The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family.

The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

Recognition rules for binding of Zn-Cys2His2 transcription factors to operator DNA.

The molecules of Zn-finger transcription factors consist of several similar small protein units. We analyzed the crystal structures 46 basic units of 22 complexes of Zn-Cys2His2 family with the fragments of operator DNA. We showed that the recognition of DNA occurs via five protein contacts. The canonical binding positions of the recognizing α-helix were -1, 3, 6, and 7, which make contacts with the tetra-nucleotide sequence ZXYZ of the coding DNA strand; here the canonical binding triplet is underlined. The non-coding DNA strand forms only one contact at α-helix position 2. We have discovered that there is a single highly conservative contact His7α with the phosphate group of nucleotide Z, which precedes each triplet XYZ of the coding DNA chain. This particular contact is invariant for the all Zn-Cys2His2 family with high frequency of occurrence 83%, which we considered as an invariant recognition rule. We have also selected a previously unreported Zn-Cys2His2-Arg subfamily of 21 Zn-finger units bound with DNA triplets, which make two invariant contacts with residues Arg6α and His7α with the coding DNA chain. These contacts show frequency of occurrence 100 and 90%, and are invariant recognition rule. Three other variable protein-DNA contacts are formed mainly with the bases and specify the recognition patterns of individual factor units. The revealed recognition rules are inherent for the Zn-Cys2His2 family and Zn-Cys2His2-Arg subfamily of different taxonomic groups and can distinguish members of these families from any other family of transcription factors.

[Influence of the orthosteric ligands binding on the conformational dynamics of the B-2-adrenergic receptor by means of essential dynamics sampling simulation].

The influence of binding of the orthosteric ligands on the conformational dynamics of the beta-2-adrenoreceptor was identified using the molecular dynamics method. It was found that there was alittle fraction of the active states of the receptor in its apo (ligand free) ensemble. Analysis of the MD trajectories indicated that such spontaneous activation of the receptor is accompanied by the motion of its VI helix. Thus receptor's constitutive activity is the direct result of its conformational dynamics. On other hand binding of the full agonist resulted in the significant shift of the initial equilibrium towards its active state. Finally binding of the inverse agonist stabilized receptor in its inactive state. Its likely that the binding of the inverse agonists might be the universal way of the constitutive activity inhibition. Our results indicate that ligand binding rather redistribute prexisting conformational degrees of freedom (in accordance to the Monod-Wyman-Changeux-Model) than cause induced fit in it. Therefore ensemble of the biological-relevant receptor conformations have been encoded in its spatial structure and individual conformations from that ensemble might be used by the cell according to the physiological behavior.

[Functionally-relevant conformational dynamics of water-soluble proteins].

A study is reported of the functional-relevant dynamics of three typical water-soluble proteins: Calmodulin, Src-tyrosine kinase as well as repressor of Trp operon. Application of the state-of-art methods of structural bioinformatics allowed to identify dynamics seen in the X-ray structures of the investigated proteins associated with their specific biological functions. In addition, Normal Mode analysis technique revealed the most probable directions of the functionally-relevant motions for all that proteins were also predicted. Importantly, overall type of the motions observed on the lowest-frequency modes was very similar to the motions seen from the analysis of the X-ray data of the examined macromolecules. Thereby it was shown that the large-scale as well as local conformational motions of the proteins might be predetermined already at the level of their tertiary structures. In particular, the determining factor might be the specific fold of the alpha-helixes. Thus functionally-relevant in vivo dynamics of the investigated proteins might be evolutionally formed by means of natural selection at the level of the spatial topology.

Study of structural dynamics of ligand-activated membrane receptors by means of principal component analysis.

The structural dynamics of three different ligand-activated G-protein coupled receptors (GPCRs) and the photoreactive receptor rhodopsin from mammals were comparatively studied. As a result, diagrams demonstrating the main structural differences between the studied membrane receptors were obtained. These diagrams represent the projection of the crystal structures of rhodopsin photointermediates and ligand-activated receptors onto the plane defined by the principal components. Thus, we were able to associate the activation process of the receptors with large-scale movements of their individual transmembrane (TM) domains. In addition, the dynamics of extracellular loops of ligand-activated receptors responsible for recognition and initial binding of ligands was studied. Based on these results, two parameters of functionally significant structural dynamics of membrane receptors can be thoroughly analyzed simultaneously - movements of individual TM helices and of extracellular loops.

[Investigation of the conformational dynamics of the adenosine A2A receptor by means of molecular dynamics simulation].

In this work structural behavior of apo form of the adenosine A2A receptor in the implicit membrane-mimicking environment was investigated by means of molecular dynamics (MD) technique. For better interpretation of the obtained data they were analyzed using principal components analysis. The principal components analysis technique was applied to both MD snapshots as well as X-ray structures of the adenosine receptor. As the result the charts were obtained which reflected an interconnection interdependence between dynamic behavior of the receptor observed on the MD trajectories as well as experimental dataset of investigated protein. The calculated MD trajectories allow to observe represent pronounced structural dynamics of the A2A receptor especially in the intracellular part loop connecting TM 5 and 6 of that protein. This observation generally corresponds to the dynamic behavior of the investigated protein seen on the experimental dataset. Therefore the pattern of the intramolecular motions might be following directly from the spatial architecture (fold) of the receptor under study.

Classification of rhodopsin structures by modern methods of structural bioinformatics.

We report a classification of the crystallographic structures of bovine and squid rhodopsins corresponding to different stages of their photocycles. Using the resource Protein (Structure) Comparison, Knowledge, Similarity, and Information server (ProCKSI, http://www.procksi.net/), selected spatial structures were compared on the basis of classification schemes (dendrograms). To compare the spatial structures of transmembrane proteins, optimal consensus was developed from methods implemented in ProCKSI. Structures were also clustered using principal component analysis, resulting in good agreement with the classification based on the ProCKSI consensus method. Analysis of the results revealed the basic movements of individual transmembrane domains of these proteins that we were able to relate to different stages of the photoactivation of rhodopsin. A combination of methods identified in this study can be used as an up-to-date analytical tool to study the conformational dynamics of membrane receptors.

Recognition rules for binding of homeodomains to operator DNA.

The spatial arrangement of interfaces between homeodomain transcription factors and operator DNA has been considered. We analyzed the binding contacts for a representative set of 22 complexes of homeodomain transcription factors with a double-stranded operator DNA in the region of the major groove. It was shown that the recognition of DNA by the recognizing _-helix of protein is governed by two contact groups. Invariant protein-DNA group of contacts includes six contacts, formed by atomic groups of coding and non-coding DNA chains with the groups of amino acids. The recognizing _-helix forms contacts by polar groups of residues Trp2 (NE1), Asn5, and Lys9 with the canonical sequence T(1)A(2)A(3)T(4) of the coding DNA chain, and contacts by residues Lys0, Arg7 and Lys11 with the sequence A(4)X(5)X(6)X(7) of a non-coding DNA chain, where X is any nucleotide. Variable protein-DNA group of contacts comprises two groups bound with the sequence T(3)A(4)X(5)X(6) of the non- coding DNA-chain. These contacts are mainly with the bases and specify the binding pattern of individual homeodomains. The invariant contact group represents a recognition pattern for transcription factors of the homeodomain family: multiple adenine-asparagine contact and six position-specific phosphate contacts mainly with lysine or arginine. Within this group, we have found three most significant invariant contacts which allow deducing the recognition rules for homeodomains. These rules are inherent for different taxonomic groups of the homeodomain family and can distinguishing members of this family from any other family of transcription factors.

Binding regularities in complexes of transcription factors with operator DNA: homeodomain family.

In order to disclose general regularities of binding in homeodomain-DNA complexes we considered five of them and extended the observed regularities over the entire homeodomain family. The five complexes have been selected by similarity of protein structures and patterns of contacting residues. Their long range interactions and interfaces were compared. The long-range stage of the recognition process was characterized by electrostatic potentials about 5 Angstrom away from molecular surfaces of protein or DNA. For proteins, clear positive potential is displayed only at the side contacting the DNA. The double-chained DNA molecule displays a rather strong negative potential, especially in their grooves. Thus, a functional role of electrostatics is a guiding of the protein into the DNA major groove, so the protein and DNA could form a loose non-specific complex. At the close-range stage, neutralization of the phosphate charges by positively charged residues is necessary for decreasing the strong electrostatic potential of DNA, allowing nucleotide bases to participate in the formation of protein-DNA atomic contacts in the interface. The recognizing alpha-helix of protein was shown to form both invariant and variable groups of contacts with DNA by means of certain specific side groups. The invariant contacts included highly specific protein-DNA hydrogen bonds between asparagine and adenine, nonpolar contacts of hydrophobic amino acids serving as a stereochemical barrier for fixing the protein factor on DNA, and an interface cluster of water molecules providing local conformational mobility necessary for the dissociation process. There is a unique water molecule within the interface that is conservative and located at the interface center. Invariant contacts of the proteins are mostly formed with the TAAT motif of the promoter DNA forward strand. While the invariant contacts specify the family of homeodomains, the variable contacts that are formed with the reverse strand of DNA provide specificity of individual complexes within the homeodomain family.

Ferrocyanide - a novel catalyst for oxymyoglobin oxidation by molecular oxygen.

A comparative study of the rates of ferrocyanide-catalyzed oxidation of several oxymyoglobins by molecular oxygen is reported. Oxidation of the native oxymyoglobins from sperm whale, horse and pig, as well as the chemically modified (MbO(2)) sperm whale oxymyoglobin, with all accessible His residues alkylated by sodium bromoacetate (CM-MbO(2)), and the mutant sperm whale oxymyoglobin [MbO(2)(His119-->Asp)], was studied. The effect of pH, ionic strength and the concentration of anionic catalyst ferrocyanide, [Fe(CN)(6)](4-), on the oxidation rate is investigated, as well as the effect of MbO(2) complexing with redox-inactive Zn(2+), which forms the stable chelate complex with functional groups of His119, Lys16 and Asp122, all located nearby. The catalytic mechanism was demonstrated to involve specific [Fe(CN)(6)](4-) binding to the protein in the His119 region, which agrees with a high local positive electrostatic potential and the presence of a cavity large enough to accommodate [Fe(CN)(6)](4-) in that region. The protonation of the nearby His113 and especially His116 plays a very important role in the catalysis, accelerating the oxidation rate of bound [Fe(CN)(6)](4-) by dissolved oxygen. The simultaneous occurrence of both these factors (i.e. specific binding of [Fe(CN)(6)](4-) to the protein and its fast reoxidation by oxygen) is necessary for the efficient ferrocyanide-catalyzed oxidation of oxymyoglobin.

[The mechanism of oxymyoglobin oxidation catalyzed by ferrocyanide ions: chemically modified and mutant sperm whale myoglobins].

A comparative study of the rate of ferrocyanide-catalyzed oxidation of native sperm whale MbO2, its chemically modified derivative in which all accessible His residues are alkylated by sodium bromoacetate, (CM-MbO2), and mutant sperm whale MbO2 with His119 replaced by Asp residiue, [MbO2(His119-->Asp)] was carried out. The influence of pH, ionic strength, and [Fe(CN)6]4- concentration on the oxidation rate was investigated, as well as the effect of complexing MbO2 with redox-inactive Zn2+ ion, which, at the equimolar Zn2+ concentration, forms a stable complex with His119(GH1) on the protein surface. It was shown that the mechanism of the catalysis involves specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) region, which is in agreement with a large positive electrostatic potential and the presence at this site of Mb of a cavity large enough to accommodate [Fe(CN)6]4- anion. The protonation of nearby His113 and His116 residiues (especially of the latter) plays a very important role in the catalysis, promoting the fast oxidation of bound [Fe(CN)6]4- by dissolved oxygen. Only the presence of these both necessary conditions in MbO2 structure provides its effective oxydation catalyzed by ferrocyanide.

[The oxidation of sperm whale, horse, and pig oxymyoglobins, catalyzed by ferrocyanide ions: kinetics and mechanism].

The influence of pH, ionic strength of the solution, and [Fe(CN)6]4- concentration on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins, which is catalyzed by ferrocyanide ions, was studied. These myoglobins have homologous spatial structures and identical redox potentials but differ by the amount of His residues located on the protein surface. The effect of the MbO2 complexing with redox-inactive Zn2+ ion on the reaction rate was also examined. At the equimolar Zn2+ concentration, zinc ions form a stable complex with His119(GH1). It was found that the kinetic behavior of horse MbO2, which lacks His12(A10) substituted for by Gln, is fully analogous to one of sperm whale MbO2, while the oxidation of pig MbO2, three histidines of which, His12, His113(G14), and His116(G17), are replaced by Gln, is strongly inhibited. The mechanism of the catalysis was shown to involve specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) site, which is in accord with the large positive electrostatic potential of this site and the presence here of a cavity large enough to accommodate [Fe(CN)6]4-. The nearby His113 and His116 residiues, which are absent in pig Mb, also play a very important role in the catalysis, because their protonation (especially of the last residue) is most likely responsible for the week oxidation of bound [Fe(CN)6]4- by dissolved oxygen.

[Catalytic effect of ferricyanide on the rate of electron transfer between myoglobin and cytochrome c]. / Kataliticheskii éffekt ferritsianida na skorost' perenosa élektrona mezhdu mioglobinom i tsitokhromom c.

The influence of small amounts of low-molecular electron acceptor, potassium ferricyanide, 1 to 20% relative to the cytohrome c concentration, on the rate of electron transfer in the sperm whale oxymyoglobin--horse heart cytochrome c and deoxymyoglobin--cytochrome c systems (under aerobic and anaerobic conditions, respectively) was studied. At low ionic strength, the redox reaction rate was found to increase proportionally to the concentration of ferricyanide in both redox systems. The effect depends on pH in the pH range 5-8, increasing sharply at pH < 6. It was shown that the enhancing of electron transfer is caused by the complexing of [Fe(CN)6]3- with cytohrome c in the Lys72 region, where one of the two strong binding sites for this anion is determined by NMR. Both the high ionic strength and the chemical modification of Lys72 residue inhibit this effect at low ionic strength, markedly decreasing the rate of reaction with myoglobin. Under the same conditions, the effect of ferricyanide in the reaction of oxy-Mb with yeast cytohrome c, which is isopotential to animal cytochromes c but possesses trimethylated Lys72, was several times smaller. In turn, the chemical modification of His residues in myoglobin and the complexing of zinc ion to His119(GH1) almost completely inhibit electron transfer in the systems. Thus, electron transfer between the proteins must proceed through the formation of the Mb.[Fe(CN)6]3-.Cyt c ternary complex, the contacting sites being localized in the His119(GH1) region of myoglobin and near Lys72 of cytohrome c. The increased electron transfer rate in the presence of [Fe(CN)6]3- can be explained by that its binding near Lys72, firstly, provides better electrostatic interactions in the electron transfer complex and, besides, decreases significantly (about 2-fold) the tunneling distance between the two hemes (two lengths of 1.7 and 1.2 nm instead of one of 2.9 nm).

[Characteristics of electrostatic interaction of Escherichia coli RNA polymerase with promoters of T4 phage DNA]. / Osobennosti élektrostaticheskogo vzaimodeistviia RNA-polymerasy Escherichia coli s promotorami DNK faga T4.

A comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken. The data obtained indicate that there are some particular elements in the patterns of electrostatic potential distribution of promoter DNA specific for promoter groups differing by their functional response to ADP-ribosylation of the alpha-subunit as well as to rpoB403- or rpoB409 mutationals of the beta-subunit of RNA-polymerase.

RNA polymerase--promoter recognition. Specific features of electrostatic potential of "early" T4 phage DNA promoters.

Comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken, along with calculation of topography of electrostatic potential around the native and ADP-ribosylated C-terminal domain of RNA polymerase alpha-subunit. The data obtained indicate that there is specific difference in the patterns of electrostatic potential distribution in far upstream regions of T4 promoters differing by their response to ADP-ribosylation of RNA polymerase. A specific change in profiles of electrostatic potential distribution for the native and ADP-ribosylated forms of RNA polymerase alpha-subunit was observed suggesting that this factor may be responsible for modulating T4 promoter activities in response to the enzyme modification.

[Oxidation of sperm whale oxymyoglobin, catalyzed by ferrocyanide ions: kinetics and mechanisms]. / Okislenie oksimioglobina kashalota, kataliziruemoe ionami ferrotsianida: kinetika i mekhanizm.

Specific catalytic oxidation of sperm whale oxymyoglobin by small amounts of potassium ferri- and ferrocyanide, from 1 to 20% in relation to the protein concentration, was studied. The mechanism of catalysis was shown to involve specific binding of the ferrocyanide anion to the protein. The influence of pH and ionic strength of the medium, the [Fe(CN)6]4- concentration and of chemical modification of Mb histidines by bromoacetate, as well as the effect of the Mb complexing with redox-inactive zinc ion on the rate of reaction was examined. The zinc ion forms a stable complex with His 119(GH1) on the Mb surface at the equimolar Zn2+ concentration. The kinetic scheme of the reaction was analyzed, and the equilibrium and kinetic parameters were obtained. It was first shown that the strong oxidant such as potassium ferricyanide is able to react with the same protein by two distinct mechanisms: (i) a simple outer sphere electron transfer over the heme edge and (ii) electron transfer after the specific binding of [Fe(CN)6]4- to oxyMb in the His 119(GH1) region, thus catalyzing the protein oxidation.

Electrostatic potentials of DNA. Comparative analysis of promoter and nonpromoter nucleotide sequences.

Distribution of electrostatic potential of DNA fragments was evaluated. A method for calculation of electrostatic potential distribution based on Coulomb's law is proposed for long DNA fragments (approximately 1000 nucleotide pairs). For short DNA sequences, this technique provides a good correlation with the results obtained using Poisson-Boltzmann equation thus justifying its application in comparative studies for long DNA fragments. Calculation was performed for several DNA fragments from E. coli and bacteriophage T7 genomes containing promoter and nonpromoter regions. The results obtained indicate that coding regions are characterized by more homogeneous distribution of electrostatic potential whereas local inhomogeneity of DNA electrostatic profile is typical for promoter regions. The possible role of electrostatic interactions in RNA polymerase-promoter recognition is discussed.

[Applicability of molecular electrostatic interaction models to describing ionic strength dependence of reaction rate between myoglobin and cytochrome c]. / Primenimost' modelei élektrostaticheskogo vzaimodeistviia molekul k opisaniiu zavisimosti skorosti reaktsii mezhdu mioglobinom i tsitokhromom s ot ionnoi sily.

The nonlinear regression method was used for the evaluation of applicability of the known model equations (Wherland-Gray, Brönsted-Debay-Hukkel and "parallel disks") which describe the ionic strength dependence of the reaction rate between charged molecules to the redox reaction of cytochrome c with sperm whale myoglobin modified at His 12(A10) with the bromoacetate spin label, and pig myoglobin. Unlike the native sperm whale Mb studied earlier [1], the objects chosen have monotonous pH-dependence of the reaction rate and are more simple with regards to electrostatic interactions in the electron-transfer complex. This allowed to study the influence of the total as well as the local protein charge on the correspondence of the ion strength dependencies to the theoretical models and optimal parameters of the equations. It was shown that the models considered, as in the case of the native sperm whale Mb-Cyt c reaction, permit satisfactory description of the experimental data, but the obtained parameters cannot be applied to the whole proteins or their contact sites. In the best case (Wherland-Gray equation) it is possible to do if the distribution of electrostatic potential in the contact area is considered. The reason can be that, unlike other protein redox-systems, the contact sites of both Mb and Cyt c have the charged residues of both signs, and the His GH1 residue located in the contact Mb site is not only involved in the electrostatic interactions in electron-transfer complex, but also participates directly in the mechanism of charge transfer.

[Electron transfer in hemoproteins. The applicability of models of electrostatic interaction of molecules for description of the relationship between the myoglobin and cytochrome reaction rate and the ionic strength]. / Izuchenie perenosa élektrona v gemoproteinakh. Primenimost' modelei élektrostaticheskogo vzaimodeistviia molekul k opisaniiu zavisimosti skorosti reaktsii mezhdu mioglobinom i tsitokhromom c ot ionnoi sily.

The nonlinear regression method was used for the evaluation of applicability of the known model equations that describe the ionic strength dependence of the rate of reaction between charged molecules to the electron transfer reaction between myoglobin and cytochrome c. The full and simplified equations of Bronsted-Debay-Hukkel and Wherland-Gray equation, as well as equations, derived from models of complementary interactions and "parallel disks" were used. The experimental dependences of reaction rate on ionic strength at all pH values in the pH interval 5-8 have a negative slope which corresponds to opposite charges of reacting particles and does not correspond (even in sign) to the total charges of Mb and Cyt c. It is found that all models considered, except for those of complementary interactions, permit a satisfactory description of the experimental data. In the case of "parallel disks" and Bronsted-Debay-Hukkel equations, however, the optimal values of Z1Z2 and R1 = R2 = R do not correspond to the real size of the Mb-Cyt c electron transfer complex and to the radii and charges at their contact sites found from the experiment. The Wherland-Gray equation allows the best approximation of the experimental ionic strength dependences assuming that Z1Z2 and R1 = R2 = R are some effective parameters that reflect the protein field distribution in the contact site. This distribution can be approximated as a monopole with parameters not coinciding with the parameters of the protein.

[Study of electron transport in heme proteins. X. Effect of pH, ionic strength, and zinc ions and the rate of ferricytochrome c reduction by oxymyoglobin from swine heart]. / Izuchenie perenosa élektrona v gemoproteidakh. X. Vliianie pH, ionnoi sily i ionov tsinka na skoros' vosstanovleniia ferritsitokhroma c oksimioglobinom iz serdtsa svin'i.

The rate of the redox reaction between porcine MbO2 and ferri-Cyt c at different ionic strengths in the pH range 5-8 has been studied. At low ionic strength (I = 0-0.1) the pH dependence curve was found to have a sigmoid shape with pKeff approximately 5.7, implying the effect of ionization of His-119(GH1) at the "active site" of myoglobin on the kinetics of the process. In this range of ionic strengths the rate of the reaction decreases sharply. The slope of the curve in the coordinates of IgKexp versus square root of I/1 + square root of I varies depending on pH. It is greater at pH less than or equal to 6 and smaller at pH 7.5, which is due to deprotonation of His(GH1). At high ionic strength (I greater than 0.1) the rate of electron transfer is negligible, independent of pH and does not practically change as I increases from 0.1 to 1. It is shown that the local electrostatic interactions play a decisive role in the formation of an efficient electron-transfer complex between Mb and Cyt c. The binding of the zinc ion to His(GH1) was found to inhibit the electron transfer at I = 0.01, similarly to what occurs at a high ionic strength, though the "reactive" charges of the proteins are not screened and the positive charge at His(GH1) is retained. This suggests that His(GH1) is directly involved in the mechanism of electron transfer from Mb to Cyt c. The data obtained are compared with earlier data on the effect of pH, ionic strength and zinc ions on the reaction between MbO2 from sperm whale and Cyt c. To explain the higher efficiency of pig MbO2 as electron donor, the electrostatic and steric properties of both myoglobins have been analyzed.