phase transition temperature. With unmodified polymyxin we observed the combined effects of electrostatic as well as hydrophobic interaction making this model system interesting for the understanding of lipid-protein interactions. Evidence is presented that the formation of the polymyxin-phosphatidic acid complex is a lateral phase separation phenomenon.     

Divalent cation binding to wheat germ calmodulin

The Ca2+ binding to plant (wheat germ) calmodulin was measured in 0.1 M NaCl by a flow-dialysis method. The four macroscopic binding constants best fitted to the data were 0.20, 0.25, 0.025, and 0.0024 microM-1. The cysteine residue of this calmodulin is located at the 27th position from the NH2-terminal (Yazawa, M. et al. (1982) Abstr. 33th Conf. Protein Structure pp. 9-12, Osaka). According to the quantitative analysis of the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with Cys 27, the calmodulin which binds 3 Ca2+ showed the minimum reactivity with DTNB. This suggests that the site for the third Ca2+ binding is located close to Cys 27. Cys 27 was spin-labeled with N-(2,2,6,6-tetramethyl-4-piperidine-1-oxyl)maleimide, and its ESR spectrum was measured in the presence of Mn2+ and/or Ca2+. The rotational relaxation time of the label (1.2 ns) was increased by about one-tenth with 1 to 2 mol of bound Ca2+, but was unchanged with Mn2+. On the other hand, Mn2+ induced a remarkable quenching of the spectrum. From the decrease in the peak heights of the ESR spectrum, the distance between the label and the first bound Mn2+ was estimated to be 0.8 nm. It is concluded that the first Mn2+ binds to a domain near the NH2-terminal. The difference UV absorption spectrum induced by Mn2+ was similar to that induced by Ca2+. However, the amount of Mn2+ needed to saturate the difference spectrum was 1 mol more than the amount of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane capacity measurements suggest a calcium-dependent insertion of synexin into phosphatidylserine bilayers

The mechanism by which synexin mediates calcium-dependent aggregation of medullary cell chromaffin granules and fusion of granule ghosts involves specific interactions with the lipid component of the membrane. To study the details of these interactions we measured synexin-induced changes in capacitance of phosphatidylserine bilayers formed at the tip of a patch pipet using the double-dip method. Provided calcium was present in the solution filling the pipet (10-50 mM) stable phosphatidylserine bilayers were easily formed. Addition of synexin (0.1 microgram/ml) to an external medium lacking added calcium induced no measurable changes in either bilayer resistance (10-30 G omega) or displacement current across the membrane. However, addition of calcium (0.1-2.5 mM) in the presence of synexin in the external solution caused a marked increase in the size and time constant of decay of the displacement current. From the steady-state value of the current we calculated a 5-fold decrease in resistance and from the charge displaced during the voltage-clamp pulses we calculated a 10-fold increase in membrane capacitance (from 20 to 200 fF). The size of the synexin-specific charge displacement in one direction during a pulse was always equal to the charge returning to the original configuration after the pulse. The synexin-specific transfer of charge reached saturation when the pipet potential was taken to a sufficient positive or negative value. These properties of the extra charge movement support our view that in the presence of calcium the cytosolic protein synexin penetrates into the bilayer. It is possible that these properties may be related to the mechanism by which synexin promotes membrane fusion in natural membranes.