Mónika Bálint

Interaction of mycotoxin zearalenone with human serum albumin

Zearalenone (ZEN) is a mycotoxin produced mainly by Fusarium species. Fungal contamination of cereals and plants can result in the formation of ZEN, leading to its presence in different foods, animal feeds, and drinks. Because ZEN is an endocrine disruptor, it causes reproductive disorders in farm animals and hyperoestrogenic syndromes in humans. Despite toxicokinetic properties of ZEN were studied in more species, we have no information regarding the interaction of ZEN with serum albumin. Since albumin commonly plays an important role in the toxicokinetics of different toxins, interaction of ZEN with albumin has of high biological importance. Therefore the interaction of ZEN with human serum albumin (HSA) was investigated using spectroscopic methods, ultrafiltration, and molecular modeling studies. Fluorescence spectroscopic studies demonstrate that ZEN forms complex with HSA. Binding constant (K) of ZEN-HSA complex was quantified with fluorescence quenching technique. The determined binding constant (logK=5.1) reflects the strong interaction of ZEN with albumin suggesting the potential biological importance of ZEN-HSA complex formation. Based on the results of the investigations with site markers as well as docking studies, ZEN occupies a non-conventional binding site on HSA. Considering the above listed observations, we should keep in mind this interaction if we would like to precisely understand the toxicokinetic behavior of ZEN.

Interaction of Citrinin with Human Serum Albumin

Citrinin (CIT) is a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. CIT occurs worldwide in different foods and drinks and causes health problems for humans and animals. Human serum albumin (HSA) is the most abundant plasma protein in human circulation. Albumin forms stable complexes with many drugs and xenobiotics; therefore, HSA commonly plays important role in the pharmacokinetics or toxicokinetics of numerous compounds. However, the interaction of CIT with HSA is poorly characterized yet. In this study, the complex formation of CIT with HSA was investigated using fluorescence spectroscopy and ultrafiltration techniques. For the deeper understanding of the interaction, thermodynamic, and molecular modeling studies were performed as well. Our results suggest that CIT forms stable complex with HSA (logK ~ 5.3) and its primary binding site is located in subdomain IIA (Sudlow’s Site I). In vitro cell experiments also recommend that CIT-HSA interaction may have biological relevance. Finally, the complex formations of CIT with bovine, porcine, and rat serum albumin were investigated, in order to test the potential species differences of CIT-albumin interactions.

Mobility-based prediction of hydration structures of protein surfaces

MOTIVATION Hydration largely determines solubility, aggregation of proteins and influences interactions between proteins and drug molecules. Despite the importance of hydration, structural determination of hydration structure of protein surfaces is still challenging from both experimental and theoretical viewpoints. The precision of experimental measurements is often affected by fluctuations and mobility of water molecules resulting in uncertain assignment of water positions. RESULTS Our method can utilize mobility as an information source for the prediction of hydration structure. The necessary information can be produced by molecular dynamics simulations accounting for all atomic interactions including water-water contacts. The predictions were validated and tested by comparison to more than 1500 crystallographic water positions in 20 hydrated protein molecules including enzymes of biomedical importance such as cyclin-dependent kinase 2. The agreement with experimental water positions was larger than 80% on average. The predictions can be particularly useful in situations where no or limited experimental knowledge is available on hydration structures of molecular surfaces. AVAILABILITY AND IMPLEMENTATION The method is implemented in a standalone C program MobyWat released under the GNU General Public License, freely accessible with full documentation at http://www.mobywat.com.

Investigation of Non-Covalent Interactions of Aflatoxins (B1, B2, G1, G2, and M1) with Serum Albumin

Aflatoxins are widely spread mycotoxins produced mainly by Aspergillus species. Consumption of aflatoxin-contaminated foods and drinks causes serious health risks for people worldwide. It is well-known that the reactive epoxide metabolite of aflatoxin B1 (AFB1) forms covalent adducts with serum albumin. However, non-covalent interactions of aflatoxins with human serum albumin (HSA) are poorly characterized. Thus, in this study the complex formation of aflatoxins was examined with HSA applying spectroscopic and molecular modelling studies. Our results demonstrate that aflatoxins form stable complexes with HSA as reflected by binding constants between 2.1 × 104 and 4.5 × 104 dm3/mol. A binding free energy value of −26.90 kJ mol−1 suggests a spontaneous binding process between AFB1 and HSA at room-temperature, while the positive entropy change of 55.1 JK−1 mol−1 indicates a partial decomposition of the solvation shells of the interacting molecules. Modeling studies and investigations with site markers suggest that Sudlow’s Site I of subdomain IIA is the high affinity binding site of aflatoxins on HSA. Interaction of AFB1 with bovine, porcine, and rat serum albumins was also investigated. Similar stabilities of the examined AFB1-albumin complexes were observed suggesting the low species differences of the albumin-binding of aflatoxins.

Exploration of Interfacial Hydration Networks of Target-Ligand Complexes.

Interfacial hydration strongly influences interactions between biomolecules. For example, drug-target complexes are often stabilized by hydration networks formed between hydrophilic residues and water molecules at the interface. Exhaustive exploration of hydration networks is challenging for experimental as well as theoretical methods due to high mobility of participating water molecules. In the present study, we introduced a tool for determination of the complete, void-free hydration structures of molecular interfaces. The tool was applied to 31 complexes including histone proteins, a HIV-1 protease, a G-protein-signaling modulator, and peptide ligands of various lengths. The complexes contained 344 experimentally determined water positions used for validation, and excellent agreement with these was obtained. High-level cooperation between interfacial water molecules was detected by a new approach based on the decomposition of hydration networks into static and dynamic network regions (subnets). Besides providing hydration structures at the atomic level, our results uncovered hitherto hidden networking fundaments of integrity and stability of complex biomolecular interfaces filling an important gap in the toolkit of drug design and structural biochemistry. The presence of continuous, static regions of the interfacial hydration network was found necessary also for stable complexes of histone proteins participating in chromatin assembly and epigenetic regulation.

Comparative investigation of the in vitro inhibitory potencies of 13-epimeric estrones and D-secoestrones towards 17β-hydroxysteroid dehydrogenase type 1.

Abstract The inhibitory effects of 13-epimeric estrones, D-secooxime and D-secoalcohol estrone compounds on human placental 17β-hydroxysteroid dehydrogenase type 1 isozyme (17β-HSD1) were investigated. The transformation of estrone to 17β-estradiol was studied by an in vitro radiosubstrate incubation method. 13α-Estrone inhibited the enzyme activity effectively with an IC50 value of 1.2 μM, which indicates that enzyme affinity is similar to that of the natural estrone substrate. The 13β derivatives and the compounds bearing a 3-hydroxy group generally exerted stronger inhibition than the 13α and 3-ether counterparts. The 3-hydroxy-13β-D-secoalcohol and the 3-hydroxy-13α-D-secooxime displayed an outstanding cofactor dependence, i.e. more efficient inhibition in the presence of NADH than NADPH. The 3-hydroxy-13β-D-secooxime has an IC50 value of 0.070 μM and is one of the most effective 17β-HSD1 inhibitors reported to date in the literature.

Mob-family kinase co-activators bind cognate Ndr/Lats kinases through conserved and modular interface

Ndr/Lats kinases bind to Mob coactivator proteins and their complexes play important roles in Hippo signaling pathways controlling cell proliferation and morphogenesis. All Ndr/Lats kinases have a 70-80 amino acid long unique N-terminal region (NTR) which binds to Mob factors. In order to gain insight into the structural basis of kinase-coactivator binding specificity, we have determined the crystal structure of Cbk1(NTR)-Mob2 and Dbf2(NTR)-Mob1 complexes from yeast (S. cerevisiae). We show that the Ndr/Lats(NTR)-Mob interface is a common structural platform through which kinase-cofactor binding is mediated, albeit amino acid variations in key positions contribute to subgroup and organism-specific differences. We further show that conserved residues at the NTR-Mob interface may participate in novel activation mechanisms likely ubiquitous in Ndr/Lats kinases. Ndr/Lats kinase activation may resemble to that of other AGC kinases but with an extra structural requirement for NTR mediated Mob binding for proper allosteric activation.Ndr/Lats kinases bind Mob coactivator proteins to form complexes that are essential and deeply conserved components of “Hippo” signaling pathways, which control cell proliferation and morphogenesis in eukaryotes. All Ndr/Lats kinases have a characteristic N-terminal region (NTR) that binds a specific Mob co-factor: Lats kinases associate with Mob1 proteins, and Ndr kinases associate with Mob2 proteins. To better understand the functional significance of Mob protein association with Ndr/Lats kinases and selective binding of Ndr and Lats to distinct Mob co-factors, we solved crystal structures of Saccharomyces cerevisiae Cbk1(NTR)-Mob2 and Dbf2(NTR)-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This significantly refines the previously determined structure of Cbk1 kinase bound to Mob2, presently the only crystallographic model of a full length Ndr/Lats kinase complexed with a Mob cofactor. Our analysis indicates that the Ndr/Lats NTR-Mob interface provides a distinctive kinase regulation mechanism, in which Mob co-factor organizes the Ndr/Lats NTR to interact with the AGC kinase C-terminal hydrophobic motif (HM) activation segment. The Mob-organized NTR appears to mediate HM association with an allosteric site on the kinase N-lobe. We also found that Cbk1 and Dbf2 associated highly specifically with Mob2 and Mob1, respectively. Alteration of specific positions in the Cbk1 NTR allows association of non-cognate Mob co-factor, indicating that cofactor specificity is restricted by discrete sites rather than broadly distributed. Overall, our analysis provides a new picture of the functional role of Mob association and indicates that the Ndr/Lats(NTR)-Mob interface overall is largely a common structural platform that mediates kinase-cofactor binding.

Interaction of 2′R-ochratoxin A with Serum Albumins: Binding Site, Effects of Site Markers, Thermodynamics, Species Differences of Albumin-binding, and Influence of Albumin on Its Toxicity in MDCK Cells

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. Roasting of OTA-contaminated coffee results in the formation of 2′R-ochratoxin A (2′R-OTA), which appears in the blood of coffee drinkers. Human serum albumin (HSA) binds 2′R-OTA (and OTA) with high affinity; therefore, albumin may influence the tissue uptake and elimination of ochratoxins. We aimed to investigate the binding site of 2′R-OTA (verses OTA) in HSA and the displacing effects of site markers to explore which molecules can interfere with its albumin-binding. Affinity of 2′R-OTA toward albumins from various species (human, bovine, porcine and rat) was tested to evaluate the interspecies differences regarding 2′R-OTA-albumin interaction. Thermodynamic studies were performed to give a deeper insight into the molecular background of the complex formation. Besides fluorescence spectroscopic and modeling studies, effects of HSA, and fetal bovine serum on the cytotoxicity of 2′R-OTA and OTA were tested in MDCK kidney cell line in order to demonstrate the influence of albumin-binding on the cellular uptake of ochratoxins. Site markers displaced more effectively 2′R-OTA than OTA from HSA. Fluorescence and binding constants of 2′R-OTA-albumin and OTA-albumin complexes showed different tendencies. Albumin significantly decreased the cytotoxicity of ochratoxins. 2′R-OTA, even at sub-toxic concentrations, increased the toxic action of OTA.

Analysis of the influence of simulation parameters on biomolecule-linked water networks

Advancement of computational molecular dynamics allows rapid calculation of large biomolecular systems in their water surroundings. New approaches of prediction of hydration networks of biomolecular surfaces and complex interfaces are also based on molecular dynamics (MD). Calculations with explicit solvent models can trace thousands of water molecules individually on a real time scale, yielding information on their mobility, and predicting their networking with biomolecular solutes and other water partners. Here, we investigate the effect of key parameters of molecular dynamics simulations on the quality of such predictions. Accordingly, systematic scans on temperature, pressure, force field, explicit water model and thermodynamic ensemble are performed. Explanations of optimal parameter values are provided using structural examples and analyses of the corresponding hydration networks.

Dynamic changes in binding interaction networks of sex steroids establish their non-classical effects

Non-classical signaling in the intracellular second messenger system plays a pivotal role in the cytoprotective effect of estradiol. Estrogen receptor is a common target of sex steroids and important in mediating estradiol-induced neuroprotection. Whereas the mechanism of genomic effects of sex steroids is fairly understood, their non-classical effects have not been elucidated completely. We use real time molecular dynamics calculations to uncover the interaction network of estradiol and activator estren. Besides steroid interactions, we also investigate the co-activation of the receptor. We show how steroid binding to the alternative binding site of the non-classical action is facilitated by the presence of a steroid in the classical binding site and the absence of the co-activator peptide. Uncovering such dynamic mechanisms behind steroid action will help the structure-based design of new drugs with non-classical responses and cytoprotective potential.