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Molecular Recognition Mechanism of Hematopoietic Prostaglandin D Synthase with its Cofactor and Substrate
494a
Wednesday, February 15, 2017
antimicrobial compounds from different classes, namely carbapenems, cephalosporins, fluoroquinolones, penicillines, tetracyclines, and beta-lactamase inhibitors. In particular, for each compound we screened for possible binding sites on the whole protein, including thus possible binding pockets other than those already known. Flexibility has been taken into account by considering ensembles of rigid structures for both the ligand and the receptor. Ligand conformations have been extracted from microsecond-long molecular dynamics (MD) simulations in explicit water [2]. For the protein we considered a large set of conformations, including structures from both high-resolution X-ray and MD simulations. We present the results of this systematic investigation by comparing the patterns of interactions observed for the different compounds. The binding properties of the transporter are discussed in terms of statistics of the most contacted residues. 1. P. Ruggerone, S. Murakami, K. M. Pos and A.V. Vargiu, Curr. Top. Med. Chem.13, 3079 (2013). 2. G. Malloci, A. V. Vargiu, G. Serra, A. Bosin, P. Ruggerone, M. Ceccarelli, Molecules20, 13997 (2015). 2430-Pos Board B37 Exploring Folate-Small Molecule Interactions in Bacterial Cells Deepika K. Nambiar, Robert Shew, Bryan Schwarz, Michael Duff, Timkhite Kulu-Berhane, Elizabeth Howell. Biochemistry, Cellular & Molecular Biology, University of Tennessee Knoxville, Knoxville, TN, USA. Folate (Vitamin B9) is involved in one carbon transfer reactions required for the synthesis of DNA and amino acids. Our current understanding of the folate pathway is mostly based on in vitro studies, which are very different from the crowded environment in the cell. E. coli produces osmoprotectants during times of osmotic stress. This leads to perturbation of water activity inside the cell, and an increase in macromolecular crowding. We have shown earlier that, in vitro, osmolytes weaken the binding of dihydrofolate to dihydrofolate reductase in the folate pathway. We hypothesize that an increased osmolyte concentration in the cell will also prevent the functioning of other folate pathway enzymes by interaction of osmolytes with the various folate redox states. In this study, we studied the effect of osmotic stress on the folate synthesizing and metabolizing enzymes such as dihydropteroate synthase (folP), dihydrofolate reductase (folA), methylenetetrahydrofolate reductase (metF) and serine hydroxymethyltransferase (glyA) in vivo. Studies were done with knockout and rescued strains. Protein expression in rescued strains was limited to very low levels using a Ptet promoter and a protein degradation tag. We can titrate the enzyme activity in the rescued strains by osmotic stress. Osmotic stress studies have indicated that the rescued strain was unable to grow in higher osmolality conditions when compared to knockout strains. We predict this is due to an increase in osmolyte concentration in vivo which leads to interaction of osmolytes with folate intermediates in the pathway. This is turn decreases the efficiency of the folate pathway enzyme. Finally, we have varied the predominant osmolyte in the cell from trehalose to glycine betaine to study if our model holds for these different conditions. 2431-Pos Board B38 Attenuating the Toxicity of Amyloid-Beta Aggregation with Specific Species Ryan Limbocker1, Benedetta Mannini1, Michele Perni1, Sean Chia1, Gabriella Heller1, Francesco S. Ruggeri1, Johnny Habchi1, Georg Meisl1, Pavan K. Challa1, Michael Zasloff2, Tuomas P.J. Knowles1, Michele Vendruscolo1, Christopher M. Dobson1. 1 Department of Chemistry, University of Cambridge, Cambridge, United Kingdom, 2Department of Surgery, MedStar Georgetown Transplant Institute, Washington, DC, USA. The deposition into amyloid plaques of the amyloid-b peptide (Ab) is a hallmark of Alzheimer’s disease (AD). Increasing evidence suggests that soluble Ab oligomers are the pathological species primarily responsible for AD onset and progression. Shifting the aggregation pathway towards the formation of non-toxic aggregated species by specific compounds could thus prove an effective therapeutic strategy for AD. Herein, we employ an array of biophysical techniques, including fluorescence-based chemical kinetics, Atomic Force Microscopy, Nuclear Magnetic Resonance, and absorbance spectroscopy, and in vivo techniques, including cells and C. elegans, to characterise compounds for their ability to mitigate the aggregation of Ab. We find structurally similar compounds that 1) regulate protein assembly by modulating the microscopic processes governing aggregation, 2) interact with primary species resulting in increased fibrilization in vitro and in vivo, 3) drive the formation of shorter, more mature fibrils, 4) generate larger, less hydrophobic, and less toxic aggregates from oligomers after incubation with pre-formed oligomers, and 5) in the case of one species, eliminates Ab42-induced toxicity in a worm model of AD.
These findings suggest reducing the toxicity associated with Ab42 aggregation could be explored to find viable therapeutic strategies for the treatment of AD. 2432-Pos Board B39 Determination of Ligand Migration Pathways in Human Cytoglobin Antonija Tangar1, Michael Goncalves2, Sophie Bernad3, Valerie Derrien3, Pierre Sebban3, Jaroslava Miksovska1. 1 Florida International University, Miami, FL, USA, 2TERRA Environmental Research Institute, Miami, FL, USA, 3Universite Paris-Sud, Orsay, France. Cytoglobin (Cygb) is one of the latest additions to the vertebrate globin family found in virtually all human tissues. Due to its low intracellular concentration, Cygb is not believed to act as a oxygen transport/storage protein and its function remains to be determined. However, in vivo studies have indicated that Cygb has a strong cytoprotective role by acting as a peroxidase, nitric oxide dioxygenase and/or reactive oxygen species scavenger. Additionally, affinity for ligands is modulated by internal disulfide bond, which is believed to act as a redox switch and impact Cygb function. In order to probe the mechanism of Cygb interactions with small diatomic ligands, we took a closer look into residues found on two main computationally-suggested ligand migration pathways: Arg84, which is proposed to act as a gate between heme cavity and solvent; and Arg33, found at the end of the tunnel enclosed by helices A, G and H. Photoacoustic calorimetry, transient absorption and stopped-flow were employed to determine kinetic and thermodynamic profiles for diatomic ligand interactions with human Cygb wild-type, hCygb Arg84Leu, hCygb Arg33Trp, and hCygb Arg33Trp Arg84Leu. Additionally, impact of mutations on Cygb stability was determined. Our results indicate that both Arg84 and Arg33 have an important role in both ligand escape and rebinding by modulating dynamics and energetics of structural changes associated with ligand binding to Cygb. Moreover, Arg84 was found to be a crucial residue in stabilizing heme pocket. Furthermore, computational approaches (locally enhanced sampling, MD pocket) were employed to determine most-sampled ligand migration pathways and occupancy of internal cavities. 2433-Pos Board B40 Molecular Recognition Mechanism of Hematopoietic Prostaglandin D Synthase with its Cofactor and Substrate Keisuke Asada1, Shigeru Shimamoto1, Tomohiro Oonoki1, Takahiro Maruno2, Yuji Kobayashi2, Kosuke Aritake3, Yoshihiro Urade3, Yuji Hidaka1. 1 Graduate School of Science and Engineering Research, University of Kindai, Higashi-Osaka, Japan, 2Graduate School of Engineering, University of Osaka, Suita, Japan, 3International Institute For Integrative Sleep Medicine, University of Tsukuba, Tsukuba, Japan. Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is a Sigma class member of the glutathione S-transferase (GST) superfamily, and requires glutathione (GSH) as a cofactor. H-PGDS catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2), which acts as an allergic and inflammatory mediator. The overproduction of PGD2 by H-PGDS causes allergic and inflammatory reactions in the necrotic muscle fibers of Duchenne muscular dystrophy (DMD) patients. Therefore, H-PGDS inhibitors are thought to have potential for use in drug therapy for DMD. The catalytic mechanism of H-PGDS remains unclear, since essential information, such as the binding affinity and stoichiometry of GSH and PGH2 to H-PGDS, have not been precisely determined yet. Therefore, to clarify the issues related to the cofactor and substrate recognition of H-PGDS, we investigated the interaction between H-PGDS and PGH2 in the presence and absence of GSH. For this purpose, isothermal titration calorimetry (ITC) was performed using a substrate mimetics U-44069 in the place of PGH2 since PGH2 is quite unstable in most solutions. Recombinant H-PGDS was prepared using an E. coli expression system and used for the ITC measurements. The results showed that U-44069 binds to H-PGDS in the presence of the cofactor, GSH, but the binding was not significant in the absence of GSH. Therefore, these results suggest that the GSH binding promotes the interaction between H-PGDS and PGH2, and that GSH plays an important role, not only for the catalytic reaction but also for substrate binding. 2434-Pos Board B41 Lipocalin-Type Prostaglandin D Synthase Possesses Two Binding Sites for its Product Shigeru Shimamoto1, Yusuke Nakagawa1, Takahiro Maruno2, Yuji Kobayashi2, Kosuke Aritake3, Urade Yoshihiro3, Yuji Hidaka1. 1 Kindai University, Higashi-osaka, Japan, 2Osaka University, Suita, Japan, 3 University of Tsukuba, Tsukuba, Japan. Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) belongs to the lipocalin superfamily which consists of transporter proteins for lipophilic ligands in
Wednesday, February 15, 2017
antimicrobial compounds from different classes, namely carbapenems, cephalosporins, fluoroquinolones, penicillines, tetracyclines, and beta-lactamase inhibitors. In particular, for each compound we screened for possible binding sites on the whole protein, including thus possible binding pockets other than those already known. Flexibility has been taken into account by considering ensembles of rigid structures for both the ligand and the receptor. Ligand conformations have been extracted from microsecond-long molecular dynamics (MD) simulations in explicit water [2]. For the protein we considered a large set of conformations, including structures from both high-resolution X-ray and MD simulations. We present the results of this systematic investigation by comparing the patterns of interactions observed for the different compounds. The binding properties of the transporter are discussed in terms of statistics of the most contacted residues. 1. P. Ruggerone, S. Murakami, K. M. Pos and A.V. Vargiu, Curr. Top. Med. Chem.13, 3079 (2013). 2. G. Malloci, A. V. Vargiu, G. Serra, A. Bosin, P. Ruggerone, M. Ceccarelli, Molecules20, 13997 (2015). 2430-Pos Board B37 Exploring Folate-Small Molecule Interactions in Bacterial Cells Deepika K. Nambiar, Robert Shew, Bryan Schwarz, Michael Duff, Timkhite Kulu-Berhane, Elizabeth Howell. Biochemistry, Cellular & Molecular Biology, University of Tennessee Knoxville, Knoxville, TN, USA. Folate (Vitamin B9) is involved in one carbon transfer reactions required for the synthesis of DNA and amino acids. Our current understanding of the folate pathway is mostly based on in vitro studies, which are very different from the crowded environment in the cell. E. coli produces osmoprotectants during times of osmotic stress. This leads to perturbation of water activity inside the cell, and an increase in macromolecular crowding. We have shown earlier that, in vitro, osmolytes weaken the binding of dihydrofolate to dihydrofolate reductase in the folate pathway. We hypothesize that an increased osmolyte concentration in the cell will also prevent the functioning of other folate pathway enzymes by interaction of osmolytes with the various folate redox states. In this study, we studied the effect of osmotic stress on the folate synthesizing and metabolizing enzymes such as dihydropteroate synthase (folP), dihydrofolate reductase (folA), methylenetetrahydrofolate reductase (metF) and serine hydroxymethyltransferase (glyA) in vivo. Studies were done with knockout and rescued strains. Protein expression in rescued strains was limited to very low levels using a Ptet promoter and a protein degradation tag. We can titrate the enzyme activity in the rescued strains by osmotic stress. Osmotic stress studies have indicated that the rescued strain was unable to grow in higher osmolality conditions when compared to knockout strains. We predict this is due to an increase in osmolyte concentration in vivo which leads to interaction of osmolytes with folate intermediates in the pathway. This is turn decreases the efficiency of the folate pathway enzyme. Finally, we have varied the predominant osmolyte in the cell from trehalose to glycine betaine to study if our model holds for these different conditions. 2431-Pos Board B38 Attenuating the Toxicity of Amyloid-Beta Aggregation with Specific Species Ryan Limbocker1, Benedetta Mannini1, Michele Perni1, Sean Chia1, Gabriella Heller1, Francesco S. Ruggeri1, Johnny Habchi1, Georg Meisl1, Pavan K. Challa1, Michael Zasloff2, Tuomas P.J. Knowles1, Michele Vendruscolo1, Christopher M. Dobson1. 1 Department of Chemistry, University of Cambridge, Cambridge, United Kingdom, 2Department of Surgery, MedStar Georgetown Transplant Institute, Washington, DC, USA. The deposition into amyloid plaques of the amyloid-b peptide (Ab) is a hallmark of Alzheimer’s disease (AD). Increasing evidence suggests that soluble Ab oligomers are the pathological species primarily responsible for AD onset and progression. Shifting the aggregation pathway towards the formation of non-toxic aggregated species by specific compounds could thus prove an effective therapeutic strategy for AD. Herein, we employ an array of biophysical techniques, including fluorescence-based chemical kinetics, Atomic Force Microscopy, Nuclear Magnetic Resonance, and absorbance spectroscopy, and in vivo techniques, including cells and C. elegans, to characterise compounds for their ability to mitigate the aggregation of Ab. We find structurally similar compounds that 1) regulate protein assembly by modulating the microscopic processes governing aggregation, 2) interact with primary species resulting in increased fibrilization in vitro and in vivo, 3) drive the formation of shorter, more mature fibrils, 4) generate larger, less hydrophobic, and less toxic aggregates from oligomers after incubation with pre-formed oligomers, and 5) in the case of one species, eliminates Ab42-induced toxicity in a worm model of AD.
These findings suggest reducing the toxicity associated with Ab42 aggregation could be explored to find viable therapeutic strategies for the treatment of AD. 2432-Pos Board B39 Determination of Ligand Migration Pathways in Human Cytoglobin Antonija Tangar1, Michael Goncalves2, Sophie Bernad3, Valerie Derrien3, Pierre Sebban3, Jaroslava Miksovska1. 1 Florida International University, Miami, FL, USA, 2TERRA Environmental Research Institute, Miami, FL, USA, 3Universite Paris-Sud, Orsay, France. Cytoglobin (Cygb) is one of the latest additions to the vertebrate globin family found in virtually all human tissues. Due to its low intracellular concentration, Cygb is not believed to act as a oxygen transport/storage protein and its function remains to be determined. However, in vivo studies have indicated that Cygb has a strong cytoprotective role by acting as a peroxidase, nitric oxide dioxygenase and/or reactive oxygen species scavenger. Additionally, affinity for ligands is modulated by internal disulfide bond, which is believed to act as a redox switch and impact Cygb function. In order to probe the mechanism of Cygb interactions with small diatomic ligands, we took a closer look into residues found on two main computationally-suggested ligand migration pathways: Arg84, which is proposed to act as a gate between heme cavity and solvent; and Arg33, found at the end of the tunnel enclosed by helices A, G and H. Photoacoustic calorimetry, transient absorption and stopped-flow were employed to determine kinetic and thermodynamic profiles for diatomic ligand interactions with human Cygb wild-type, hCygb Arg84Leu, hCygb Arg33Trp, and hCygb Arg33Trp Arg84Leu. Additionally, impact of mutations on Cygb stability was determined. Our results indicate that both Arg84 and Arg33 have an important role in both ligand escape and rebinding by modulating dynamics and energetics of structural changes associated with ligand binding to Cygb. Moreover, Arg84 was found to be a crucial residue in stabilizing heme pocket. Furthermore, computational approaches (locally enhanced sampling, MD pocket) were employed to determine most-sampled ligand migration pathways and occupancy of internal cavities. 2433-Pos Board B40 Molecular Recognition Mechanism of Hematopoietic Prostaglandin D Synthase with its Cofactor and Substrate Keisuke Asada1, Shigeru Shimamoto1, Tomohiro Oonoki1, Takahiro Maruno2, Yuji Kobayashi2, Kosuke Aritake3, Yoshihiro Urade3, Yuji Hidaka1. 1 Graduate School of Science and Engineering Research, University of Kindai, Higashi-Osaka, Japan, 2Graduate School of Engineering, University of Osaka, Suita, Japan, 3International Institute For Integrative Sleep Medicine, University of Tsukuba, Tsukuba, Japan. Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is a Sigma class member of the glutathione S-transferase (GST) superfamily, and requires glutathione (GSH) as a cofactor. H-PGDS catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2), which acts as an allergic and inflammatory mediator. The overproduction of PGD2 by H-PGDS causes allergic and inflammatory reactions in the necrotic muscle fibers of Duchenne muscular dystrophy (DMD) patients. Therefore, H-PGDS inhibitors are thought to have potential for use in drug therapy for DMD. The catalytic mechanism of H-PGDS remains unclear, since essential information, such as the binding affinity and stoichiometry of GSH and PGH2 to H-PGDS, have not been precisely determined yet. Therefore, to clarify the issues related to the cofactor and substrate recognition of H-PGDS, we investigated the interaction between H-PGDS and PGH2 in the presence and absence of GSH. For this purpose, isothermal titration calorimetry (ITC) was performed using a substrate mimetics U-44069 in the place of PGH2 since PGH2 is quite unstable in most solutions. Recombinant H-PGDS was prepared using an E. coli expression system and used for the ITC measurements. The results showed that U-44069 binds to H-PGDS in the presence of the cofactor, GSH, but the binding was not significant in the absence of GSH. Therefore, these results suggest that the GSH binding promotes the interaction between H-PGDS and PGH2, and that GSH plays an important role, not only for the catalytic reaction but also for substrate binding. 2434-Pos Board B41 Lipocalin-Type Prostaglandin D Synthase Possesses Two Binding Sites for its Product Shigeru Shimamoto1, Yusuke Nakagawa1, Takahiro Maruno2, Yuji Kobayashi2, Kosuke Aritake3, Urade Yoshihiro3, Yuji Hidaka1. 1 Kindai University, Higashi-osaka, Japan, 2Osaka University, Suita, Japan, 3 University of Tsukuba, Tsukuba, Japan. Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) belongs to the lipocalin superfamily which consists of transporter proteins for lipophilic ligands in