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Conformational Evolution of Three Regulator of G-Protein Signaling Proteins (RGS4, RGS8, RGS19) in Microsecond-Scale Simulations
Tuesday, February 14, 2017 1732-Pos Board B52 What Makes Enzymes Work? Exploring Protein Dynamics in P-T-X Qi Huang1, Jocelyn M. Rodgers2, Russell J. Hemley3, Toshiko Ichiye1. 1 Chemistry, Georgetown University, Washington, DC, USA, 2Geophysical, Carnege Institute for Science, Washington, DC, USA, 3Civil and Environmental Engineering, George Washington University, Washington, DC, USA. Although both stability and flexibility are necessary for enzymes to function, general quantitative measures of flexibility have yet to emerge so that design of functional enzymes is difficult. Clarifying what type of flexibility is important may help to resolve this question. For instance, collective motions are important, but are the atomic fluctuations that give rise to the collective motions more fundamentally important? Exploring ‘‘Nature’s laboratory’’ has become a powerful method, namely, by examining enzymes from ‘‘extremophilic’’ organisms that live under extreme conditions. Extremophiles must maintain enzyme activity under extreme conditions to grow, and often the adaptations necessary for stability and flexibility conflict. While enzymes from extremophiles who live under extremes of temperature (T) and even chemical composition (X) have been well-studied, enzymes from piezophiles who live at high pressure (P) have not been, in part because high pressure is a difficult variable in the field and in the laboratory. In addition, pressure apparently affects proteins differently than temperature, and new high-pressure instrumentation makes experimental studies of high pressure effects on enzymes possible. Here, flexibility measures are assessed in homologous enzymes from mesophiles and piezophiles under different P-T-X conditions using molecular dynamics simulations in light of results from high pressure experiments. 1733-Pos Board B53 Extreme Biophysics: Enzymes under Pressure Qi Huang1, Jocelyn M. Rodgers2, Russell J. Hemley3, Toshiko Ichiye1. 1 Georgetown University, Washington, DC, USA, 2Georgetown University, Carnegie Institution of Washington, Washington, DC, USA, 3George Washington University, Washington, DC, USA. The discoveries of ‘‘extremophilic’’ microbes that thrive under extremes of temperature, pressure, pH, etc. raise many questions including the adaptations made in their constituent molecules such as enzymes in order to function under conditions where their counterparts in mesophiles fail. Of extreme conditions, the effects of high pressure have been relatively unexplored, both because of the difficulty in producing high pressure in the lab and because of the difficulty in collecting samples of ‘‘piezophiles’’, microbes adapted for high pressure environments. Here, factors are examined that may lead to the increased activity under pressure in dihydrofolate reductase from the piezophilic Moritella profunda compared to the homologous enzyme from the mesophilic Escherichia coli. Molecular dynamics simulations are performed at various temperatures and pressures to examine how pressure affects the flexibility of the enzymes from these two microbes, since both stability and flexibility are necessary for enzyme activity. The results suggest that collective motions on the 10 ns timescale are responsible for the flexibility necessary for ‘‘corresponding states’’ activity at the growth conditions of the parent organism. The results also suggest that while the lower stability of many enzymes from deep-sea microbes may be an adaptation for greater flexibility at low temperatures, it may also enable the enzymes to withstand high pressure. Determination of the adaptations of enzymes for extreme conditions can lead to a greater understanding of enzyme structure-function relationships. In addition, understanding these adaptations can be used in biotechnology so that enzymes can be bioengineered to function under specific conditions. Moreover, determining the limiting conditions where enzyme activity can be maintained is one of the factors in determining the ‘‘limits of life’’, which could guide the search for life in extreme environments such as beneath the oceanic and continental surface or even extraterrestrially. 1734-Pos Board B54 Improved Relaxation Mode Analysis of a Hen Egg-White Lysozyme Protein Naoyuki Karasawa1, Ayori Mitsutake1,2, Hiroshi Takano1. 1 Keio University, Yokohama, Japan, 2JST PRESTO, Chiyoda, Japan. Proteins have various functions within living organisms and play important roles in life phenomena. Their functions are related to not only structures but also structural fluctuations around them. Molecular dynamics (MD) simulation is an effective tool to investigate protein dynamics by tracing motions of all atoms in a system. However, structural fluctuations of proteins include various time-scale dynamics and are complex. Therefore, the motions related
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to functions must be extracted from the time-series data. One of the multivariate analysis methods, principal component analysis (PCA), is widely used for analysing static properties of systems. On the other hand, relaxation mode analysis (RMA) has been used to elucidate dynamic properties in a random spin system [1], a homo-polymer system [2], and hetero-polymer systems [3, 4, 5]. This method, on the basis of statistical physics, finds slow relaxation modes in systems with relaxational dynamics. In its previous application to a protein system [4], however, the long-time behavior was not described well. Two-step RMA has recently been developed in a homo-polymer system in order to improve description of long-time behavior [6]. We applied this method to a hetero-polymer system, hen egg-white lysozyme (HEWL) protein. We found that two-step RMA not only describes long-time behavior well, but also extracts slow structural transitions of a protein more effectively. [1] H. Takano, S. Miyashita, J. Phys. Soc. Jpn. 64, 3688 (1995). [2] H. Hirao, S. Koseki, H. Takano, J. Phys. Soc. Jpn. 66, 3399 (1997). [3] A. Mitsutake, H. Iijima, H. Takano, J. Chem. Phys.135, 164102 (2011). [4] T. Nagai, A. Mitsutake, and H. Takano, J. Phys. Soc. Jpn. 82, 023803 (2013). [5] A. Mitsutake, H. Takano, J. Chem. Phys. 143, 124111 (2015). [6] S. Natori, H. Takano, submitted J. Phys. Soc. Jpn. 1735-Pos Board B55 Importance of Protein Vibration Directionality on Function Katherine A. Niessen1, Mengyang Xu1, Yanting Deng1, Edward H. Snell2,3, Andrea G. Markelz1. 1 Physics, SUNY University at Buffalo, Buffalo, NY, USA, 2HauptmanWoodward Medical Research Institute, Buffalo, NY, USA, 3Structural Biology, SUNY University at Buffalo, Buffalo, NY, USA. Global protein vibrations have long been associated with protein functionality. Long-range intramolecular vibrations have been measured in proteins using anisotropic absorption in the terahertz frequency range [1,2]. These measurements directly correspond to the directionality of vibrations and show large changes in the directionality of the vibrational displacements for free chicken egg white lysozyme (CEWL) and inhibitor bound CEWL. Normal mode ensemble analysis (NMEA) and quasiharmonic analysis (QHA) calculations of the free and tri-acetylglucosamine (3NAG) bound CEWL dynamics were performed and also show large changes in vibration directionality with binding. This in spite of the calculated energy distribution showing very little change, in agreement with neutron scattering measurements. We investigate the importance of the protein directionality by extending the calculations to a double deletion mutant (DD CEWL) with an activity rate 1.4 times that of WT [3]. The deletions are far from the active site, indicating that the increased activity arises from changes in the dynamics. The calculated anisotropic spectra show large changes with mutation, whereas the energy distribution show practically no change from the WT. Similarities in the bound WT, free DD, and bound DD CEWL NMEA spectra indicate the mutation may be changing the directionality of the vibrations toward more efficient motions. Projections of the low frequency vibrations, from QHA, on the functional displacement show higher overlap of residues around Glu35 and Asp52 in the mutant system. These residues are known to be critical in CEWL functionality. The results reveal that the mutation may be steering the protein toward more functional dynamics without significantly affecting the energy distribution. [1] K.A. Niessen, et al. (2015) DOI: 10.1007/s12551-015-0168-4 [2] G. Acbas, et al. (2014) DOI: 10.1038/ncomms4076 [3] S. Mine, et al. (1999) DOI: 10.1006/jmbi.1999.2572. Supported by NSF (DBI 1556359, MCB 1616529) and DOE (DE-SC0016317). 1736-Pos Board B56 Conformational Evolution of Three Regulator of G-Protein Signaling Proteins (RGS4, RGS8, RGS19) in Microsecond-Scale Simulations Hossein Mohammadiarani, Harish Vashisth. Chemical Engineering, University of New Hampshire, Durham, NH, USA. Regulators of G protein signaling (RGS) proteins modulate GPCR signaling by binding to Ga-subunits of heterotrimeric G proteins and accelerating hydrolysis of GTP. Therefore, RGS proteins are becoming increasingly important therapeutic targets to be directly or allosterically inhibited from binding to Ga-subunits. While structures of several known RGS proteins are highly similar and largely contain a-helical motifs, some thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for closely related proteins thereby suggesting intrinsic differences in dynamics of these proteins. In this work, we have
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Tuesday, February 14, 2017
studied the dynamics of three different RGS proteins (apo-RGS4, apo-RGS8, and apo-RGS19) using microsecond-scale classical molecular dynamics (MD) simulations with CHARMM and AMBER force-fields. Analyses of these trajectories reveal high fluctuations in a5 and a6 helices and the loops connecting them. These fluctuations lead to perturbations in residues in the RGS-Ga interface and in the vicinity of cysteines that are targets of allosteric inhibitors. These findings have significant implications for understanding differences in potencies and specificities of inhibitory small-molecules. 1737-Pos Board B57 Comparative Structural Dynamic Analysis of GTPases Hongyang Li, Xin-Qiu Yao, Barry Grant. Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA. GTPases regulate a multitude of essential cellular processes ranging from movement and division to differentiation and neuronal activity. These ubiquitous enzymes operate by hydrolyzing GTP to GDP with associated conformational changes that modulate affinity for family specific binding partners. There are three major GTPase superfamilies: Ras-like GTPases, heterotrimeric G proteins and protein-synthesizing GTPases. Although they contain similar nucleotide-binding sites, the detailed mechanisms by which these structurally and functionally diverse superfamilies operate remain unclear. Here we compare and contrast the structural dynamic mechanisms of each superfamily using extensive molecular dynamics (MD) simulations and subsequent network analysis approaches. In particular, dissection of the cross-correlations of atomic displacements in both the GTP and GDP-bound states of Ras, transducin and elongation factor EF-Tu reveals analogous dynamic features. This includes similar dynamic communities and subdomain structures (termed lobes). For all three proteins the GTP-bound state has stronger couplings between equivalent lobes. Network analysis further identifies common and family specific residues mediating the state specific coupling of distal functional sites. Mutational simulations demonstrate how disrupting these couplings leads to distal dynamic effects at the nucleotide binding site of each family. Collectively our studies extend current understanding of GTPase allosteric mechanisms and highlight previously unappreciated similarities across functionally diverse families. 1738-Pos Board B58 The Molecular Mechanism of Entropic Allostery of Hemoglobin Takashi Yonetani1, Kenji Kanaori2. 1 Biophys/Biochem, Univ. Pennsylvania, Philadelphia, PA, USA, 2Biomol Engineering, Kyoto Inst Tech, Kyoto, Japan. Protohemes in Hb (Hb) are reversible low-affinity binding sites for diatomic ligands such as O2, CO, and NO. The ligand-affinity of Hb is regulated by controlling two distal effects, i.e., distal H-bonding and geminate-recombination of the ligand, which enhance retention of the ligand at the hemes to increase apparent ligand-affinity of Hb. Ligands, dissociating from hemes, encounter physical barriers of globin matrix (‘‘Caged’’ states), before either geminately recombining to the hemes to regenerate the respective ligated hemes [Route A] or migrating through globin matrices, followed by bimolecular dissociation to form deoxy-Hb [Route B]. Therefore, only a fraction of the dissociating ligands moves out of Hb (bi-molecular quantum yield [Q]). The remainder reverts back to the hemes to form the ligated Hb (geminate quantum yield [1-Q]). This is resulted in increases in the apparent ligand-affinity of Hb in comparison with those of free hemes (P50a [Q]/ [1-Q]). Binding of heterotropic effectors reduces the O2-affinity of Hb (increases in P50). Effectors cause enhancement of very high-frequency (>tera-Hz) thermal fluctuations of globin in both in deoxy- and oxy-Hb (1), resulting in increases in apparent transparency/porosity of globin matrix toward migrating ligands, enhancing bi-molecular dissociation (Route B) and reducing geminate-rebinding (Route A) This results in substantial decrease in the apparent O2-affinity (KT and KR) without detectable changes in static crystallographic structures of deoxy- and oxy-Hb as well as the coordination/electronic structures of the hemes in T(deoxy)- and R(oxy)Hb (2). References: [1] Laberge & Yonetani, Molecular dynamics simulations of hemoglobin A in different states and bound to DPG, Biophys. J. 94 (2008) 2737; [2] Yonetani & Kanaori, How does hemoglobin generate such diverse functionality of physiological relevance?, BBA 1834 (2013) 1873. 1739-Pos Board B59 Steered Molecular Dynamic Simulations Reveal Critical Residues for (Un) Binding of Substrates, Inhibitors and a Product of the Malarial PFM1AAP Daniel S. Moore, John P. Dalton, Irina G. Tikhonova. Molecular Therapuitics, Queen‘s University, Belfast, United Kingdom.
Malaria is one of the most prevalent and fatal infectious diseases of the world and is compounded by wide spread drug resistance, with some strains developing resistance to current front-line anti-malarials. Plasmodium falciparum M1 alanyl aminopeptidase (PFM1AAP) is involved in the terminal stages of haemoglobin degradation and the generation of an amino acid pool to support parasitic growth and development. This represents a promising new antimalarial target where inhibition of this enzyme is lethal to the malarial parasite. To start understanding ligand recognition and binding in PFM1AAP for future drug design efforts, we explored (un)binding pathways of substrates, inhibitors and a product in the enzyme using steered molecular dynamics (sMD) simulations. The results of our simulations further clarify the substrate entrance and exit pathways to the buried active site of PFM1AAP and identify their respective physiochemical profiles. Our SMD simulations reveal several binding characteristics: (1) A substrate/inhibitor recognition mechanism, (2) active migration into the entrance channel, (3) a water reservoir which facilitates correct substrate orientation, (4) a molecular gate controlling product egress. Furthermore, several critical residues have been identified that in concert, facilitate these processes. These residues are now the focus of a mutagenesis study to fortify the in silico predictions. We observe differences in (un)binding pathways of substrates, inhibitors and product. A novel residue-based network analysis has been developed to highlight repetitive rather than sporadic events in the multiple sMD simulation trajectories of ligand (un)binding. Our work paves the way toward designing novel potent PFM1AAP inhibitors for the treatment of malaria. 1740-Pos Board B60 Dynamic Allostery in Regulated Entry of Newcastle Disease Virus into Host Cells Nalvi D. Duro, Sameer Varma. University of South Florida, Seminole, FL, USA. Newcastle disease virus (NDV) belongs to the family of paramyxoviruses that cause numerous fatal diseases in humans and farm animals. Here we focus on mechanisms that underlie its regulated entry into host cells. To gain entry, NDV utilizes two of its membrane proteins: Hemagglutinin-Neuraminidase (HN) and the fusion (F) protein. HN binds to sialic acids expressed on the host cell, and this binding stimulates HN to activate F, which, in turn, mediates virus-host membrane fusion. HN interacts with sialic acid and F through separate sites located on two different domains, however, no models explain this allosteric coupling. In fact, the analogous mechanisms in other paramyxoviruses also remain undetermined. Starting with X-ray structures of HN‘s receptor binding domain (bound to sialic acid derivatives), we examine using molecular dynamics how sialic acid affects HN’s receptor binding domain (RBD) as well as HN’s RBD-RBD dimeric interface. We note first that sialic acid induces only minor structural changes in individual RBDs - an observation similar to proteins like GPCRs that are regulated by dynamic allostery. Despite inducing minor changes in individual RBDs, we find that sialic acid reorients the RBDRBD interface, and that this reorientation contributes to HN stimulation. Finally, we note that the induced RBD-RBD reorientation is unlike what we observed in the case Nipah’s HN homolog (Dutta et al. Biophys. J. 2016), suggesting that fusion stimulation mechanisms are not conserved across paramyxoviruses. 1741-Pos Board B61 The Allostery Landscape: Quantifying Thermodynamic Couplings in Biomolecular Systems Michel A. Cuendet1,2, Harel Weinstein1,2, Michael V. LeVine1,2. 1 Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, NY, USA, 2HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College of Cornell University, New York, NY, USA. Allostery plays a fundament role in most biological processes. However, little theory is available to describe it outside of two-state models. Here we use a statistical mechanical approach to show that the allosteric coupling between two collective variables is not a single number, but instead a twodimensional thermodynamic coupling function that is directly related to the mutual information from information theory and the copula density function from probability theory. On this basis, we demonstrate how to quantify the contribution of specific energy terms to this thermodynamic coupling function, enabling a decomposition that reveals the mechanism of allostery. We illustrate the thermodynamic coupling function and its use by showing how allosteric coupling in the alanine dipeptide molecule contributes to the overall shape of the F/J free energy surface, and by identifying the interactions that are necessary for this coupling.
353a
to functions must be extracted from the time-series data. One of the multivariate analysis methods, principal component analysis (PCA), is widely used for analysing static properties of systems. On the other hand, relaxation mode analysis (RMA) has been used to elucidate dynamic properties in a random spin system [1], a homo-polymer system [2], and hetero-polymer systems [3, 4, 5]. This method, on the basis of statistical physics, finds slow relaxation modes in systems with relaxational dynamics. In its previous application to a protein system [4], however, the long-time behavior was not described well. Two-step RMA has recently been developed in a homo-polymer system in order to improve description of long-time behavior [6]. We applied this method to a hetero-polymer system, hen egg-white lysozyme (HEWL) protein. We found that two-step RMA not only describes long-time behavior well, but also extracts slow structural transitions of a protein more effectively. [1] H. Takano, S. Miyashita, J. Phys. Soc. Jpn. 64, 3688 (1995). [2] H. Hirao, S. Koseki, H. Takano, J. Phys. Soc. Jpn. 66, 3399 (1997). [3] A. Mitsutake, H. Iijima, H. Takano, J. Chem. Phys.135, 164102 (2011). [4] T. Nagai, A. Mitsutake, and H. Takano, J. Phys. Soc. Jpn. 82, 023803 (2013). [5] A. Mitsutake, H. Takano, J. Chem. Phys. 143, 124111 (2015). [6] S. Natori, H. Takano, submitted J. Phys. Soc. Jpn. 1735-Pos Board B55 Importance of Protein Vibration Directionality on Function Katherine A. Niessen1, Mengyang Xu1, Yanting Deng1, Edward H. Snell2,3, Andrea G. Markelz1. 1 Physics, SUNY University at Buffalo, Buffalo, NY, USA, 2HauptmanWoodward Medical Research Institute, Buffalo, NY, USA, 3Structural Biology, SUNY University at Buffalo, Buffalo, NY, USA. Global protein vibrations have long been associated with protein functionality. Long-range intramolecular vibrations have been measured in proteins using anisotropic absorption in the terahertz frequency range [1,2]. These measurements directly correspond to the directionality of vibrations and show large changes in the directionality of the vibrational displacements for free chicken egg white lysozyme (CEWL) and inhibitor bound CEWL. Normal mode ensemble analysis (NMEA) and quasiharmonic analysis (QHA) calculations of the free and tri-acetylglucosamine (3NAG) bound CEWL dynamics were performed and also show large changes in vibration directionality with binding. This in spite of the calculated energy distribution showing very little change, in agreement with neutron scattering measurements. We investigate the importance of the protein directionality by extending the calculations to a double deletion mutant (DD CEWL) with an activity rate 1.4 times that of WT [3]. The deletions are far from the active site, indicating that the increased activity arises from changes in the dynamics. The calculated anisotropic spectra show large changes with mutation, whereas the energy distribution show practically no change from the WT. Similarities in the bound WT, free DD, and bound DD CEWL NMEA spectra indicate the mutation may be changing the directionality of the vibrations toward more efficient motions. Projections of the low frequency vibrations, from QHA, on the functional displacement show higher overlap of residues around Glu35 and Asp52 in the mutant system. These residues are known to be critical in CEWL functionality. The results reveal that the mutation may be steering the protein toward more functional dynamics without significantly affecting the energy distribution. [1] K.A. Niessen, et al. (2015) DOI: 10.1007/s12551-015-0168-4 [2] G. Acbas, et al. (2014) DOI: 10.1038/ncomms4076 [3] S. Mine, et al. (1999) DOI: 10.1006/jmbi.1999.2572. Supported by NSF (DBI 1556359, MCB 1616529) and DOE (DE-SC0016317). 1736-Pos Board B56 Conformational Evolution of Three Regulator of G-Protein Signaling Proteins (RGS4, RGS8, RGS19) in Microsecond-Scale Simulations Hossein Mohammadiarani, Harish Vashisth. Chemical Engineering, University of New Hampshire, Durham, NH, USA. Regulators of G protein signaling (RGS) proteins modulate GPCR signaling by binding to Ga-subunits of heterotrimeric G proteins and accelerating hydrolysis of GTP. Therefore, RGS proteins are becoming increasingly important therapeutic targets to be directly or allosterically inhibited from binding to Ga-subunits. While structures of several known RGS proteins are highly similar and largely contain a-helical motifs, some thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for closely related proteins thereby suggesting intrinsic differences in dynamics of these proteins. In this work, we have
354a
Tuesday, February 14, 2017
studied the dynamics of three different RGS proteins (apo-RGS4, apo-RGS8, and apo-RGS19) using microsecond-scale classical molecular dynamics (MD) simulations with CHARMM and AMBER force-fields. Analyses of these trajectories reveal high fluctuations in a5 and a6 helices and the loops connecting them. These fluctuations lead to perturbations in residues in the RGS-Ga interface and in the vicinity of cysteines that are targets of allosteric inhibitors. These findings have significant implications for understanding differences in potencies and specificities of inhibitory small-molecules. 1737-Pos Board B57 Comparative Structural Dynamic Analysis of GTPases Hongyang Li, Xin-Qiu Yao, Barry Grant. Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA. GTPases regulate a multitude of essential cellular processes ranging from movement and division to differentiation and neuronal activity. These ubiquitous enzymes operate by hydrolyzing GTP to GDP with associated conformational changes that modulate affinity for family specific binding partners. There are three major GTPase superfamilies: Ras-like GTPases, heterotrimeric G proteins and protein-synthesizing GTPases. Although they contain similar nucleotide-binding sites, the detailed mechanisms by which these structurally and functionally diverse superfamilies operate remain unclear. Here we compare and contrast the structural dynamic mechanisms of each superfamily using extensive molecular dynamics (MD) simulations and subsequent network analysis approaches. In particular, dissection of the cross-correlations of atomic displacements in both the GTP and GDP-bound states of Ras, transducin and elongation factor EF-Tu reveals analogous dynamic features. This includes similar dynamic communities and subdomain structures (termed lobes). For all three proteins the GTP-bound state has stronger couplings between equivalent lobes. Network analysis further identifies common and family specific residues mediating the state specific coupling of distal functional sites. Mutational simulations demonstrate how disrupting these couplings leads to distal dynamic effects at the nucleotide binding site of each family. Collectively our studies extend current understanding of GTPase allosteric mechanisms and highlight previously unappreciated similarities across functionally diverse families. 1738-Pos Board B58 The Molecular Mechanism of Entropic Allostery of Hemoglobin Takashi Yonetani1, Kenji Kanaori2. 1 Biophys/Biochem, Univ. Pennsylvania, Philadelphia, PA, USA, 2Biomol Engineering, Kyoto Inst Tech, Kyoto, Japan. Protohemes in Hb (Hb) are reversible low-affinity binding sites for diatomic ligands such as O2, CO, and NO. The ligand-affinity of Hb is regulated by controlling two distal effects, i.e., distal H-bonding and geminate-recombination of the ligand, which enhance retention of the ligand at the hemes to increase apparent ligand-affinity of Hb. Ligands, dissociating from hemes, encounter physical barriers of globin matrix (‘‘Caged’’ states), before either geminately recombining to the hemes to regenerate the respective ligated hemes [Route A] or migrating through globin matrices, followed by bimolecular dissociation to form deoxy-Hb [Route B]. Therefore, only a fraction of the dissociating ligands moves out of Hb (bi-molecular quantum yield [Q]). The remainder reverts back to the hemes to form the ligated Hb (geminate quantum yield [1-Q]). This is resulted in increases in the apparent ligand-affinity of Hb in comparison with those of free hemes (P50a [Q]/ [1-Q]). Binding of heterotropic effectors reduces the O2-affinity of Hb (increases in P50). Effectors cause enhancement of very high-frequency (>tera-Hz) thermal fluctuations of globin in both in deoxy- and oxy-Hb (1), resulting in increases in apparent transparency/porosity of globin matrix toward migrating ligands, enhancing bi-molecular dissociation (Route B) and reducing geminate-rebinding (Route A) This results in substantial decrease in the apparent O2-affinity (KT and KR) without detectable changes in static crystallographic structures of deoxy- and oxy-Hb as well as the coordination/electronic structures of the hemes in T(deoxy)- and R(oxy)Hb (2). References: [1] Laberge & Yonetani, Molecular dynamics simulations of hemoglobin A in different states and bound to DPG, Biophys. J. 94 (2008) 2737; [2] Yonetani & Kanaori, How does hemoglobin generate such diverse functionality of physiological relevance?, BBA 1834 (2013) 1873. 1739-Pos Board B59 Steered Molecular Dynamic Simulations Reveal Critical Residues for (Un) Binding of Substrates, Inhibitors and a Product of the Malarial PFM1AAP Daniel S. Moore, John P. Dalton, Irina G. Tikhonova. Molecular Therapuitics, Queen‘s University, Belfast, United Kingdom.
Malaria is one of the most prevalent and fatal infectious diseases of the world and is compounded by wide spread drug resistance, with some strains developing resistance to current front-line anti-malarials. Plasmodium falciparum M1 alanyl aminopeptidase (PFM1AAP) is involved in the terminal stages of haemoglobin degradation and the generation of an amino acid pool to support parasitic growth and development. This represents a promising new antimalarial target where inhibition of this enzyme is lethal to the malarial parasite. To start understanding ligand recognition and binding in PFM1AAP for future drug design efforts, we explored (un)binding pathways of substrates, inhibitors and a product in the enzyme using steered molecular dynamics (sMD) simulations. The results of our simulations further clarify the substrate entrance and exit pathways to the buried active site of PFM1AAP and identify their respective physiochemical profiles. Our SMD simulations reveal several binding characteristics: (1) A substrate/inhibitor recognition mechanism, (2) active migration into the entrance channel, (3) a water reservoir which facilitates correct substrate orientation, (4) a molecular gate controlling product egress. Furthermore, several critical residues have been identified that in concert, facilitate these processes. These residues are now the focus of a mutagenesis study to fortify the in silico predictions. We observe differences in (un)binding pathways of substrates, inhibitors and product. A novel residue-based network analysis has been developed to highlight repetitive rather than sporadic events in the multiple sMD simulation trajectories of ligand (un)binding. Our work paves the way toward designing novel potent PFM1AAP inhibitors for the treatment of malaria. 1740-Pos Board B60 Dynamic Allostery in Regulated Entry of Newcastle Disease Virus into Host Cells Nalvi D. Duro, Sameer Varma. University of South Florida, Seminole, FL, USA. Newcastle disease virus (NDV) belongs to the family of paramyxoviruses that cause numerous fatal diseases in humans and farm animals. Here we focus on mechanisms that underlie its regulated entry into host cells. To gain entry, NDV utilizes two of its membrane proteins: Hemagglutinin-Neuraminidase (HN) and the fusion (F) protein. HN binds to sialic acids expressed on the host cell, and this binding stimulates HN to activate F, which, in turn, mediates virus-host membrane fusion. HN interacts with sialic acid and F through separate sites located on two different domains, however, no models explain this allosteric coupling. In fact, the analogous mechanisms in other paramyxoviruses also remain undetermined. Starting with X-ray structures of HN‘s receptor binding domain (bound to sialic acid derivatives), we examine using molecular dynamics how sialic acid affects HN’s receptor binding domain (RBD) as well as HN’s RBD-RBD dimeric interface. We note first that sialic acid induces only minor structural changes in individual RBDs - an observation similar to proteins like GPCRs that are regulated by dynamic allostery. Despite inducing minor changes in individual RBDs, we find that sialic acid reorients the RBDRBD interface, and that this reorientation contributes to HN stimulation. Finally, we note that the induced RBD-RBD reorientation is unlike what we observed in the case Nipah’s HN homolog (Dutta et al. Biophys. J. 2016), suggesting that fusion stimulation mechanisms are not conserved across paramyxoviruses. 1741-Pos Board B61 The Allostery Landscape: Quantifying Thermodynamic Couplings in Biomolecular Systems Michel A. Cuendet1,2, Harel Weinstein1,2, Michael V. LeVine1,2. 1 Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, NY, USA, 2HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College of Cornell University, New York, NY, USA. Allostery plays a fundament role in most biological processes. However, little theory is available to describe it outside of two-state models. Here we use a statistical mechanical approach to show that the allosteric coupling between two collective variables is not a single number, but instead a twodimensional thermodynamic coupling function that is directly related to the mutual information from information theory and the copula density function from probability theory. On this basis, we demonstrate how to quantify the contribution of specific energy terms to this thermodynamic coupling function, enabling a decomposition that reveals the mechanism of allostery. We illustrate the thermodynamic coupling function and its use by showing how allosteric coupling in the alanine dipeptide molecule contributes to the overall shape of the F/J free energy surface, and by identifying the interactions that are necessary for this coupling.