A human FVIII inhibitor modulates FVIII surface electrostatics at a VWF-binding site distant from its epitope.

SUMMARY BACKGROUND: BO2C11 is a human monoclonal factor (F) VIII inhibitor. When bound to the C2 domain of FVIII, the Fab fragment of BO2C11 (Fab(BO2C11)) buries a surface of C2 that contains residues participating in a binding site for von Willebrand factor (VWF). BO2C11 has thus been proposed to neutralize FVIII by steric hindrance. OBJECTIVES: The BO2C11 epitope on C2 overlaps with residues located at the periphery of the putative VWF binding site; hence, most of the residues that constitute the VWF binding site on C2 and a3 remain accessible for VWF interaction following BO2C11/FVIII complex formation. We thus investigated the contribution of alternative molecular mechanisms to FVIII inactivation by BO2C11. METHODS: Continuum electrostatic calculations were applied to the crystal structure of C2, free or Fab(BO2C11)-complexed. In silico predictions were confirmed by site-directed mutagenesis and VWF-binding assays of the mutated FVIII. RESULTS: Binding of Fab(BO2C11) to C2 induced perturbations in the electrostatic potential of C2 and in the local electrostatic parameters of 18 charged residues in C2, which are distant from the BO2C11 epitope. Nine of the predicted electrostatic hotspots clustered on the VWF-binding site of C2. Mutation of some of the predicted electrostatic hotspots has been associated with hemophilia A and reduced VWF binding in vitro. CONCLUSIONS: Inhibitors may neutralize FVIII by alteration of protein surface electrostatics at a long distance from their epitope. Perturbation of the electrostatic environment of C2, either upon binding by anti-FVIII antibodies or consecutive to missense mutations in the F8 gene, may lead to hampered VWF binding and reduced FVIII residence time in circulation.

Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases.

The pH dependence of the pK(a) values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 beta-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates. By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction intermediate, we found that acid-catalyzed protonation of the beta-lactam nitrogen is energetically favored as the initiating event, followed by base-catalyzed nucleophilic attack on the carbonyl carbon of the beta-lactam group. N-protonation is catalyzed through a hydrogen-bonded cluster involving the 2-carboxylate group of the substrate, the side chains of S130 and K234, and a solvent molecule. Nucleophilic attack on the carbonyl carbon is carried out by the side chain of S70 with proton abstraction catalyzed by a water molecule hydrogen-bonded to the side chain of E166. Stabilization of ion pairs in the active site through interactions with distant clusters of charged residues in the enzyme was concluded to be an important driving force of the catalytic mechanism.

Local electrostatic potentials in pyridoxal phosphate labelled horse heart cytochrome c.

The present work shows the application of an optical label pyridoxal phosphate (PLP) for the experimental determination of local electrostatic potentials in singly substituted cytochromes c modified by pyridoxal phosphate at Lys 79 (PLP-Lys-79-cyt.c) or at Lys 86 (PLP-Lys-86-cyt.c). PLP has also been used to calculate the pKa values of all ionizable groups and the electrostatic potentials in the modified proteins and to analyse their properties. The experimental pKa values for the pyridine nitrogen and phenolic hydroxyl of the bound label were obtained from pH-dependent absorbance and fluorescence measurements, as follows: in PLP-Lys-79-cyt.c for pyridine nitrogen 4.5 (absorbance) and 5.1 (fluorescence), for phenolic hydroxyl 8.6 (absorbance) and 8.3 (fluorescence); in PLP-Lys-86-cyt.c for pyridine nitrogen 4.7 (absorbance) and 5.8 (fluorescence), for phenolic hydroxyl 8.3 (absorbance) and 8.5 (fluorescence). The differences between absorbance and fluorescence data are related to differences in the behaviour of the bound label in the ground and excited electronic states and to intermolecular charge-charge interactions. Molecular modelling was used to generate the atomic co-ordinates of the PLP-modified horse heart cytochrome c necessary for the theoretical calculations of the pKa values and electrostatic potentials.

[X-ray fluorescent study of the ionic composition of lysozyme crystals]. / Rentgenofluorestsentnoe issledovanie ionnogo sostava kristallov lizotsima.

The method of the studying of the ionic content in the protein crystals by X-ray fluorescence spectroscopy is proposed. The ionic content of the tetragonal glutaraldehyde--cross-linked lysozyme crystals in the 2- 11 pH range and upon the low and high ionic strengths is studied. The acid-base titration of the lysozyme crystals is carried out for determination of the pH-dependence of the net charge of the lysozyme molecule within protein crystal. It has been shown that ionic content in the protein crystal channels is determined mainly by the Donnan potential. The specific binding of the bromide-ions with lysozyme molecules is revealed and possible binding sites are discussed.

Spatial optimization of electrostatic interactions between the ionized groups in globular proteins.

A model approach is suggested to estimate the degree of spatial optimization of the electrostatic interactions in protein molecules. The method is tested on a set of 44 globular proteins, representative of the available crystallographic data. The theoretical model is based on macroscopic computation of the contribution of charge-charge interactions to the electrostatic term of the free energy for the native proteins and for a big number of virtual structures with randomly distributed on protein surface charge constellations (generated by a Monte-Carlo technique). The statistical probability of occurrence of random structures with electrostatic energies lower than the energy of the native protein is suggested as a criterion for spatial optimization of the electrostatic interactions. The results support the hypothesis that the folding process optimizes the stabilizing effect of electrostatic interactions, but to very different degree for different proteins. A parallel analysis of ion pairs shows that the optimization of the electrostatic term in globular proteins has increasingly gone in the direction of rejecting the repulsive short contacts between charges of equal sign than of creating of more salt bridges (in comparison with the statistically expected number of short-range ion pairs in the simulated random structures). It is observed that the decrease in the spatial optimization of the electrostatic interactions is usually compensated for by an appearance of disulfide bridges in the covalent structure of the examined proteins.

Structural modeling and electrostatic properties of aspartate transcarbamylase from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequences so far, suggesting that this region is interacting with another domain of the multifunctional protein.

Urea unfolding and stability of gamma-II crystallin.

The conformational stability of gamma-II crystallin at pH 7.0 was estimated by studying its urea denaturation at isothermal conditions. The conformational states were monitored by far UV-CD and fluorescence measurements. Gamma-II crystallin shows sigmoidal order-disorder transition curves by both methods. The presence of more than one intermediate was confirmed but at neutral pH. The experiment results were critically analyzed in terms of both linear extrapolation and Tanford's models. The Gibbs free energy of unfolding delta G u,H2O = -36 kcal mol-1 was obtained. This value corresponds to the high conformational stability of the protein predicted qualitatively by its crystal structure.

pH-dependence of photo-induced electron transfer in zinc-substituted sperm whale myoglobin.

The electron transfer between the excited triplet state of zinc-substituted sperm whale myoglobin and Cu2+ has been studied by following the decay rate of delayed fluorescence. The Cu2+ bound on the surface of the myoglobin molecule are efficient quenchers of the excited electron state of Zn-myoglobin. Two bimolecular rate constants of quenching (KQ) for every pH investigated have been calculated. The pH-dependence of KQ1 indicates that the protonation of one amino acid residue (His-GH1 (119] is important for the process. Our results support the idea of the common nature of the mechanism of quenching by Cu2+ and oxidation of oxymyoglobin by Cu2+.

Pyridoxal phosphate modified cytochromes c. Identification and electron transfer properties.

The preparation, purification and characterization of the three singly, three doubly and one triply substituted derivatives of cytochrome c modified by pyridoxal phosphate (PLP) at lysine residues are reported. The PLP positions in PLP derivatives were determined by the amino acid analysis and sequence of PLP peptides. The results identified the lysine at position 86 in one of the singly substituted, lysine 79 in the other singly substituted and lysines 86 and 79 in the third doubly substituted cytochrome c derivatives. The area surrounding phenylalanine 82 forms the predominant PLP binding site on the cytochrome c molecule. The visible, CD and proton NMR spectra, the full intensity of the conformation-sensitive 695 nm band and the oxidation-reduction properties provide evidence to confirm the conclusion that singly and doubly substituted PLP cytochromes c retain the native conformation. The ability to restore both succinate and ascorbate/TMPD oxidation in cytochrome c-depleted mitochondria decreases in the order: native cytochrome c greater than PLP-Lys-79-cytochrome c greater than PLP-Lys-86-cytochrome c greater than PLP-Lys-79,86-cytochrome c greater than triply substituted derivative.

Charge interactions of cytochrome c with cytochrome c oxidase.

The pyridoxal phosphate (PLP) modification of the lysine amino groups in cytochrome c causes decrease in the reaction rate with cytochrome c oxidase. The rate constants for (PLP)2-cyt. c, PLP(Lys 86)-cyt. c, PLP(Lys 79)-cyt. c and native cytochrome c (at pH 7.4, I = 0.02) are 3.6 X 10(-3) sec-1, 5.5 X 10(-3) sec-1, 5.2 X 10(-3) sec-1 and 9.8 X 10(-3) sec-1, respectively. In spite of the same positive charge of singly PLP-cytochromes c the reaction between PLP(Lys 86)-cyt. c and cyt. c oxidase exhibits the ionic strength dependence that differs from those of the PLP(Lys 79)-cyt. c. The rate constants at zero and infinite ionic strength for PLP(Lys 86)-cyt. c is 2-fold less than that for PLP(Lys 79)-cyt. c. The positively charged cytochrome c lysines 86 and 79 form two from four or five predicted complementary charge interactions with carboxyl groups on cytochrome c oxidase.

[Electron transport in hemoproteins. VIII. The effect of chemical modification of ferricytochrome C on the rate of its reduction by oxymyoglobin]. / Izuchenie perenosa élektrona v gemoproteinakh. VIII. Vliianie khimicheskoi modifikatsii ferritsitokhroma C na skorost' vosstanovleniia ego oksimioglobinom.

The influence of chemical modification of separate amino acid residues in ferricytochrome c (Cyt c) on the rate of the redox reaction with MbO2 has been studied at various pH and ionic strength values. It is shown that alkylation of His-33 and Met-65 by bromacetate does not affect the reaction rate. On the contrary, acylation of Tyr-74 or one of the neighbouring lysines, Lys-72 or Lys-73, by the spin-label N-(2,2',5,5'-tetramethyl-3-carboxypyrrolin-1-oxy)-imidazol diminishes sharply the efficiency of electron transfer in the redox system studied. Besides, unlike the reaction between native proteins, the rate of electron transfer in this case does not depend on ionic strength. The modification of Tyr-74 or Lys 72/43 does not alter the midpoint potential and the entire conformation of Cyt c. The observed effects can therefore be explained by essential disturbance of interactions, first of all, the electrostatic ones in the active complex, which is induced by the attachment of the bulky reagent to the site of "active contact" of Cyt c. Based on the obtained findings and the atomic coordinates of Cyt c, the positions of all charge and some uncharged groups on the surface of Cyt c interacting with myoglobin during electron transfer are presented.

[Electron transfer in hemoproteins. VIII. Influence of ionic strength on the rate of reduction of ferricytochrome c by oxymyoglobin derivatives, chemically modified at histidine residues]. / Izuchenie perenosa élektrona v gemoproteinakh. VII. Vliianie ionnoi sily na skorost' vosstanovleniia ferritsitokhroma c khimicheski modifitsirovannymi po ostatkam gistidina proisvodnymi oksimioglobina.

The influence of chemical modification of His residues in Mb on the rate of redox reaction in system MbO2--Cyt c has been studied at different ionic strengths and pH medium. The products of alkylation of all available His by bromacetate and iodacetamide, CM-Mb and CA-Mb, respectively, and myoglobin, modified by spin label 2,2', 6,6'-tetramethyl-4-bromoacetoxypiperidine-1-oxyl (SL) at His residue A10--Sl (His-A10)--Mb have been studied. It has been shown, that the character of the ionic strength dependence of reaction SL(His-A10)--MbO2 with Cyt c at pH 6.0 ann 7.0 is basically analogues to that, observed for intact protein. It means that only His-GH1 of two His residues, His-A10 and His-GH1, situated in the region of "active contact" of Mg with Cyt c molecule, participates in the interactions, essential for electron transfer. The interaction of the charge of this His with the negatively charged group of Cyt c is necessary, probably for the proper arrangement of other interactions in the active complex, because the deprotonation of His-GHl in the studied pH interval decreases the rate of the process by more than one order of magnitude. The rate of oxidation of MC-MbO2 and CA-MbO2 by ferricytochrome c, in contrast to intact protein, shows a weak dependence on the ionic strength and does not depend on the pH medium, throughout the range of ionic strengths from 0.005 to 1.0. The cause of the radical change in the ionic strength dependence is, probably, nearly entire disturbance of electrostatic interactions in the active complex due to chemical modification of His residues in the site of "active contact", and first of all, the His-CHl residue. The fact, that during alkylation of all available His in Mb the electron transfer persists in the system, points to that in the process of electron transfer to cytochrome c, uncharged group, most probably "inner" His-B5, participates. Based on the data on spatial structure and the obtained results, the positions of the charged groups in the site of "active contact" of Mb with Cyt c molecule are presented.

[Electron transfer in hemoproteins. VI. The dependence of the reduction rate of ferricytochrome c by oxymyoglobin on ionic strength]. / Izuchenie perenosa élektrona v gemoproteinakh. VI. Zavisimost' skorosti vosstanovleniia ferritsitokhroma c oksimioglobinom ot ionnoi sily rastvora.

The rate of the redox-reaction between MbO2 and ferri-Cyt c has been investigated in the pH range 5-8 under different ionic strength of the solution. The influence of various anions-phosphate, chloride, sulfate and acetat on the rate of the reaction were also studied. It has been shown that under the low ionic strength, I less than 0.1, all pH-dependence curves have pronounced maximum near pH 6.0. While the ionic strength values increase in this interval the reaction rate falls markedly, the profile of lg k versus square root of I/1 + square root of I is linear. Under high ionic strength values, I greater than 0.1, the reaction rate in MbO2-Cyt c system is only slightly influenced by increasing salt concentrations and by pH changing. The results obtained support the idea that the local interactions of charged groups in " active sites" of MbO2 and Cyt c play the most important role in the mechanism of electron transfer. On the contrary net charges of the molecules have a negligible effect on the rate of the reaction. Compared to anions Cl-, SO42- and CH3COO- which influence the reaction rate in an analogous way, phosphate ions have essential inhibiting effect. This is most likely explained by the specific bonding of the phosphate ions to Cty c in the immediate vicinity from the site of the "active contact" with Mb molecule.

[Study of electron transfer in hemoproteins. IV. Oxidation of sperm whale oxymoglobin catalyzed by copper ions]. / Izuchenie perenosa élektrona v gemoproteinakh. IV. Kataliziruemoe ionami medi okislenie oksimioglobina kashalota.

The influence of cupric ions on the oxidation of sperm whale oxymoglobin and soybean oxyleghemoglobin at pH 4.8--7.5 and 10--40 degrees C has been studied. The stability constants of cupric ions to myoglobin were determined: K1 = 3.4 . 10(5) M(-1) for the more reactive center, and K2 = 2.1 . 10(3) M(-1) for the next 5--7 binding centers. The role of ionization of His-GH1 in the process of oxidation of sperm whale myoglobin is discussed. A mechanism of electron transfer from myoglobin molecule to the external acceptor is presented.

[Pyridoxalphosphate-modified derivatives of cytochrome c. Mono- and disubstituted derivatives: characteristics and effect on electron transport in cytochrome c-depleted mitochondria]. / Modifitsirovannye piridoksal'fosfatom proizvodnye tsitokhroma c. Mono- i dizameshchennye proizvodnye: kharakteristika i vliianie na perenos élektronov v obednennykh tsitokhromom c mitokhondriiakh.

Cytochrome c is modified by covalent binding of pyridoxal phosphate (PLP) to lysine residues. One di-substituted [(PLP)2--C] and two mono-substituted derivatives [(PLP)--c and (PLP)''--c] were obtained and precisely purified. The peak at 695 nm and CD-spectra in 190--600 nm region show that all derivatives have native conformation. The differential UV-spectra of the derivatives against native protein show that in (PLP)2--c there is a contact dipole-dipole interaction between PLP chromophores. It is calculated that the N-atoms of the two PLP-substituted lysines must be at a distance less than or equal to 12 A. Analysing our and literature data, one may suppose that Lys-13 and Lys-87 are the most probable candidates for modification with PLP. (PLP)---c and (PLP)''--c behave differently during ion-exchange chromatography and when added to cytochrom c-depleted mitochondria. (PLP)''--c restores electron transfer at higher concentrations than (PLP)'--c. Both they restore fully succinate and ascorbate oxidation but at considerably higher concentrations than the native protein, i. e. modification of any one of the reactive towards PLP lysines descreases but does not exclude the interaction with its reductase and oxidase. The effective equilibrium constants of binding of modified derivatives to cytochrome c-depleted mitochondria are lower than the constant for native protein. Together with decrease in binding activity, Hill coefficients increase. From our results it may be supposed that probably the binding sites of cytochrome c for its reductase and oxidase partially overlap.

[Electron transfer in hemoproteins. III. Physico-chemical characteristics of autoxidation of sperm whale oxymyoglobin]. / Izuchenie perenosa élektrona v gemoproteinakh. III. Fiziko-khimicheskaia kharakteristika avtookisleniia oksimioglobina kashalota.

The influence of pH, temperature and ionic strength on the autoxidation of sperm whale oxy-Mb is described and the data obtained are discussed. The process depends on the ionization of two groups with pKeff approximately or equal to 6. They are not accessible histidines of Mb. There are two parallel processes of autoxidation and each of them have a different temperature dependence. Two regions of ionic strength of opposite influence on the rats of autoxidation both at "low" (10 degrees C) and "high" (30 degrees C) temperatures were found.

[Lupine leghemoglobin affinity to ligands. The effect of pH and buffer nature]. / Izuchenie srodstva leggemoglibina liupina k ligandam. Bliianie rN i prirody bufera.

The myoglobin-like haemoprotein leghaemoglobin (Lb I) from lupine root nodules has a great affinity to molecular oxygen and seems to be involved in O2-transport. Some ligands of low molecular weight are supposed to affect the haemoglobin (Hb) and myoglobin (Mo) function in O2-transport. To investigate this possibility for lupine Lb I, the affinity of this protein to cyanide (CN-), azide (N3-), fluoride (F-), thiocyanate (NCS-), imidazole (Im), nicotinic acid (NA), acetic acid has been investigated, using: 0.05 M MES, pH 5.2-6.5; 0.1 M Na-phosphate in 0.05 M Tris-buffer, pH 6.5-9.0. The affinity for Lb I to N3-, CN-, F- and NA (the Bohr effect) was found to be pH-dependent. The values of PK ionization for the groups affecting the ligands binding were determined. The positive correlation between the ligand affinity and the ligand power was found. Lb I appears to have the greatest ligand affinity constants when compared with other haemoproteins of this class.