Recombinant Bet v 1-BanLec chimera modulates functional characteristics of peritoneal murine macrophages by promoting IL-10 secretion.

Allergen-specific immunotherapy (AIT) is a desensitizing treatment for allergic diseases that corrects the underlined pathological immune response to innocuous protein antigens, called allergens. Recombinant allergens employed in the AIT allowed the production of well-defined formulations that possessed consistent quality but were often less efficient than natural allergen extracts. Combining recombinant allergens with an adjuvant or immunomodulatory agent could improve AIT efficacy. This study aimed to perform structural and functional characterization of newly designed recombinant chimera composed of the Bet v 1, the major birch pollen allergen, and Banana Lectin (BanLec), TLR2, and CD14 binding protein, for the application in AIT. rBet v 1-BanLec chimera was designed in silico and expressed as a soluble fraction in Escherichia coli. Purified rBet v 1-BanLec (33.4 kDa) retained BanLec-associated biological activity of carbohydrate-binding and preserved IgE reactive epitopes of Bet v 1. The chimera revealed secondary structures with predominant ß sheets. The immunomodulatory capacity of rBet v 1-BanLec tested on macrophages showed changes in myeloperoxidase activity, reduced NO production, and significant alterations in the production of cytokines when compared to both rBanLec and rBet v 1. Comparing to rBet v 1, rBet v 1-BanLec was demonstrated to be more efficient promoter of IL-10 production as well as weaker inducer of NO production and secretion of pro-inflammatory cytokines TNFα, and IL-6. The ability of rBet v 1-BanLec to promote IL-10 in together with the preserved 3D structure of Bet v 1 part implies that the construct might exert a beneficial effect in the allergen-specific immunotherapy.

Self-Assembly and Biorecognition of a Spirohydantoin Derived from α-Tetralone: Interplay between Chirality and Intermolecular Interactions.

A racemic spirohydantoin derivative with two aromatic substituents, a tetralin and a 4-methoxybenzyl unit, was synthesized and its crystal structure was determined. To define the relationship between molecular stereochemistry and spatial association modes, development of the crystal packing was analyzed through cooperativity of intermolecular interactions. Homo and heterochiral dimeric motifs were stabilized by intermolecular N-H⋅⋅⋅O, C-H⋅⋅⋅O, C-H⋅⋅⋅π interactions and parallel interactions at large offsets (PILO), thus forming alternating double layers. The greatest contribution to the total stabilization came from a motif of opposite enantiomers linked by N-H⋅⋅⋅O bonds (interaction energy=-13.72 kcal/mol), followed by a homochiral motif where the 4-methoxybenzyl units allowed C-H⋅⋅⋅π, C-H⋅⋅⋅O interactions and PILO (interaction energy=-11.56 kcal/mol). The number of the contact fragments in the environment of the tetralin unit was larger, but the 4-methoxybenzyl unit had greater contribution to the total stabilization. The statistical analysis of the data from the Cambridge Structural Database (CSD) showed that this is a general trend. The compound is a potential inhibitor of kinase enzymes and antigen protein-coupled receptors. A correlation between the docking study and the results of the CSD analysis can be drawn. Due to a greater flexibility, the 4-methoxybenzyl unit is more adaptable for interactions with the biological targets than the tetralin unit.

Current advances in research of cytochrome c oxidase.

The function of cytochrome c oxidase as a biomolecular nanomachine that transforms energy of redox reaction into protonmotive force across a biological membrane has been subject of intense research, debate, and controversy. The structure of the enzyme has been solved for several organisms; however details of its molecular mechanism of proton pumping still remain elusive. Particularly, the identity of the proton pumping site, the key element of the mechanism, is still open to dispute. The pumping mechanism has been for a long time one of the key unsolved issues of bioenergetics and biochemistry, but with the accelerating progress in this field many important details and principles have emerged. Current advances in cytochrome oxidase research are reviewed here, along with a brief discussion of the most complete proton pumping mechanism proposed to date, and a molecular basis for control of its efficiency.

Coupled electron and proton transfer reactions during the O→E transition in bovine cytochrome c oxidase.

A combined DFT/electrostatic approach is employed to study the coupling of proton and electron transfer reactions in cytochrome c oxidase (CcO) and its proton pumping mechanism. The coupling of the chemical proton to the internal electron transfer within the binuclear center is examined for the O→E transition. The novel features of the His291 pumping model are proposed, which involve timely well-synchronized sequence of the proton-coupled electron transfer reactions. The obtained pK(a)s and E(m)s of the key ionizable and redox-active groups at the different stages of the O→E transition are consistent with available experimental data. The PT step from E242 to H291 is examined in detail for various redox states of the hemes and various conformations of E242 side-chain. Redox potential calculations of the successive steps in the reaction cycle during the O→E transition are able to explain a cascade of equilibria between the different intermediate states and electron redistribution between the metal centers during the course of the catalytic activity. All four electrometric phases are discussed in the light of the obtained results, providing a robust support for the His291 model of proton pumping in CcO.

Improved density functional theory/electrostatic calculation of the His291 protonation state in cytochrome C oxidase: self-consistent charges for solvation energy calculation.

The protonation state of His291 in cytochrome c oxidase (CcO), a ligand to the Cu(B) center of the enzyme, has been recently studied in this group by using combined density functional theory (DFT)/electrostatic (QM/MM) calculations. On the basis of these calculations, a model of the proton pumping mechanism of CcO has been proposed. Due to certain technical difficulties, the procedure used in the previous calculation to find partial atomic charges of the QM system for the solvation energy evaluation was not entirely satisfactory; i.e., it was not self-consistent. Here, we describe a procedure that resolves the problem and report on the improved calculations of the protonation state of the His residue. The new procedure fits the protein and reaction field potentials in the region of the QM system with artificial point charges placed on a surface of a sphere surrounding the QM system and a few charges inside the sphere and allows one to perform DFT calculations that involve an inhomogeneous dielectric environment in a self-consistent way. The procedure improves the accuracy of calculations in comparison with previous work. The improved results show, however, that although the absolute energies change significantly the relative energies of the protonated and deprotonated states of His291 remain close to the previously reported ones and therefore do not change significantly the pK(a) values reported earlier. Therefore, our new improved calculations support for the proposed His291 model of the CcO pump.

Two conformational states of Glu242 and pKas in bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain of aerobic organisms. It catalyses the reduction of atmospheric oxygen to water, and couples this reaction to proton pumping across the membrane; this process generates the electrochemical gradient that subsequently drives the synthesis of ATP. The molecular details of the mechanism by which electron transfer is coupled to proton pumping in CcO is poorly understood. Recent calculations from our group indicate that His291, a ligand of the Cu(B) center of the enzyme, may play the role of the pumping element. In this paper we describe calculations in which a DFT/continuum electrostatic method is used to explore the coupling of the conformational changes of Glu242 residue, the main proton donor of both chemical and pump protons, to its pKa, and the pKa of His291, a putative proton loading site of our pumping model. The computations are done for several redox states of metal centers, different protonation states of Glu242 and His291, and two well-defined conformations of the Glu242 side chain. Thus, in addition to equilibrium redox/protonation states of the catalytic cycle, we also examine the transient and intermediate states. Different dielectric models are employed to investigate the robustness of the results, and their viability in the light of the proposed proton pumping mechanism of CcO. The main results are in agreement with the experimental measurements and support the proposed pumping mechanism. Additionally, the present calculations indicate a possibility of gating through conformational changes of Glu242; namely, in the pumping step, we find that Glu242 needs to be reprotonated before His291 can eject a proton to the P-site of membrane. As a result, the reprotonation of Glu can control proton release from the proton loading site.

Combined DFT and electrostatics study of the proton pumping mechanism in cytochrome c oxidase.

Cytochrome c oxidase is a redox-driven proton pump which converts atmospheric oxygen to water and couples the oxygen reduction reaction to the creation of a membrane proton gradient. The structure of the enzyme has been solved; however, the mechanism of proton pumping is still poorly understood. Recent calculations from this group indicate that one of the histidine ligands of enzyme's CuB center, His291, may play the role of the pumping element. In this paper, we report on the results of calculations that combined first principles DFT and continuum electrostatics to evaluate the energetics of the key energy generating step of the model-the transfer of the chemical proton to the binuclear center of the enzyme, where the hydroxyl group is converted to water, and the concerted expulsion of the proton from delta-nitrogen of His291 ligand of CuB center. We show that the energy generated in this step is sufficient to push a proton against an electrochemical membrane gradient of about 200 mV. We have also re-calculated the pKa of His291 for an extended model in which the whole Fe(a3)-CuB center with their ligands is treated by DFT. Two different DFT functionals (B3LYP and PBE0), and various dielectric models of the protein have been used in an attempt to estimate potential errors of the calculations. Although current methods of calculations do not allow unambiguous predictions of energetics in proteins within few pKa units, as required in this case, the present calculation provides further support for the proposed His291 model of CcO pump and makes a specific prediction that could be targeted in the experimental test.

Proton exit channels in bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal transmembrane enzyme of the respiratory electron transport chain in aerobic cells. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping, a process which results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping and proton exit pathways remain unknown. In our previous work, the continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine cytochrome c oxidase for different redox states of the enzyme. A possible mechanism of oxygen reduction and proton pumping via His291 was proposed. In this paper, using electrostatic calculations, we examine the proton exit pathways in the enzyme. By monitoring the changes of the protonation states, proton affinities, and energies of electrostatic interactions between the titratable groups in different redox states of CcO, we identified the clusters of strongly interacting residues. Using these data, we detected four possible proton exit points on the periplasmic side of the membrane (Lys171B/Asp173B, His24B/Asp25B, Asp51, and Asp300). We then were able to trace the proton exit pathways and to evaluate the energy profiles along the paths. On the basis of energetic considerations and the conservation of the residues in a protein sequence, the most likely exit pathway is one via the Lys171B/Asp173B site. The obtained results are fully consistent with our His291 model of proton pumping, and provide a rationale for the absence of proton leaking in CcO between the pumping strokes.

DFT/electrostatic calculations of pK(a) values in cytochrome c oxidase.

Using classical electrostatic calculations, earlier we examined the dependence of the protonation state of bovine cytochrome c oxidase (CcO) on its redox state. Based on these calculations, we have proposed a model of CcO proton pumping that involves His291, one of the Cu(B) histidine ligands, which was found to respond to redox changes of the enzyme Fe(a)(3)-Cu(B) catalytic center. In this work, we employ combined density functional and continuum electrostatic calculations to evaluate the pK(a)() values of His291 and Glu242, two key residues of the model. The pK(a) values are calculated for different redox states of the enzyme, and the influence of different factors on the pK(a)'s is analyzed in detail. The calculated pK(a)() values of Glu242 are between 9.4 and 12.0, depending on the redox state of the protein, which is in excellent agreement with recent experimental measurements. Assuming the reduced state of heme a(3), His291 of the oxidized Cu(B) center possesses a pK(a)() between 2.1 and 4.0, while His291 of the reduced Cu(B) center has a pK(a) above 17. The obtained results support the proposal that the His291 ligand of the Cu(B) center in CcO is a proton pump element.

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating.

Using electrostatic calculations, we have examined the dependence of the protonation state of cytochrome c oxidase from bovine heart on its redox state. Based on these calculations, we propose a possible scheme of redox-linked proton pumping. The scheme involves His291 - one of the ligands of the Cu(B) redox center - which plays the role of the proton loading site (PLS) of the pump. The mechanism of pumping is based on ET reaction between two hemes of the enzyme, which is coupled to a transfer of two protons. Upon ET, the first proton (fast reaction) is transferred to the PLS (His291), while subsequent transfer of the second "chemical" proton to the binuclear center (slow reaction) is accompanied by the ejection of the first (pumped) proton. Within the proposed model, we discuss the catalytic cycle of the enzyme.

Electrostatic study of the proton pumping mechanism in bovine heart cytochrome C oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme of the cell respiratory chain in mitochondria and aerobic bacteria. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping across the inner-mitochondrial membrane, a process that results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping remains unknown. In the present paper, continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine heart cytochrome c oxidase for different redox states of the enzyme along its catalytic cycle. Three different computational models of the enzyme were employed to test the stability of the results. The energetics and pH dependence of the P-->F, F-->O, and O-->E steps of the cycle have been investigated. On the basis of electrostatic calculations, two possible schemes of redox-linked proton pumping are discussed. The first scheme involves His291 as a pump element, whereas the second scheme involves a group linked to propionate D of heme a(3). In both schemes, loading of the pump site is coupled to ET between the two hemes of the enzyme, while transfer of a chemical proton is accompanied by ejection of the pumped H(+). The two models, as well as the energetics results are compared with recent experimental kinetic data. The proton pumping across the membrane is an endergonic process, which requires a sufficient amount of energy to be provided by the chemical reaction in the active site. In our calculations, the conversion of OH(-) to H(2)O provides 520 meV of energy to displace pump protons from a loading site and overall about 635 meV for each electron passing through the system. Assuming that the two charges are translocated per electron against the membrane potential of 200 meV, the model predicts an overall efficiency of 63%.

Energetics of radical transfer in DNA photolyase.

Charge separation and radical transfer in DNA photolyase from Escherichia coli is investigated by computing electrostatic free energies from a solution of the Poisson-Boltzmann equation. For the initial charge separation 450 meV are available. According to recent experiments [Aubert et al. Nature 2000, 405, 586-590] the flavin receives an electron from the proximal tryptophan W382, which consequently forms a cationic radical WH(*)(+)382. The radical state is subsequently transferred along the triad W382-W359-W306 of conserved tryptophans. The radical transfer to the intermediate tryptophan W359 is nearly isoenergetic (58 meV uphill); the radical transfer from the intermediate W359 to the distal W306 is 200 meV downhill in energy, funneling and stabilizing the radical state at W306. The resulting cationic radical WH(*)(+)306 is further stabilized by deprotonation, yielding the neutral radical W(*)306, which is 214 meV below WH(*)(+)306. The time scale of the charge recombination process yielding back the resting enzyme with FADH(*) is governed by reprotonation of W306, with a calculated lifetime of 1.2 ms that correlates well with the measured lifetime of 17 ms. In photolyase from Anacystis nidulans the radical state is partially transferred to a tyrosine [Aubert et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5423-5427]. In photolyase from Escherichia coli, there is a tyrosine (Y464) close to the distal tryptophan W306 that could play this role. We show that this tyrosine cannot be involved in radical transfer, because the electron transfer from tyrosine to W306 is much too endergonic (750 meV) and a direct hydrogen transfer is likely too slow. Coupling of specific charge states of the tryptophan triad with protonation patterns of titratable residues of photolyase is small.