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Exploring Molecular Interactions between Escherichia coli RNA Polymerase and Topoisomerase I by Molecular Simulations
Monday, February 13, 2017 the phenotypic classification of the compounds. Dimensional reduction and clustering of behaviors based on image analysis successfully recapitulates the phenomenological differences between the compounds, suggesting that bioinformatic approaches may be able to successfully pick out interesting compounds from large behavioral screens without extensive manual analysis of the behavioral data.
Molecular Dynamics II 1405-Pos Board B473 Investigating the Influence of Sequence on Protein Folding Mechanism using a Structure Based Model Elizabeth Gichana, Charles L. Brooks III. University of Michigan, Ann Arbor, MI, USA. There are two major sources of frustration in a protein folding - energetic and topological. Energy landscape theory posits that main-chain topology is the major determinant in the folding mechanism of a protein. What is less well understood is the role that sequence effects play. To gain insight into this challenging problem, we utilize ancestral sequences reconstructed along mesophilic and thermophilic lineages of ribonuclease H (RNaseH) leading back to a common ancestor. This family of proteins share function and topology and have high sequence homology but differ in their biophysical properties. We investigate the role that variation in sequence over these evolutionary timescales alters the energy landscape of RNaseH to give rise to these differences by employing coarse-grained molecular dynamics simulations using a structure-based Golike model to investigate folding mechanisms. 1406-Pos Board B474 Modulation of Protein Flexibility with Changes in Sequence and Complexation State of Ubiquitin Family Proteins Sanjoy Paul. Tata Institute of Fundamental Research, Mumbai, India. Ubiquitin (Ub) and small ubiquitin-related modifier (SUMO) proteins are structurally homologous molecular tags which regulate diverse cellular processes in eukaryotes. Here, we provide a conceptual basis to understand how nature harnesses protein flexibility, a molecular descriptor, to modulate the diversity of macromolecular interactions and cellular responses seen for ubiquitin family proteins. We hypothesize that the flexibility of ubiquitin family proteins is modulated by both changes in protein sequence and complexation with other proteins. To validate our hypothesis, we developed computational measures of protein flexibility, directional and overall spring constants, from microsecond molecular dynamics simulations and examined these measures for ubiquitin and SUMO proteins in free form and within complexes. Our results show ubiquitin to be stiffer than SUMO proteins and comparable stiffness for SUMO1 and SUMO2 proteins. Complexation with an E2 enzyme which attaches the ubiquitin tag (UBCH5A) to target proteins in the ubiquitylation cascade increases the intrinsic stiffness of ubiquitin. In contrast, complexation with a motif that recognizes ubiquitin tagged molecular cargo (TSG101 UEV domain) decreases intrinsic ubiquitin stiffness. Directional spring constants for ubiquitin (along N-C and 48-C directions), SUMO2 (N-C direction) and SUMO1 (N-C direction) show excellent correlations with spring constants extracted from single-molecule force spectroscopy pulling experiments and we provide experimentally testable predictions for stiffness in other directions. Our computational framework is transferable and can be applied to study subtle changes in protein flexibility as a function of sequence and complexation state in topologically similar proteins and structural motifs. Finally, our computational framework offers detailed insights on the nature of the underlying energy landscape sampled by proteins in molecular dynamics simulations. 1407-Pos Board B475 Characterization of Profilin Binding Kinetics using Ensemble Molecular Dynamics Simulations Jocelyn Sunseri1, David R. Koes1, Partha Roy2, David Gau2. 1 Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA, 2Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA. We investigate the dynamics of profilin binding, including differences in binding for the loading and recruiting subregions of VASP, the effect of binding site mutations on peptide affinity for profilin, and the effect of actin binding on profilin dynamics. State-of-the-art computational methods are used and their accuracy, resource requirements, and reproducibility are evaluated. These include the use of ensemble molecular dynamics simulations with bootstrapping of MM/PBSA and MM/GBSA-derived free energy calculations to robustly estimate binding affinities. The essential features of the interaction between profilin and poly-proline peptides is explored through statistical analyses of simulation data. Results are compared to available experimental evidence.
287a
1408-Pos Board B476 Exploring Molecular Interactions between Escherichia coli RNA Polymerase and Topoisomerase I by Molecular Simulations Purushottam Tiwari1, Prem Chapagain2, Srikanth Banda2, Yesim Darici2, ¨ ren1. Yuk-Ching Tse-Dinh2, Aykut U 1 Georgetown University, Washington, DC, USA, 2Florida International University, Miami, FL, USA. During transcription, RNA polymerase (RNAP) generates hypernegative DNA supercoiling. Efficient transcription in bacteria requires relaxation of such a DNA supercoiling. In Escherichia coli, this task is performed by topoisomerase I (EctopoI), which is a type IA DNA topoisomerase. The exact nature of the RNAP-EctopoI interaction remains unresolved due to the lack of structural information of the complex. Utilizing in silico molecular docking, we obtained the RNAP-EctopoI complex, which showed the RNAP b0 subunit interacting with the C-terminal domain of EctopoI, in agreement with a previously published experimental result. Using surface plasmon resonance (SPR) based affinity technique, we have determined a KD value of ~93 nM for the RNAP-EctopoI complex formation. All-atom molecular dynamics (MD) simulations were performed for optimizing the conformational integrity of the docked complex. MD simulations revealed major conformational changes in the RNAP b0 subunit for the RNAP-EctopoI complex formation and stabilization. Our results suggest that the RNAP-EctopoI complex is stabilized via salt-bridges, E1009(RNAP)R609(EctopoI) and E874(RNAP)-K627(EctopoI), as well as hydrogen bond interactions, S1117(RNAP)-K664(EctopoI) and V967(EctopoI)-K664(EctopoI). Simulations of the complex with the mutant EctopoI showed that the complex is unstable with the mutation K166A, highlighting the importance of the lysine residue at this position. Our investigations provide molecular insights for the RNAP-EctopoI interactions and open the door for further protein-protein and protein-DNA interaction studies in this important system. Acknowledgements This work is supported by National Institutes of Health (NIH) grant R01GM054226 (Y. T.). Experimental SPR sensorgrams were measured by using Biacore T200 instrument available in Biacore Molecular Interaction Shared Resource (BMISR) facility at Georgetown University. The BMISR is supported by NIH grant P30CA51008. 1409-Pos Board B477 Computational Study on a Variety of Pathways for Conformational Changes of a Protein Sotaro Fuchigami. Yokohama City University, Yokohama, Japan. Conformational changes of proteins usually play a crucial role in their function. Protein dynamics during conformational changes have been revealed by various experimental and computational techniques. Interestingly, when a large conformational change occurs, its pathway is not always identical but rather shows variability because of the intrinsic flexibility of a protein. However, the underlying molecular details remain unclear. In the present study, we selected lysine-, arginine-, ornithine-binding protein (LAO) as a target protein. LAO comprises two domains and undergoes slow and large-amplitude domain motions. All-atom molecular dynamics simulations of apo-LAO from the closed conformation in explicit water were performed many times using MARBLE and the CHARMM22/CMAP force field parameters. Almost all trajectories showed conformational changes from the closed form to the open form, some of which occurred immediately and others slowly. We will discuss the similarity and differences in the pathways of conformational change. 1410-Pos Board B478 Molecular Dynamics Study for Streptavidin Mutant With/Without Biotin Analog Keiko Shinoda, Hideaki Fujitani. LSBM, RCAST, The Univ. of Tokyo, Tokyo, Japan. The streptavidin (SA) is a tetrameric protein with four identical subunits. Each subunit has a biotin-binding pocket. The biotin-SA system is known to have a strongest noncovalent biological interaction, and has been widely used as not only a molecular detection tool in many biotechnological applications but also medical applications such as pre-targeting system. One of pre-targeting approach to cancer radioimmunotherapy uses an antibody-streptavidin conjugate that is first localized to the tumor. The excess reagent is then cleared from blood and the biotinylated radiation carriers are administered. So far, many trials for the clinical application have not been successful due to mainly two problems, the immunogenicity of a bacterium-derived SA and the endogenous BTN species. Recently, a pre-targeting system, which overcomes such the two problems, has been developed by kawato et al. In the system, the SA is modified to decrease the immunogenicity, and the SA mutant binds to an artificial biotin analog while abolishing affinity for natural biocytin. Here, we have
Molecular Dynamics II 1405-Pos Board B473 Investigating the Influence of Sequence on Protein Folding Mechanism using a Structure Based Model Elizabeth Gichana, Charles L. Brooks III. University of Michigan, Ann Arbor, MI, USA. There are two major sources of frustration in a protein folding - energetic and topological. Energy landscape theory posits that main-chain topology is the major determinant in the folding mechanism of a protein. What is less well understood is the role that sequence effects play. To gain insight into this challenging problem, we utilize ancestral sequences reconstructed along mesophilic and thermophilic lineages of ribonuclease H (RNaseH) leading back to a common ancestor. This family of proteins share function and topology and have high sequence homology but differ in their biophysical properties. We investigate the role that variation in sequence over these evolutionary timescales alters the energy landscape of RNaseH to give rise to these differences by employing coarse-grained molecular dynamics simulations using a structure-based Golike model to investigate folding mechanisms. 1406-Pos Board B474 Modulation of Protein Flexibility with Changes in Sequence and Complexation State of Ubiquitin Family Proteins Sanjoy Paul. Tata Institute of Fundamental Research, Mumbai, India. Ubiquitin (Ub) and small ubiquitin-related modifier (SUMO) proteins are structurally homologous molecular tags which regulate diverse cellular processes in eukaryotes. Here, we provide a conceptual basis to understand how nature harnesses protein flexibility, a molecular descriptor, to modulate the diversity of macromolecular interactions and cellular responses seen for ubiquitin family proteins. We hypothesize that the flexibility of ubiquitin family proteins is modulated by both changes in protein sequence and complexation with other proteins. To validate our hypothesis, we developed computational measures of protein flexibility, directional and overall spring constants, from microsecond molecular dynamics simulations and examined these measures for ubiquitin and SUMO proteins in free form and within complexes. Our results show ubiquitin to be stiffer than SUMO proteins and comparable stiffness for SUMO1 and SUMO2 proteins. Complexation with an E2 enzyme which attaches the ubiquitin tag (UBCH5A) to target proteins in the ubiquitylation cascade increases the intrinsic stiffness of ubiquitin. In contrast, complexation with a motif that recognizes ubiquitin tagged molecular cargo (TSG101 UEV domain) decreases intrinsic ubiquitin stiffness. Directional spring constants for ubiquitin (along N-C and 48-C directions), SUMO2 (N-C direction) and SUMO1 (N-C direction) show excellent correlations with spring constants extracted from single-molecule force spectroscopy pulling experiments and we provide experimentally testable predictions for stiffness in other directions. Our computational framework is transferable and can be applied to study subtle changes in protein flexibility as a function of sequence and complexation state in topologically similar proteins and structural motifs. Finally, our computational framework offers detailed insights on the nature of the underlying energy landscape sampled by proteins in molecular dynamics simulations. 1407-Pos Board B475 Characterization of Profilin Binding Kinetics using Ensemble Molecular Dynamics Simulations Jocelyn Sunseri1, David R. Koes1, Partha Roy2, David Gau2. 1 Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA, 2Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA. We investigate the dynamics of profilin binding, including differences in binding for the loading and recruiting subregions of VASP, the effect of binding site mutations on peptide affinity for profilin, and the effect of actin binding on profilin dynamics. State-of-the-art computational methods are used and their accuracy, resource requirements, and reproducibility are evaluated. These include the use of ensemble molecular dynamics simulations with bootstrapping of MM/PBSA and MM/GBSA-derived free energy calculations to robustly estimate binding affinities. The essential features of the interaction between profilin and poly-proline peptides is explored through statistical analyses of simulation data. Results are compared to available experimental evidence.
287a
1408-Pos Board B476 Exploring Molecular Interactions between Escherichia coli RNA Polymerase and Topoisomerase I by Molecular Simulations Purushottam Tiwari1, Prem Chapagain2, Srikanth Banda2, Yesim Darici2, ¨ ren1. Yuk-Ching Tse-Dinh2, Aykut U 1 Georgetown University, Washington, DC, USA, 2Florida International University, Miami, FL, USA. During transcription, RNA polymerase (RNAP) generates hypernegative DNA supercoiling. Efficient transcription in bacteria requires relaxation of such a DNA supercoiling. In Escherichia coli, this task is performed by topoisomerase I (EctopoI), which is a type IA DNA topoisomerase. The exact nature of the RNAP-EctopoI interaction remains unresolved due to the lack of structural information of the complex. Utilizing in silico molecular docking, we obtained the RNAP-EctopoI complex, which showed the RNAP b0 subunit interacting with the C-terminal domain of EctopoI, in agreement with a previously published experimental result. Using surface plasmon resonance (SPR) based affinity technique, we have determined a KD value of ~93 nM for the RNAP-EctopoI complex formation. All-atom molecular dynamics (MD) simulations were performed for optimizing the conformational integrity of the docked complex. MD simulations revealed major conformational changes in the RNAP b0 subunit for the RNAP-EctopoI complex formation and stabilization. Our results suggest that the RNAP-EctopoI complex is stabilized via salt-bridges, E1009(RNAP)R609(EctopoI) and E874(RNAP)-K627(EctopoI), as well as hydrogen bond interactions, S1117(RNAP)-K664(EctopoI) and V967(EctopoI)-K664(EctopoI). Simulations of the complex with the mutant EctopoI showed that the complex is unstable with the mutation K166A, highlighting the importance of the lysine residue at this position. Our investigations provide molecular insights for the RNAP-EctopoI interactions and open the door for further protein-protein and protein-DNA interaction studies in this important system. Acknowledgements This work is supported by National Institutes of Health (NIH) grant R01GM054226 (Y. T.). Experimental SPR sensorgrams were measured by using Biacore T200 instrument available in Biacore Molecular Interaction Shared Resource (BMISR) facility at Georgetown University. The BMISR is supported by NIH grant P30CA51008. 1409-Pos Board B477 Computational Study on a Variety of Pathways for Conformational Changes of a Protein Sotaro Fuchigami. Yokohama City University, Yokohama, Japan. Conformational changes of proteins usually play a crucial role in their function. Protein dynamics during conformational changes have been revealed by various experimental and computational techniques. Interestingly, when a large conformational change occurs, its pathway is not always identical but rather shows variability because of the intrinsic flexibility of a protein. However, the underlying molecular details remain unclear. In the present study, we selected lysine-, arginine-, ornithine-binding protein (LAO) as a target protein. LAO comprises two domains and undergoes slow and large-amplitude domain motions. All-atom molecular dynamics simulations of apo-LAO from the closed conformation in explicit water were performed many times using MARBLE and the CHARMM22/CMAP force field parameters. Almost all trajectories showed conformational changes from the closed form to the open form, some of which occurred immediately and others slowly. We will discuss the similarity and differences in the pathways of conformational change. 1410-Pos Board B478 Molecular Dynamics Study for Streptavidin Mutant With/Without Biotin Analog Keiko Shinoda, Hideaki Fujitani. LSBM, RCAST, The Univ. of Tokyo, Tokyo, Japan. The streptavidin (SA) is a tetrameric protein with four identical subunits. Each subunit has a biotin-binding pocket. The biotin-SA system is known to have a strongest noncovalent biological interaction, and has been widely used as not only a molecular detection tool in many biotechnological applications but also medical applications such as pre-targeting system. One of pre-targeting approach to cancer radioimmunotherapy uses an antibody-streptavidin conjugate that is first localized to the tumor. The excess reagent is then cleared from blood and the biotinylated radiation carriers are administered. So far, many trials for the clinical application have not been successful due to mainly two problems, the immunogenicity of a bacterium-derived SA and the endogenous BTN species. Recently, a pre-targeting system, which overcomes such the two problems, has been developed by kawato et al. In the system, the SA is modified to decrease the immunogenicity, and the SA mutant binds to an artificial biotin analog while abolishing affinity for natural biocytin. Here, we have