Boris P. Atanasov

Mutational Analyses of the Recombinant Globular Regions of Human C1q A, B, and C Chains Suggest an Essential Role for Arginine and Histidine Residues in the C1q-IgG Interaction

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the globular domain (gC1q) of C1q to the Fc regions of aggregated IgG or IgM. Each gC1q domain is a heterotrimer of the C-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Our recent studies have suggested a modular organization of gC1q, consistent with the view that ghA, ghB, and ghC are functionally autonomous modules and have distinct and differential ligand-binding properties. Although C1q binding sites on IgG have been previously identified, the complementary interacting sites on the gC1q domain have not been precisely defined. The availability of the recombinant constructs expressing ghA, ghB, and ghC has allowed us, for the first time, to engineer single-residue substitution mutations and identify residues on the gC1q domain, which are involved in the interaction between C1q and IgG. Because C1q is a charge pattern recognition molecule, we have sequentially targeted arginine and histidine residues in each chain. Consistent with previous chemical modification studies and the recent crystal structure of gC1q, our results support a central role for arginine and histidine residues, especially Arg114 and Arg129 of the ghB module, in the C1q-IgG interaction.

Electrostatic interactions in proteins. A theoretical analysis of lysozyme ionization

Abstract A semi-empirical method is proposed for calculations of the electrostatic interactions in proteins. The minimum of needed input data are the atomic coordinates of the protein and its potentiometric titration curve. On the basis of experimental data for potentiometric titration, an effective potential function of charge pair interactions is determined by parametric identification. A linear correlation of intrinsic pK with the solvent accessibility of ionizing atoms is assumed for accounting the influence of water-protein vicinity of the titratable groups. An analysis of electrostatic interactions in lysozyme was carried out. The calculated characteristics (titration curve, p K 1 2 of the individual ionic groups and pI) are in good agreement with the experimental data. It is found that the influence of the helix macro-dipoles is essential for high pK value of Glu-35 from the active site.

Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin

Molluscan hemocyanins are very large biological macromolecules and they act as oxygen-transporting glycoproteins. Most of them are glycoproteins with molecular mass around 9000 kDa. The oligosaccharide structures of the structural subunit RvH2 of Rapana venosa hemocyanin (RvH) were studied by sequence analysis of glycans using MALDI-TOF-MS and tandem mass spectrometry on a Q-Trap mass spectrometer after enzymatical liberation of the N-glycans from the polypeptides. Our study revealed a highly heterogeneous mixture of glycans of the compositions Hex(0-9) HexNAc(2-4) Hex(0-3) Pent(0-3) Fuc(0-3). A novel type of N-glycan, with an internal fucose residue connecting one GalNAc(β1-2) and one hexuronic acid, was detected, as also occurs in subunit RvH1. A glycan with the same structure but with two deoxyhexose residues was observed as a doubly charged ion. Antiviral effects of the native molecules of RvH and also of Helix lucorum hemocyanin (HlH), of their structural subunits, and of the glycosylated functional unit RvH2-e and the non-glycosylated unit RvH2-c on HSV virus type 1 were investigated. Only glycosylated FU RvH2-e exhibits this antiviral activity. The carbohydrate chains of the FU are likely to interact with specific regions of glycoproteins of HSV, through van der Waals interactions in general or with certain amino acid residues in particular. Several clusters of these residues can be identified on the surface of RvH2-e.

Electrostatic interactions in proteins: Calculations of the electrostatic term of free energy and the electrostatic potential field

The pH-dependence of the electrostatic energy of interactions between titratable groups is calculated for some well studied globular proteins: basic pancreatic trypsin inhibitor, sperm whale myoglobin and tuna cytochrome c. The calculations are carried out using a semi-empirical appraach in terms of the macroscopic model based on the Kirkwood-Tanford theory. The results are discussed in the light of their physicochemical and biological properties. It was found that the pH-dependence of the electrostatic energy correlates with the III–IV transition of cytochrome c. The electrostatic field of the cysteine proteinase inhibitor, cystatin, was calculated in two ways. In the first one, the electrostatic field created by the pH dependent charges of the ionizable groups and peptide dipoles was calculated using the approach proposed. In the second one, the finite-difference method was used. The results obtained by the two methods are in overall agreement. The calculated field was discussed in terms of the binding of cystatin to papain.

Heme Interacts with C1q and Inhibits the Classical Complement Pathway

C1q is the recognition subunit of the first component of the classical complement pathway. It participates in clearance of immune complexes and apoptotic cells as well as in defense against pathogens. Inappropriate activation of the complement contributes to cellular and tissue damage in different pathologies, urging the need for the development of therapeutic agents that are able to inhibit the complement system. In this study, we report heme as an inhibitor of C1q. Exposure of C1q to heme significantly reduced the activation of the classical complement pathway, mediated by C-reactive protein (CRP) and IgG. Interaction analyses revealed that heme reduces the binding of C1q to CRP and IgG. Furthermore, we demonstrated that the inhibition of C1q interactions results from a direct binding of heme to C1q. Formation of complex of heme with C1q caused changes in the mechanism of recognition of IgG and CRP. Taken together, our data suggest that heme is a natural negative regulator of the classical complement pathway at the level of C1q. Heme may play a role at sites of excessive tissue damage and hemolysis where large amounts of free heme are released.

PHEPS: web-based pH-dependent Protein Electrostatics Server

PHEPS (pH-dependent Protein Electrostatics Server) is a web service for fast prediction and experiment planning support, as well as for correlation and analysis of experimentally obtained results, reflecting charge-dependent phenomena in globular proteins. Its implementation is based on long-term experience (PHEI package) and the need to explain measured physicochemical characteristics at the level of protein atomic structure. The approach is semi-empirical and based on a mean field scheme for description and evaluation of global and local pH-dependent electrostatic properties: protein proton binding; ionic sites proton population; free energy electrostatic term; ionic groups proton affinities (pKa,i) and their Coulomb interaction with whole charge multipole; electrostatic potential of whole molecule at fixed pH and pH-dependent local electrostatic potentials at user-defined set of points. The speed of calculation is based on fast determination of distance-dependent pair charge-charge interactions as empirical three exponential function that covers charge–charge, charge–dipole and dipole–dipole contributions. After atomic coordinates input, all standard parameters are used as defaults to facilitate non-experienced users. Special attention was given to interactive addition of non-polypeptide charges, extra ionizable groups with intrinsic pKas or fixed ions. The output information is given as plain-text, readable by ‘RasMol’, ‘Origin’ and the like. The PHEPS server is accessible at .

PHEMTO: protein pH-dependent electric moment tools

PHEMTO (protein pH-dependent electric moment tools) is released in response to the high demand in protein science community for evaluation of electrostatic characteristics in relations to molecular recognition. PHEMTO will serve protein scientists with new advanced features for analysis of protein molecular interactions: Electric/dipole moments, their pH-dependence and in silico charge mutagenesis effects on these properties as well as alternative algorithms for electric/dipole moment computation—Singular value decomposition of electrostatic potential (EP) to account for reaction field. The implementation is based on long-term experience—PHEI mean field electrostatics and PHEPS server for evaluation of global and local pH-dependent properties. However, PHEMTO is not just an update of our PHEPS server. Besides standard electrostatics, we offer new, advanced and useful features for analysis of protein molecular interactions. In addition our algorithms are very fast. Special emphasis is given to the interface—intuitive and user-friendly. The input is comprised of the atomic coordinate file in Protein Data Bank format. The advanced user is provided with a special input section for addition of non-polypeptide charges. The output covers actually full electrostatic characteristics but special emphasis is given to electric/dipole moments and their interactive visualization. PHEMTO server can be accessed at http://phemto.orgchm.bas.bg/.

Subfractionation and recombination of a neurotoxic complex from the venom of the bulgarian viper (Vipera ammodytes ammodytes)

In our previous studies [ I] we isolated and purified the strongest toxic protein (NW) of the venom from Bulgarian viper. It was shown that NVC is an electrophoretically homogeneous protein complex consisting of two components: one TC with an isoelectric point in the alkaline region and the other NC with an isoelectric point in the acidic region [2]. Systematic studies on the isolated NVC components have shown that TC is a phospholipase AZ while NC has not got an enzymatic activity but partially inhibits the phospholipase AZ activity of the TC when bound (1: 1 ratio) in NVC [3,4] . The properties of NVC have been compared with those of the analogous crotoxin complex [3,4] . Toxic phospholipases AZ have been isolated from Vipera ammodytes venom [5] and Vipera berus venom [6] but up to now their binding in any kind of protein complex has not been established.

Complete amino acid sequence of poplar plastocyanin b

A second type of plastocyanin, plastocyanin b (PCb) was isolated from leaves of poplar Populus nigra var. Italica. The complete amino acid sequence of 99 residues in the single polypeptide chain of plastocyanin b has been determined: DVLLGADDG SLAFVPSEFS GEKIVFK NNAGFPHN FDED PSGV D SKISMSEE DLLNAKGETF EVALS KGEY FYCSPHQGA GMVGKV VN The sequence obviously demonstrates, that PCb, in comparison with the known plastocyanin, plastocyanin a (PCa), has 12 amino acid replacements (underlined letters): Ile 1→Val, Ile 21‐Ser 22‐Pro 23→Val‐Pro‐Ala, Ile 39‐Val 40→Val‐Leu, Ser 45‐Ile 46→ Ala‐Val, Ala 52→Val, Asn 76→Asp, Ser 81→Thr and Thr 97→Ile. The replacements at positions 40 and 76 of PCb are probably essential for differences in its redox and electrochemical properties, respectively.

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 (FabBO2C11) 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 FabBO2C11‐complexed. In silico predictions were confirmed by site‐directed mutagenesis and VWF‐binding assays of the mutated FVIII. Results: Binding of FabBO2C11 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.