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Hydrophobicity of Poly(A)-Binding Protein's Intrinsically Disordered Region Determines its Conformation and Organism Thermotolerance
558a
Wednesday, March 2, 2016
2751-Pos Board B128 Probing the Conformational Ensemble of a Bacterial Antitoxin through Molecular Dynamics Simulations and Mass Spectrometry Virginia M. Burger1, Albert Konijnenberg2, Alexandra Vanderwelde3,4, Frank Sobott2,5, Remy Loris3,4, Collin M. Stultz6,7. 1 Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA, 2Department of Chemistry, University of Antwerp, Antwerp, Belgium, 3Department of Biotechnology, Department of Structural Biology, Vrije Universiteit Brussel, Brussels, Belgium, 4Structural Biology Brussels, Brussels, Belgium, 5UA-VITO Centre for Proteomics, University of Antwerp, Antwerp, Belgium, 6Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA, 7Research Laboratory of Electronics, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA. Intrinsic disorder plays a key role in the regulation of cell death by bacterial toxin-antitoxin (TA) modules. In TA modules, an unstable antitoxin normally inhibits a protein toxin. Cellular stressors trigger increased degradation of the labile antitoxin, thereby releasing the toxin. When not inhibited, the toxin disrupts essential cellular processes, causing cell death or quiescence. The CcdACcdB TA module in E. coli consists of the antitoxin CcdA and the toxin CcdB. CcdA is comprised of a folded DNA-binding domain and two intrinsically disordered regions (IDRs), which regulate binding to CcdB. NMR studies suggest that CcdA, in the absence of CcdB, predominantly samples conformations belonging to either a closed or an open state, distinguished by the distance from the IDR termini to the folded domain. The potential for CcdA to sample multiple partially stable states provokes the question of the role these states play in facilitating the IDRs’ functions. We use all-atom explicit-water molecular dynamics (MD) simulations and native ion mobility-mass spectrometry (MS) to determine the conformational ensemble of unbound CcdA, with the goal of inferring functional roles from structural details. Both MD and MS indicate that CcdA samples metastable states of varying compactness. In one state CcdA can adopt compact, relatively closed conformations, as predicted by NMR. In a separate state, that has similar energy, one IDR extends away from the central folded domain. Further analysis of IDR helicity and solvent exposure within each substate provides insight into the functional role of these states. As intrinsically disordered antitoxin proteins like CcdA are plentiful in bacteria, understanding how disorder facilitates their functions could lead to novel antibiotics that harness TA modules to kill bacterial cells. 2752-Pos Board B129 The Role of Intrinsic Disorder in the Molecular Mechanism of Nuclear Transport Laura K. Maguire1, Kathryn Wall1, Geoff Armstrong1, Kaushik Dutta2, Samuel Sparks3, Deniz B. Temel3, Alia Kamal4, Jaclyn Tetenbaum-Novatt4, Michael P. Rout4, David Cowburn3, Loren Hough1. 1 CU Boulder, Boulder, CO, USA, 2New York Structural Biology Center, New York, NY, USA, 3Albert Einstein College of Medicine, Bronx, NY, USA, 4Rockefeller University, New York, NY, USA. Nuclear pore complexes form a selective filter that allows the rapid passage of transport factors and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG) rich repeats line the pore and interact with transport factors. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with transport factors. We report on recently published (Hough et. al. eLife 2015) as well as subsequent experiments to probe the molecular mechanism of transport. We used biophysical characterization and nuclear magnetic resonance both in solution and within the bacterial and yeast cellular environments to probe the molecular mechanism of transport and the role of disorder in this process. 2753-Pos Board B130 Elucidating the Mechanism of Recognition and Binding of Protein Kinase Inhibitor by Protein Kinase a using NMR and Fluorescence Spectroscopy Geoffrey Li1, Cristina Olivieri2, Matthew Neibergall3, Jonggul Kim1, Susan Taylor4, Joseph Muretta2, Gianluigi Veglia1,2. 1 Chemistry, University of Minnesota, Minneapolis, MN, USA, 2 Biochemistry, Molecular Biology, Biophysics, University of Minnesota, Minneapolis, MN, USA, 3Chemistry, Bethel University, St. Paul, MN, USA, 4 Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA. Cyclic AMP-dependent protein kinase A (PKA) mediates many crucial cellular events including gene expression, metabolism, and cardiac contractility by catalyzing phosphoryl transfer. The endogenous thermostable protein kinase inhibitor (PKI) is a key protein that regulates the activity and intracellular localization of PKA through its two functional motifs: the kinase inhibitory domain and nu-
clear export signal domain. The kinase inhibitory domain binds specifically with the catalytic subunit of PKA (PKA-C) with high affinity while the nuclear export signal is responsible for shuttling the kinase out of the nucleus. PKI is intrinsically disordered in its free form but adopts residual lowly populated structure through the two distinct domains. Upon PKA-C binding, PKI folds into a more ordered conformation with chemical shift changes propagated throughout the inhibitory domain, however the remainder of the inhibitor is disordered. Here, we aim to elucidate the mechanism by which PKA recognizes and binds PKI by a combination of NMR and fluorescence spectroscopy. Our findings reveal the molecular mechanism of interaction that will help us understand how an intrinsically disordered protein can perform its dual regulatory functions. Our future work will focus on the structural basis of nuclear export of PKA-C upon PKI binding, revealing the mechanistic details of regulation of PKA outside of cAMP control. 2754-Pos Board B131 Conformations and Exchange Dynamics of FlgM, an Intrinsically Disordered Protein, in Dilute and Crowded Conditions Studied by NMR Spectroscopy Pieter E.S. Smith, Huan-Xiang Zhou. Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA. The majority of biochemical and biophysical studies are performed using dilute solutions of macromolecules. In contrast, the cellular environment contains a high total concentration (up to 400 g/L) of biomacromolecules. Concentrated bystander molecules (i.e. crowders) can exert important effects on the dynamics and conformational ensembles of proteins, especially intrinsically disordered proteins (IDPs). In particular, FlgM, a regulator of the ordered synthesis of flagellar proteins, is an IDP with transient helices in the C-terminal region and becomes structured upon binding its sigma factor target. Structure formation can be induced by high concentrations of glucose and bovine serum albumin. To investigate the conformations and exchange dynamics of FlgM in dilute and crowded conditions, we carried out backbone 15N NMR relaxation and CPMG relaxation dispersion experiments. In a dilute condition, elevated transverse relaxation rates (R2) in the C-terminal region are consistent with transient secondary structure. CPMG relaxation dispersion data indicate conformational exchange throughout the protein sequence, possibly between extended conformations with few tertiary contacts and more compact conformations with some tertiary contacts. The addition of 100 g/L dextran resulted in elevation of R2 throughout the protein, suggesting an increase in helical content in both the C-terminal and N-terminal regions. Moreover, dextran led to an increase in the amplitude of dispersion, suggesting an increase in the population of compact conformations. The NMR studies lay the foundation for quantitative characterization of an IDP in dilute and crowded conditions. 2755-Pos Board B132 Hydrophobicity of Poly(A)-Binding Protein’s Intrinsically Disordered Region Determines its Conformation and Organism Thermotolerance Joshua Riback1, Chris Katanski2, Tobin R. Sosnick2, D. Allan Drummond2. 1 Institute of Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 2Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA. Many intrinsically disordered regions (IDRs) are involved in promoting and regulating protein-protein interactions and liquid-liquid phase separations. How the amino acid composition and conformation of an IDR translates into biological functions is not well understood. Here, we study the proline-rich IDR (P domain) of budding yeast’s poly(A)-binding protein (Pab1), a canonical marker and implicated promoter of stress granules. The P domain has a conserved composition containing low charge, and near-average hydrophobicity (compared to other IDRs), and hence, is expected to be prone to non-specific collapse and aggregation due to a preference for H-bonding. We found that the domain collapses but is disordered by SAXS, CD and NMR. Collapse is robust, being retained even upon sequence randomization or substitution of all glycines (14% of residues) to prolines (19% to 33%) or alanines (12% to 26%). Only substitutions to hydrophobic residues such as methionine and valine (10% and 5% respectively) that weaken hydrophobicity perturb collapse. The mutational data suggest that hydrophobicity rather then H-bonding or specific packing drives collapse. We also found that the aggregation propensity of Pab1 bearing substitutions to the P domain correlates with collapse suggesting that both intra- and inter- molecular contacts are influential. Furthermore, we find that yeast natively expressing Pab1 with these substitutions in the P domain have thermotolerance that correlates with collapse and aggregation propensity. The correlation suggests that the biological function of the P domain is related to its hydrophobicity, that interaction between hydrophobic contacts of an IDR can be relevant for weak protein interactions such as those of membrane-less organelles such as stress granules, and establishes that even in IDRs, hydrophobicity can determine conformation and function.
Wednesday, March 2, 2016
2751-Pos Board B128 Probing the Conformational Ensemble of a Bacterial Antitoxin through Molecular Dynamics Simulations and Mass Spectrometry Virginia M. Burger1, Albert Konijnenberg2, Alexandra Vanderwelde3,4, Frank Sobott2,5, Remy Loris3,4, Collin M. Stultz6,7. 1 Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, USA, 2Department of Chemistry, University of Antwerp, Antwerp, Belgium, 3Department of Biotechnology, Department of Structural Biology, Vrije Universiteit Brussel, Brussels, Belgium, 4Structural Biology Brussels, Brussels, Belgium, 5UA-VITO Centre for Proteomics, University of Antwerp, Antwerp, Belgium, 6Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA, 7Research Laboratory of Electronics, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA. Intrinsic disorder plays a key role in the regulation of cell death by bacterial toxin-antitoxin (TA) modules. In TA modules, an unstable antitoxin normally inhibits a protein toxin. Cellular stressors trigger increased degradation of the labile antitoxin, thereby releasing the toxin. When not inhibited, the toxin disrupts essential cellular processes, causing cell death or quiescence. The CcdACcdB TA module in E. coli consists of the antitoxin CcdA and the toxin CcdB. CcdA is comprised of a folded DNA-binding domain and two intrinsically disordered regions (IDRs), which regulate binding to CcdB. NMR studies suggest that CcdA, in the absence of CcdB, predominantly samples conformations belonging to either a closed or an open state, distinguished by the distance from the IDR termini to the folded domain. The potential for CcdA to sample multiple partially stable states provokes the question of the role these states play in facilitating the IDRs’ functions. We use all-atom explicit-water molecular dynamics (MD) simulations and native ion mobility-mass spectrometry (MS) to determine the conformational ensemble of unbound CcdA, with the goal of inferring functional roles from structural details. Both MD and MS indicate that CcdA samples metastable states of varying compactness. In one state CcdA can adopt compact, relatively closed conformations, as predicted by NMR. In a separate state, that has similar energy, one IDR extends away from the central folded domain. Further analysis of IDR helicity and solvent exposure within each substate provides insight into the functional role of these states. As intrinsically disordered antitoxin proteins like CcdA are plentiful in bacteria, understanding how disorder facilitates their functions could lead to novel antibiotics that harness TA modules to kill bacterial cells. 2752-Pos Board B129 The Role of Intrinsic Disorder in the Molecular Mechanism of Nuclear Transport Laura K. Maguire1, Kathryn Wall1, Geoff Armstrong1, Kaushik Dutta2, Samuel Sparks3, Deniz B. Temel3, Alia Kamal4, Jaclyn Tetenbaum-Novatt4, Michael P. Rout4, David Cowburn3, Loren Hough1. 1 CU Boulder, Boulder, CO, USA, 2New York Structural Biology Center, New York, NY, USA, 3Albert Einstein College of Medicine, Bronx, NY, USA, 4Rockefeller University, New York, NY, USA. Nuclear pore complexes form a selective filter that allows the rapid passage of transport factors and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG) rich repeats line the pore and interact with transport factors. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with transport factors. We report on recently published (Hough et. al. eLife 2015) as well as subsequent experiments to probe the molecular mechanism of transport. We used biophysical characterization and nuclear magnetic resonance both in solution and within the bacterial and yeast cellular environments to probe the molecular mechanism of transport and the role of disorder in this process. 2753-Pos Board B130 Elucidating the Mechanism of Recognition and Binding of Protein Kinase Inhibitor by Protein Kinase a using NMR and Fluorescence Spectroscopy Geoffrey Li1, Cristina Olivieri2, Matthew Neibergall3, Jonggul Kim1, Susan Taylor4, Joseph Muretta2, Gianluigi Veglia1,2. 1 Chemistry, University of Minnesota, Minneapolis, MN, USA, 2 Biochemistry, Molecular Biology, Biophysics, University of Minnesota, Minneapolis, MN, USA, 3Chemistry, Bethel University, St. Paul, MN, USA, 4 Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA. Cyclic AMP-dependent protein kinase A (PKA) mediates many crucial cellular events including gene expression, metabolism, and cardiac contractility by catalyzing phosphoryl transfer. The endogenous thermostable protein kinase inhibitor (PKI) is a key protein that regulates the activity and intracellular localization of PKA through its two functional motifs: the kinase inhibitory domain and nu-
clear export signal domain. The kinase inhibitory domain binds specifically with the catalytic subunit of PKA (PKA-C) with high affinity while the nuclear export signal is responsible for shuttling the kinase out of the nucleus. PKI is intrinsically disordered in its free form but adopts residual lowly populated structure through the two distinct domains. Upon PKA-C binding, PKI folds into a more ordered conformation with chemical shift changes propagated throughout the inhibitory domain, however the remainder of the inhibitor is disordered. Here, we aim to elucidate the mechanism by which PKA recognizes and binds PKI by a combination of NMR and fluorescence spectroscopy. Our findings reveal the molecular mechanism of interaction that will help us understand how an intrinsically disordered protein can perform its dual regulatory functions. Our future work will focus on the structural basis of nuclear export of PKA-C upon PKI binding, revealing the mechanistic details of regulation of PKA outside of cAMP control. 2754-Pos Board B131 Conformations and Exchange Dynamics of FlgM, an Intrinsically Disordered Protein, in Dilute and Crowded Conditions Studied by NMR Spectroscopy Pieter E.S. Smith, Huan-Xiang Zhou. Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA. The majority of biochemical and biophysical studies are performed using dilute solutions of macromolecules. In contrast, the cellular environment contains a high total concentration (up to 400 g/L) of biomacromolecules. Concentrated bystander molecules (i.e. crowders) can exert important effects on the dynamics and conformational ensembles of proteins, especially intrinsically disordered proteins (IDPs). In particular, FlgM, a regulator of the ordered synthesis of flagellar proteins, is an IDP with transient helices in the C-terminal region and becomes structured upon binding its sigma factor target. Structure formation can be induced by high concentrations of glucose and bovine serum albumin. To investigate the conformations and exchange dynamics of FlgM in dilute and crowded conditions, we carried out backbone 15N NMR relaxation and CPMG relaxation dispersion experiments. In a dilute condition, elevated transverse relaxation rates (R2) in the C-terminal region are consistent with transient secondary structure. CPMG relaxation dispersion data indicate conformational exchange throughout the protein sequence, possibly between extended conformations with few tertiary contacts and more compact conformations with some tertiary contacts. The addition of 100 g/L dextran resulted in elevation of R2 throughout the protein, suggesting an increase in helical content in both the C-terminal and N-terminal regions. Moreover, dextran led to an increase in the amplitude of dispersion, suggesting an increase in the population of compact conformations. The NMR studies lay the foundation for quantitative characterization of an IDP in dilute and crowded conditions. 2755-Pos Board B132 Hydrophobicity of Poly(A)-Binding Protein’s Intrinsically Disordered Region Determines its Conformation and Organism Thermotolerance Joshua Riback1, Chris Katanski2, Tobin R. Sosnick2, D. Allan Drummond2. 1 Institute of Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 2Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA. Many intrinsically disordered regions (IDRs) are involved in promoting and regulating protein-protein interactions and liquid-liquid phase separations. How the amino acid composition and conformation of an IDR translates into biological functions is not well understood. Here, we study the proline-rich IDR (P domain) of budding yeast’s poly(A)-binding protein (Pab1), a canonical marker and implicated promoter of stress granules. The P domain has a conserved composition containing low charge, and near-average hydrophobicity (compared to other IDRs), and hence, is expected to be prone to non-specific collapse and aggregation due to a preference for H-bonding. We found that the domain collapses but is disordered by SAXS, CD and NMR. Collapse is robust, being retained even upon sequence randomization or substitution of all glycines (14% of residues) to prolines (19% to 33%) or alanines (12% to 26%). Only substitutions to hydrophobic residues such as methionine and valine (10% and 5% respectively) that weaken hydrophobicity perturb collapse. The mutational data suggest that hydrophobicity rather then H-bonding or specific packing drives collapse. We also found that the aggregation propensity of Pab1 bearing substitutions to the P domain correlates with collapse suggesting that both intra- and inter- molecular contacts are influential. Furthermore, we find that yeast natively expressing Pab1 with these substitutions in the P domain have thermotolerance that correlates with collapse and aggregation propensity. The correlation suggests that the biological function of the P domain is related to its hydrophobicity, that interaction between hydrophobic contacts of an IDR can be relevant for weak protein interactions such as those of membrane-less organelles such as stress granules, and establishes that even in IDRs, hydrophobicity can determine conformation and function.