Studies on the peptide corresponding to residues 34-47 of bovine factor X.

Calcium binding studies of a 14-residue peptide corresponding to the 37-46 sequence of bovine factor X were performed using calcium ion selective electrode titrations and equilibrium dialysis. The presence of gamma-carboxyglutamic acid residues at positions 36 and 40 coupled with the assumption that the peptide would bind calcium ions also prompted an investigation of possibly secondary conformational changes in the peptide by use of circular dichroism spectroscopy. Equilibrium dialysis revealed a single relatively weak calcium binding site (log Ka = 2.39); an ion selective electrode experiment confirmed this result (log Ka = 2.17). The peptide maintained a random coil conformation throughout the calcium ion titrations as measured by circular dichroism.

Refinement of the NMR solution structure of the gamma-carboxyglutamic acid domain of coagulation factor IX using molecular dynamics simulation with initial Ca2+ positions determined by a genetic algorithm.

A genetic algorithm (GA) successfully identified the calcium positions in the crystal structure of bovine prothrombin fragment 1 bound with calcium ions (bf1/Ca). The same protocol was then used to determine the calcium positions in a closely related fragment, the Gla domain of coagulation factor IX, the structure of which had previously been determined by NMR spectroscopy in the presence of calcium ions. The most frequently occurring low-energy structure found by GA was used as the starting structure for a molecular dynamics refinement. The molecular dynamics simulation was performed using explicit water and the Particle-Mesh Ewald method to accommodate the long-range electrostatic forces. While the overall conformation of the NMR structure was preserved, significant refinement is apparent when comparing the simulation average structure with its NMR precursor in terms of the N-terminal (Tyr1-N) network, the total number of hydrogen bonds, the calcium ion coordinations, and the compactness of the structure. It is likely that the placement of calcium ions in the protein is critical for refinement. The calcium ions apparently induce structural changes during the course of the simulation that result in a more compact structure.

Investigation of the 35-62 conserved helix and disulfide loop of bovine prothrombin using an anti-peptide antibody.

A 28-residue peptide corresponding to the 35-62 region of bovine prothrombin fragment 1 (BF1) was synthesized by solid-phase methods. In BF1 this region consists of three conserved aromatic residues within an alpha-helical region followed by a disulfide loop. This synthetic peptide was used to produce murine monoclonal antibodies (MAbs) that would recognize and bind native BF1. Antibody AH.Ab.E3, an IgG1 antibody that was isolated and cloned, recognized and bound to both the synthetic peptide and the BF1 molecule. Residues 55-59 (REKLN) were shown to be critical for antibody binding. This MAb was subsequently used to study the 48-62 disulfide loop region of BF1. MAb AH.Ab.E3, which has been shown to bind the BF1 calcium-dependent conformation (BF1:Ca), does not appear to perturb the binding interaction between BF1:Ca and phospholipid (PL) vesicles as studied by light scattering methods.

Is Ca(II) ion binding to prothrombin fragment 1 intrinsically cooperative, or is the cooperative binding accounted for by self-association?

The recent suggestion that the apparent cooperativity seen in the binding of Ca(II) ions to prothrombin fragment 1 is due to protein aggregation is evaluated. Since (1) we find that the Ca(II) ion binding is not dependent upon protein concentration, (2) the analytical expression for the equilibrium constant of the aggregation model is unrealistically large when evaluated at realistic Ca(II) ion concentrations, and (3) a very simple allosteric cooperative binding model (Monod) can be shown to fit the experimental data, we conclude that the aggregation explanation for the apparent cooperativity in the Ca(II) ion binding by prothrombin fragment 1 is not correct.

The first solvation shell of magnesium ion in a model protein environment with formate, water, and X-NH3, H2S, imidazole, formaldehyde, and chloride as ligands: an Ab initio study.

The first coordination shell of an Mg(II) ion in a model protein environment is studied. Complexes containing a model carboxylate, an Mg(II) ion, various ligands (NH3, H2S, imidazole, and formaldehyde) and water of hydration about the divalent metal ion were geometry optimized. We find that for complexes with the same coordination number, the unidentate carboxylate-Mg(II) ion is greater than 10 kcal mol-1 more stable than the bidentate orientation. Imidazole was found to be the most stable ligand, followed in order by NH3, formaldehyde, H2O, and H2S.

Predicted secondary structure of bovine prothrombin fragment 1 and related proteins in different environments by circular dichroism spectroscopy.

Circular dichroism spectroscopy was used to investigate the structure of bovine prothrombin fragment 1 (BF1) and related proteins in several environments. The conformational change induced in BF1 by the addition of Mg[II] ions was found to be different from that induced by Ca[II] or Sr[II]. The Ca[II] and Sr[II] conformations appear to differ only slightly from the apo-metal conformation. The conformation of the 1-45 fragment of prothrombin, however, is markedly different than the conformation of the same fragment in the presence of either Ca[II] of Mg[II]; both of the latter structures differ substantially from one another. The presence of phospholipids has almost no effect on the structure of either BF1 or the 1-45 fragment; in the presence of both phospholipids and Ca[II] a structural change is seen for the 1-45 fragment but not BF1 (relative to the protein alone). The addition of phospholipids to the Mg[II]/BF1 structure did not induce a CD-detectable conformational change, while the addition of phospholipids to the Ca[II]/BF1 or Sr[II]/BF1 structures induced a change to a conformation similar in secondary structure composition to the relative apometal structures.

Surface binding kinetics of prothrombin fragment 1 on planar membranes measured by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRFM) has been employed to investigate the Ca(2+)-dependent membrane-binding characteristics of fluorescein-labeled bovine prothrombin-fragment 1 (F-BF1). Light scattering measurements demonstrated that F-BF1 bound to small unilamellar phosphatidylserine/phosphatidylcholine (25/75, mol/mol) vesicles with an apparent dissociation constant (1.5 +/- 0.2 microM) similar to that of unlabeled protein (1.1 +/- 0.1 microM). Negatively charged supported planar membranes were constructed by fusing small unilamellar vesicles at quartz surfaces. TIRFM measurements under equilibrium conditions showed that F-BF1 bound to planar membranes with an apparent dissociation constant (0.9 +/- 0.2 microM) approximately equal to that on vesicles. Total internal reflection/fluorescence photobleaching recovery (TIR/FPR) curves for F-BF1 on 25 mol% PS planar surfaces were diffusion-influenced at F-BF1 solution concentrations less than or equal to 5 microM. Fluorescence recovery rates from samples of high F-BF1 concentrations were slowed by increasing the solution viscosity with glycerol, thus providing further support for a diffusion-limited effect at low F-BF1 concentrations. Analysis of the reaction-limited fluorescence recovery curves at F-BF1 solution concentrations greater than or equal to 10 microM gave average association and dissociation kinetic rates of approximately 10(5) M-1 s-1 and approximately 0.1 s-1, respectively. Kinetic association rates increased significantly with increasing PS, whereas kinetic dissociation rates increased only slightly. Fluorescence recovery curves were nonmonoexponential; possible mechanisms for this behavior are described.

Modifications of bovine prothrombin fragment 1 in the presence and absence of Ca(II) ions. Loss of positive cooperativity in Ca(II) ion binding for the modified proteins.

Chemical modification of bovine prothrombin fragment 1 according to the procedure of D. J. Welsch and G. L. Nelsestuen (1988) [Biochemistry 27, 4946-4952 and ealier papers] provided a series of fragment 1 derivatives in which various nitrogen-containing side chains were N-acetylated and/or N-2,4,6-trinitrophenylated. In addition the des-[Ala-1,Asn-2]- and des-[Ala-1,Asn-2,Lys-3]-fragment 1 derivatives were prepared by limited enzymatic hydrolysis of fragment 1 using cathepsin C and plasmin, respectively. Quantitative studies on the Ca(II) binding of these proteins have been accomplished using 45Ca(II) equilibrium dialysis. Binding of these fragment 1 derivatives to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles (25:75) in the presence of Ca(II) ions has been studied using the light-scattering technique. Acylation of the 5 lysine residues of fragment 1 by the action of acetic anhydride (500-fold molar excess) in the presence of 75 mM Ca(II), pH 8.0, results in loss of positive cooperativity in Ca(II) binding (Scatchard plot) and an increase in the number of Ca(II) ions bound. The Ca(II)-dependent PS/PC binding of the acylated protein is reduced. Removal of 2 and 3 residues from the amino terminus likewise leads to loss of positive cooperativity in Ca(II) binding and reduced binding affinity to PS/PC vesicles. The important role of the amino-terminal 1-10 sequence is discussed. We conclude that positive cooperativity in Ca(II) binding is not a prerequisite for the Ca(II)-dependent binding of bovine prothrombin fragment 1 to PS/PC vesicles.

A metal ion-binding site in the kringle region of bovine prothrombin fragment 1.

45Ca(II) binding studies (equilibrium dialysis) on the kringle domain of bovine prothrombin fragment 1 were conducted using a mixture of peptides (residues 43-156 and 46-156) resulting from limited alpha-chymotryptic hydrolysis of fragment 1. Analysis of the Scatchard plot of these data indicates a single, low affinity Ca(II)-binding site to be present. Similar results were obtained from studies on the decarboxylated fragment 1 derivative, 10-gamma-MGlu-fragment 1. Acetylation of bovine fragment 1 in the absence of Ca(II) or Mg(II) ions results in the loss of the metal ion-promoted quenching of the intrinsic Trp fluorescence of the protein and the Ca(II)-mediated binding to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles. The acetylation of the NH2 alpha-group of Ala-1 has been shown (Welsch, D. J., and Nelsestuen, G. L. (1988) Biochemistry 27, 4946-4952) to abolish the PS/PC binding property of fragment 1. The present study demonstrates that acetylation of a second site possibly Ser-79 or Thr-81 using the conditions described in the preceding paper results in loss of both the fluorescence transition and the Ca(II)-mediated PS/PC binding of the resulting protein derivative. Removal of the O-acetyl group at the Ser-79/Thr-81 site is accomplished by aminolysis with 0.2 M hydroxylamine, pH 10, 50 degrees C; the fluorescence transition is partially restored. PS/PC binding is partially restored if the NH2 alpha-group of Ala-1 is trinitrophenylated but is not restored if the NH2 alpha-group of Ala-1 is acetylated. We conclude that the Ser-79/Thr-81 site may represent a portion of the metal ion-binding site within the kringle domain of fragment 1. Occupancy of this site by a Ca(II) ion appears to be important in the binding of the protein to PS/PC vesicles.

Determination of strontium binding to macromolecules.

An equilibrium dialysis technique for determining the binding of strontium to macromolecules is described. The major difficulty to be overcome is that 90Sr has a decay product, 90Y, which is also a beta-emitter. The described protocol is used to determine the Sr binding isotherm to bovine prothrombin fragment 1. The binding is found to be cooperative, somewhat weaker than Ca binding, and to involve approximately nine strontium sites. The stoichiometric equilibrium constants are determined by nonlinear regression. The procedure should be of great utility for many macromolecules that show strontium affinity.

Proton, calcium, and magnesium binding by peptides containing gamma-carboxyglutamic acid.

gamma-Carboxyglutamic acid (Gla) is believed to bind Ca [II] ions and Mg [II] ions in prothrombin and other coagulation proteins. Binding constants for H+, Ca [II] ions, and Mg [II] ions to Gla-containing peptides are determined using pH and ion selective electrode titrations. The binding constants for peptides containing a single Gla residue are similar to the constants for malonic acid. Peptides containing two Gla residues in sequence (di-Gla peptides) bind Ca [II] ions and Mg [II] ions more strongly. KMgL for the di-Gla peptides is similar to the site-binding constant for Ca [II] ions in denatured BF1. These di-Gla peptides may be useful analogs for metal binding by the disordered Gla domain in BF1.

Solution conformations of the gamma-carboxyglutamic acid domain of bovine prothrombin fragment 1, residues 1-65.

Molecular dynamics simulations have been performed (AMBER version 3.1) on solvated residues 1-65 of bovine prothrombin fragment 1 (BF1) by using the 2.8-A resolution crystallographic coordinates as the starting conformation for understanding calcium ion-induced conformational changes that precede experimentally observable phospholipid binding. Simulations were performed on the non-metal-bound crystal structure, the form resulting from addition of eight calcium ions to the 1-65 region of the crystal structure, the form resulting from removal of calcium ions after 107 ps and continuing the simulation, and an isolated hexapeptide loop (residues 18-23). In all cases, the 100-ps time scale seemed adequate to sample an ensemble of solution conformers within a particular region of conformation space. The non-metal-containing BF1 did not unfold appreciably during a 106-ps simulation starting from the crystallographic geometry. The calcium ion-containing structure (Ca-BF1) underwent an interesting conformational reorganization during its evolution from the crystal structure: during the time course of a 107-ps simulation, Ca-BF1 experienced a trans----cis isomerization of the gamma-carboxyglutamic acid-21 (Gla-21)-Pro-22 peptide bond. Removal of the calcium ions from this structure followed by 114 ps of additional molecular dynamics showed significant unfolding relative to the final 20-ps average structure of the 107-ps simulation; however, the Gla-21-Pro-22 peptide bond remained cis. A 265-ps simulation on the termini-protected hexapeptide loop (Cys-18 to Cys-23) containing two calcium ions also did not undergo a trans----cis isomerization. It is believed that the necessary activation energy for the transitional event observed in the Ca-BF1 simulation was largely supplied by global conformational events with a possible assist from relief of intermolecular crystal packing forces. The presence of a Gla preceding Pro-22, the inclusion of Pro-22 in a highly strained loop structure, and the formation of two long-lived salt bridges prior to isomerization may all contribute to this finding.

Calcium ion binding to human and bovine factor X.

Human and bovine factor X contain 11 and 12 glutamyl residues, respectively, within the first 40 amino terminal residues that are post-translationally modified to gamma-carboxyglutamyl (Gla) residues. We have measured calcium ion binding to human factor X by equilibrium dialysis. This is the first examination of calcium ion binding to human factor X. We have also re-examined the equilibrium dialysis binding of calcium ions to bovine facor X in order to compare the two species. The data was analysed using a variety of models that allow for more than one class of binding site and for co-operativity among binding sites. Calcium ion binding to human factor X fits a model that had two classes of sites: one class with a single site that had an affinity of 0.1 mM and a second class with 19 equivalent, non-interacting sites with an average affinity of 3.5 mM. There was no evidence for co-operativity in calcium ion binding. Calcium ion binding to bovine factor X was best stimulated by a model that assumed one tight site, four co-operative sites, and 18 equivalent, non-interacting sites. To examine the co-operativity seen in calcium ion binding to bovine factor X, calcium ion binding to isolated Gla region (residues 1-44) and Gla-domainless factor X was measured by equilibrium dialysis. Calcium ion binding to Gla-domainless factor X was simulated by a model that had two classes of sites: one class with a single site that had an affinity of 0.25 mM, and a second class that had 15 sites with very low affinity sites (greater than 15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and characterization of 1,1,4,4-butanetetracarboxylic acid. A di-gamma-carboxyglutamic acid (GlaGla) analogue.

1,1,4,4-Butanetetracarboxylic acid (BTCA) is evaluated as an analogue for the metal binding site in dipeptides of gamma-carboxyglutamic acid (Gla). Molecular modeling suggests that the four carboxylic acid groups in BTCA can assume a similar conformation to the four gamma-carboxylic acid groups in GlaGla and thus provides the impetus for the synthesis and metal binding determinations. BTCA is synthesized via the tert.-butyl ester and characterized via NMR, mass spectroscopy, and elemental composition. Equilibrium binding constants with protons, Ca(II) and Mg(II) are determined via pH and Ca(II) ion-selective electrode titrations and are found to be similar to those for GlaGla peptides with blocked termini.

Divalent metal ion mediated interaction of proteins with negatively charged membranes. A model study employing molecular mechanics.

Previous molecular mechanics calculations on the effect of Ca(II) and Mg(II) ions on the conformation of the 18-23 loop of bovine prothrombin [Maynard et al. 1988, Int. J. Peptide Protein Res. 31, 137-149] are extended to include the effect of a model phospholipid head group methyl[L-seryl] phosphate. Whereas the conformation of the Gla-21 Pro-22 amide bond remains decidedly trans in the absence of the model head group, in its presence, the cis Ca(II) ion induced (but not Mg(II] form is significantly lowered in relative energy. The low energy Ca(II) structures establish a coordination sphere with more ligands than do the low energy Mg(II) ion structures.

The role of hydrated divalent metal ions in the bridging of two anionic groups. An ab initio quantum chemical and molecular mechanics study of dimethyl phosphate and formate bridged by calcium and magnesium ions.

Ab initio quantum chemical (Gaussian82) and molecular mechanics (AMBER2.0) computational techniques are employed to investigate the interaction of two anions (formate an dimethylphosphate) and a central divalent metal cation (magnesium or calcium). These systems are models for the essential GDP binding unit of the G-proteins (e.g., EF-Tu or the ras oncogene proteins) and for protein/phospholipid interactions, both of which are mediated by divalent metal cations. Various levels of hydration are utilized to examine coordination of differences between magnesium and calcium ions. Two different orientations of formate and dimethyl phosphate in direct ion contact with a magnesium ion and two waters of hydration were energy minimized with both quantum and molecular mechanics techniques. The structures and energy differences between the two orientations determined by either of the computational techniques are similar. Magnesium ion has a strong propensity to assume six coordination whereas calcium ion preferentially assumes a coordination greater than six. Likewise, water molecules attached to magnesium ion are held more rigidly than those of calcium ion, thus calcium ion is more accommodating in the exchange of water for negative ligands.

Salt or ion bridges in biological systems: a study employing quantum and molecular mechanics.

Equilibrium geometries and binding energies of model "salt" or "ion" bridge systems have been computed by ab initio quantum chemistry techniques (GAUSSIAN82) and by empirical force field techniques (AMBER2.0). Formate and dimethyl phosphate served as anions in the model compounds while interacting with several organic cations, including methyl ammonium, methyl guanidinium, and divalent metal ion (either Mg2+ or Ca2+) without and with an additional chloride; and a divalent metal ion (either Mg2+ or Ca2+), chloride, and four water molecules of hydration about the metal ion. The majority of the quantum chemical computations were performed using a split-valence basis set. For the model compounds studied we find that the ab initio optimized geometries are in remarkably good agreement with the molecular mechanics geometries. Several calculations were also performed using diffuse fractions. The formate anion binds these model cations more strongly than does dimethyl phosphate, while the organic cation methyl ammonium binds model anions more strongly than does methyl guanidinium. Finally, in model compounds including organic anions, Mg2+ or Ca2+ and four molecules of water, and a chloride anion, we find that the equilibrium structure of the magnesium complex involves a solvent separated ion pair (the magnesium ion is six coordinate), whereas the calcium ion complex remains seven coordinate. Molecular mechanics overestimates binding energies, but the estimates may be close enough to actual binding energies to give useful insight into the details of salt bridges in biological systems.

Phospholipid binding properties of bovine prothrombin peptide residues 1-45.

The present study investigates the unique contribution of the NH2-terminal 33 residues of prothrombin, the gamma-carboxyglutamic acid (Gla) domain, to the Ca(II) and phospholipid-binding properties of prothrombin. Two Gla domain peptides, 1-42 and 1-45, produced by chymotryptic cleavage of prothrombin fragment 1 (residues 1-156 of the amino terminus of bovine prothrombin) and isolated by anion-exchange chromatography were utilized to characterize the Gla domain of prothrombin. This investigation utilized several experimental approaches to examine the properties of the Gla domain peptides. These studies were somewhat hampered by the metal ion-induced insolubility of the peptides. However, the 1-45 peptide was specifically radioiodinated, which facilitated the study of this peptide at low concentrations. In contrast to prothrombin fragment 1, the intrinsic fluorescence of both 1-42 and 1-45 was not quenched upon the addition of 1 mM Ca(II) or any concentration of Mg(II). Equilibrium dialysis studies revealed that the 1-42 peptide bound three Ca(II) ions noncooperatively, whereas fragment 1 binds seven Ca(II) ions in a positive cooperative manner. Ca(II)-promoted conformational changes are observed by comparison of electrophoretic mobility changes in the presence of increasing Ca(II) concentrations. Prothrombin, fragment 1, and the Gla domain peptides 1-42 and 1-45 exhibited similar electrophoretic mobility behavior in the presence of Ca(II) ions. The radiolabeled 1-45 peptide was found to comigrate with phospholipid vesicles on gel permeation chromatography in the presence of Ca(II). Fragment 1 was shown to inhibit this Ca(II)-dependent phospholipid binding of 1-45, demonstrating that the 1-45 peptide does possess the necessary phospholipid-binding structure. Furthermore, a metal ion-dependent conformational monoclonal antibody, F9.29, was inhibited from binding fragment 1 by the 1-42 peptide.

The determination of a calcium-dependent binding constant of the bovine prothrombin Gla domain (residues 1-45) to phospholipid vesicles.

Calcium-mediated binding of the radioiodinated peptide representing residues 1-45 of bovine prothrombin to single bilayer phospholipid vesicles composed of phosphatidylserine from bovine brain and synthetic 1-palmitoyl-2-oleoyl-phosphatidylcholine (25:75 PS/PC) has been studied over peptide concentrations from 0.33 microM to 3.75 microM and at a calcium concentration of 1.0 mM. The binding isotherm for the interaction between the radioiodinated peptide and PS/PC vesicles fits a model in which there is noncooperative binding of the peptide to non-interacting sites on the phospholipid bilayer. A dissociation constant determined at these conditions is 11.8 microM compared to 1.0 microM for prothrombin fragment 1.

Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions. Sequence studies on 3-gamma-MGlu-fragment.

Chemical modification of the gamma-carboxyglutamyl (Gla) residues of bovine prothrombin fragment 1 using the formaldehyde-morpholine method in the presence of 100 Kappm Tb3+ ions at pH 5.0 provided a modified protein containing 3 gamma-methyleneglutamyl residues (gamma-MGlu) and 7 Gla residues (bovine 3-gamma-MGlu-fragment 1). The modified protein bound the same number of Ca2+ ions as the native protein (six to seven), exhibited 28Mg2+-binding properties identical to native fragment 1 (five Mg2+ ions bound), exhibited the metal ion-promoted quenching of the intrinsic fluorescence in a manner similar to the native protein, but did not bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles in the presence of Ca2+ ions. Modification of the bovine protein using [14C]formaldehyde-morpholine provided a 14C-labeled 3-gamma-MGlu-fragment 1 suitable for sequence analysis. Edman sequencing of the peptides released by a tryptic digest of the reduced and carboxymethylated bovine [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7, 8, and 33 had been converted to [14C]gamma-methyleneglutamyl residues. In addition Lys97 was found to contain a 14C label. Similar analysis of the human [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7 and 32 were major modification sites and that Gla residues at positions 6 and 14 were partially modified. Lysine 96 was also modified in the human protein. The incorporation of a 14C label at Lys97 in bovine 3-gamma-MGlu-fragment 1 protein is not responsible for the loss of Ca2+-promoted binding to PS/PC vesicles. We suggest that Gla residues 7, 8, and 33 are elements of the first Ca2+-binding site; occupancy of this site establishes the Ca2+-specific conformation which is essential for the Ca2+-promoted interaction of the bovine protein with PS/PC vesicles. These studies also suggest that the loss of Gla residues at positions 7 and 32 prevents the formation of the initial Ca2+-binding site in the human protein.