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smFRET Reveals Structural Basis for Conformational Misfolding of a Cystic Fibrosis Mutation in CFTR
204a
Monday, February 13, 2017
concentrations of monomeric subunits. However, the structure of the pore domain as a monomer before tetramerization is not clear. Recently, thiollabeling experiments have shown that isolated monomers engineered from the voltage-gated potassium channel Kv1.3 can readily adopt their native fold as they are being synthesized. Mutations at certain critical positions make the p-helix become more solvent-exposed; however, the mechanism by how the p-helix becomes more solvent-exposed is unclear. Here, our MD simulations and NMR measurements to show that the monomeric pore domains of potassium channels are heterogeneous and dynamic. According to the simulations, WT Kv1.2 monomer is unstable in its native-like structure and the 3 helices are very dynamic in the membrane. While the 3 helices separate from each other and form many non-native interactions, all helices retain their helical structure and remain buried in the membrane. Simulations of mutants (W384A/W385A/V387E, V387E, and V388E) also show the separation of all 3 helices, yet the p-helix loses helicity and becomes less stable, leading to higher solvent-exposure of the p-helix. We found that the helicity and membrane burial of the p-helix depends on the locations of the inserted charged groups. When a charged group is introduced to the opposite face of the helix as the native aspartate D381, the p-helix leaves the membrane and loses its helicity but when the mutation is on the same face, the helix goes into the interfacial region and retains its helical nature. In addition, our NMR spectroscopy and hydrogen exchange measurements suggest that the WT monomer of KcsA in lipid bicelles may be highly heterogeneous, but are all buried in the membrane. Together, we suggest that the WT monomers are highly dynamic before tetramerization and the locations of mutations on the p-helix highly dictate its helicity and burial. 1007-Pos Board B75 Role of Lipids in the Reaction Coordinate of GlpG Rhomboid Protease Ana-Nicoleta Bondar. Department of Physics, Freie Universit€at Berlin, Berlin, Germany. Rhomboid proteases function in hydrated lipid membrane environments that can impact significantly the catalytic activity of the enzyme. How lipids participate in the reaction coordinate of rhomboid proteases is, however, unclear. We address this question by performing all-atom molecular dynamics simulations of the rhomboid protease from Escherichia coli, GlpG. We study GlpG starting from three different protein crystal structures, and explore interactions with lipids by considering five one-component lipid membranes with different lipid headgroups or alkyl chains, and in mixed membranes of phosphatidylethanolamine and phosphatidylgylcerol lipids. Lipid alkyl chains can visit inter-helical regions of the protein close to the gate helix, which can indicate regions of potential interest for substrate binding. Variability in how the protein can interact with lipids closest to the helical gate region suggests that membrane environments may impact proteolysis by participating in the substrate-binding step of the reaction coordinate. Research was supported in part by funding from the Excellence Initiative of the German Federal and State Governments via the Freie Universit€at Berlin, and by an allocation of computing time from HLRN, The North-German Supercomputing Center (bec00076). 1008-Pos Board B76 smFRET Reveals Structural Basis for Conformational Misfolding of a Cystic Fibrosis Mutation in CFTR Georg Krainer1, Antoine Treff1, Andreas Hartmann1, Henry Chang2, Tracy Stone2, Ariana Rath2, Charles Deber2, Michael Schlierf1. 1 B CUBE - Molecular Bioengineering, TU Dresden, Dresden, Germany, 2 University of Toronto, Toronto, ON, Canada. Cystic fibrosis (CF) is the most common lethal genetic disease among the Western population caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The development of effective therapeutic agents against CF requires a molecular understanding of how mutationrelated structural alterations lead to impaired functioning of CFTR. The main cause for CF are processing mutations frequently found within CFTRtransmembrane (TM) segments which affect co-translational folding and insertion. However, the underlying molecular mechanisms for misfolding remain obscure, mainly because of the unavailability of high-resolution structures and a lack of methods suitable for studying membrane protein structurefunction relationships. Devising a divide-and-conquer approach in combination with single-molecule FRET (smFRET), we present here a strategy to gain novel insights into the structural basis for conformational misfolding in CFTR. Using this approach, we study the CF-phenotypic polar mutation V232D located within the fourth TM helix (TM4) of CFTR. In vivo folding and insertion of TM4 is functionally coupled with TM3 where both TM segments are co-translationally inserted into the membrane as a TM helix-loop-helix hairpin motif (TM3/4). Utilizing this
minimal folding unit, we employ smFRET as a molecular ruler to readout structural alterations imposed on the helical packing of the TM3/4 hairpin upon mutation. In contrast to earlier findings that suggested a TM lock between TM3 and TM4 by a non-native H-bond, our results reveal that V232D TM3/4 associates with membranes in an open conformation. This implies that the charged residue imposes an energy penalty on membrane insertion of the hairpin. We propose a model for CF pathogenesis where partitioning of V232D TM3/4 into the interfacial region leads to misfolding of CFTR during cotranslational membrane insertion and inhibits maturation by trapping the protein as a partially folded intermediate. 1009-Pos Board B77 Investigating the Insertion and Folding of Membrane Transporters into Lipid Bilayers using a Cell Free Expression System Nicola J. Harris, Paula J. Booth. King’s College London, London, United Kingdom. Membrane proteins play a vital role in many biological processes, and yet due to their instability in vitro remain poorly understood. The Major Facilitator Superfamily (MFS) is a large group of transport proteins responsible for transporting a wide range of substrates. This project aims to investigate the insertion and folding of MFS proteins into lipid bilayers, using a commercial cell free expression system (PURExpress) in combination with synthetic liposomes of defined lipid composition. These studies will aid understanding of cooperative folding, folding intermediates, and the effects of the lipid bilayer on folding and insertion. We have chosen model prokaryotic transporters, as they can offer important insights into other proteins, and facilitate the study of more biologically relevant targets. We have found that the MFS transporters LacY, XylE, PepTso, GalP and GlpT spontaneously insert into liposomes, without the aid of an insertase such as SecYEG. This indicates that the innate hydrophobicity of these transporters is sufficient for insertion into a bilayer. Additionally, spontaneously inserted LacY is able to transport a fluorescent substrate across a Droplet Interface Bilayer, a good indication that it is folded correctly once inserted. This spontaneous insertion is highly influenced by the lipid composition of the liposomes. All the transporters tested to date prefer a lipid headgroup composition at least 50 % phosphatidylglycerol (PG), whereas bilayers containing phosphatidylcholine (PC) headgroups have the lowest spontaneous insertion yield. On-going work will investigate whether the two helical domains of MFS transporters fold independently or cooperatively when expressed as two separate polypeptides. Preliminary results indicate that they are able to fold independently. 1010-Pos Board B78 Folding Membrane Proteins by Contacts Inferred from Non-Membrane Proteins and Near-Atomic Level Refinement Sheng Wang1,2, Zongan Wang3,4, John Jumper3,4, Karl F. Freed3,4, Tobin R. Sosnick5,6, Jinbo Xu1,7. 1 Toyota Technological Institute at Chicago, Chicago, IL, USA, 2Department of Human Genetics, University of Chicago, Chicago, IL, USA, 3Department of Chemistry, University of Chicago, Chicago, IL, USA, 4James Franck Institute, University of Chicago, Chicago, IL, USA, 5Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA, 6Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 7Computation Institute, University of Chicago, Chicago, IL, USA. Computational prediction of membrane protein structure is very challenging due to lack of sufficient solved structures as templates or training data. Recently direct evolutionary coupling (EC) analysis has shed some light on protein contact prediction and accordingly contact-assisted folding. Limited by information within a single protein family, EC analysis is effective only on some very large-sized families. We have developed a deep transfer learning method to predict contacts for membrane proteins not in a large family, by making use of information in thousands of non-membrane protein families. Deep learning can learn complex patterns from large datasets and has recently revolutionized object and speech recognition and the GO game. Our deep learning method learns contact patterns and the complex relationship between contacts and protein features (amino acid property, residue conservation level and residue coevolution strength) from non-membrane proteins, but can predict membrane protein contacts very well. Tested on 398 non-redundant membrane proteins, the average top L/10 long-range contact prediction accuracy of our method is 0.78, much better than the representative EC method CCMpred (0.52), the CASP11 winner MetaPSICOV (0.61) and the methods trained by only membrane proteins (0.60). When some membrane proteins are added into the training data, we can further improve accuracy by ~4%. Using only our predicted contacts, we can correctly fold 160 test membrane proteins with ˚ while homology modeling, CCMpred and MetaPSICOV contacts RMSD <8A can fold only 10, 60 and 87 proteins, respectively. We further refine the 3D
Monday, February 13, 2017
concentrations of monomeric subunits. However, the structure of the pore domain as a monomer before tetramerization is not clear. Recently, thiollabeling experiments have shown that isolated monomers engineered from the voltage-gated potassium channel Kv1.3 can readily adopt their native fold as they are being synthesized. Mutations at certain critical positions make the p-helix become more solvent-exposed; however, the mechanism by how the p-helix becomes more solvent-exposed is unclear. Here, our MD simulations and NMR measurements to show that the monomeric pore domains of potassium channels are heterogeneous and dynamic. According to the simulations, WT Kv1.2 monomer is unstable in its native-like structure and the 3 helices are very dynamic in the membrane. While the 3 helices separate from each other and form many non-native interactions, all helices retain their helical structure and remain buried in the membrane. Simulations of mutants (W384A/W385A/V387E, V387E, and V388E) also show the separation of all 3 helices, yet the p-helix loses helicity and becomes less stable, leading to higher solvent-exposure of the p-helix. We found that the helicity and membrane burial of the p-helix depends on the locations of the inserted charged groups. When a charged group is introduced to the opposite face of the helix as the native aspartate D381, the p-helix leaves the membrane and loses its helicity but when the mutation is on the same face, the helix goes into the interfacial region and retains its helical nature. In addition, our NMR spectroscopy and hydrogen exchange measurements suggest that the WT monomer of KcsA in lipid bicelles may be highly heterogeneous, but are all buried in the membrane. Together, we suggest that the WT monomers are highly dynamic before tetramerization and the locations of mutations on the p-helix highly dictate its helicity and burial. 1007-Pos Board B75 Role of Lipids in the Reaction Coordinate of GlpG Rhomboid Protease Ana-Nicoleta Bondar. Department of Physics, Freie Universit€at Berlin, Berlin, Germany. Rhomboid proteases function in hydrated lipid membrane environments that can impact significantly the catalytic activity of the enzyme. How lipids participate in the reaction coordinate of rhomboid proteases is, however, unclear. We address this question by performing all-atom molecular dynamics simulations of the rhomboid protease from Escherichia coli, GlpG. We study GlpG starting from three different protein crystal structures, and explore interactions with lipids by considering five one-component lipid membranes with different lipid headgroups or alkyl chains, and in mixed membranes of phosphatidylethanolamine and phosphatidylgylcerol lipids. Lipid alkyl chains can visit inter-helical regions of the protein close to the gate helix, which can indicate regions of potential interest for substrate binding. Variability in how the protein can interact with lipids closest to the helical gate region suggests that membrane environments may impact proteolysis by participating in the substrate-binding step of the reaction coordinate. Research was supported in part by funding from the Excellence Initiative of the German Federal and State Governments via the Freie Universit€at Berlin, and by an allocation of computing time from HLRN, The North-German Supercomputing Center (bec00076). 1008-Pos Board B76 smFRET Reveals Structural Basis for Conformational Misfolding of a Cystic Fibrosis Mutation in CFTR Georg Krainer1, Antoine Treff1, Andreas Hartmann1, Henry Chang2, Tracy Stone2, Ariana Rath2, Charles Deber2, Michael Schlierf1. 1 B CUBE - Molecular Bioengineering, TU Dresden, Dresden, Germany, 2 University of Toronto, Toronto, ON, Canada. Cystic fibrosis (CF) is the most common lethal genetic disease among the Western population caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The development of effective therapeutic agents against CF requires a molecular understanding of how mutationrelated structural alterations lead to impaired functioning of CFTR. The main cause for CF are processing mutations frequently found within CFTRtransmembrane (TM) segments which affect co-translational folding and insertion. However, the underlying molecular mechanisms for misfolding remain obscure, mainly because of the unavailability of high-resolution structures and a lack of methods suitable for studying membrane protein structurefunction relationships. Devising a divide-and-conquer approach in combination with single-molecule FRET (smFRET), we present here a strategy to gain novel insights into the structural basis for conformational misfolding in CFTR. Using this approach, we study the CF-phenotypic polar mutation V232D located within the fourth TM helix (TM4) of CFTR. In vivo folding and insertion of TM4 is functionally coupled with TM3 where both TM segments are co-translationally inserted into the membrane as a TM helix-loop-helix hairpin motif (TM3/4). Utilizing this
minimal folding unit, we employ smFRET as a molecular ruler to readout structural alterations imposed on the helical packing of the TM3/4 hairpin upon mutation. In contrast to earlier findings that suggested a TM lock between TM3 and TM4 by a non-native H-bond, our results reveal that V232D TM3/4 associates with membranes in an open conformation. This implies that the charged residue imposes an energy penalty on membrane insertion of the hairpin. We propose a model for CF pathogenesis where partitioning of V232D TM3/4 into the interfacial region leads to misfolding of CFTR during cotranslational membrane insertion and inhibits maturation by trapping the protein as a partially folded intermediate. 1009-Pos Board B77 Investigating the Insertion and Folding of Membrane Transporters into Lipid Bilayers using a Cell Free Expression System Nicola J. Harris, Paula J. Booth. King’s College London, London, United Kingdom. Membrane proteins play a vital role in many biological processes, and yet due to their instability in vitro remain poorly understood. The Major Facilitator Superfamily (MFS) is a large group of transport proteins responsible for transporting a wide range of substrates. This project aims to investigate the insertion and folding of MFS proteins into lipid bilayers, using a commercial cell free expression system (PURExpress) in combination with synthetic liposomes of defined lipid composition. These studies will aid understanding of cooperative folding, folding intermediates, and the effects of the lipid bilayer on folding and insertion. We have chosen model prokaryotic transporters, as they can offer important insights into other proteins, and facilitate the study of more biologically relevant targets. We have found that the MFS transporters LacY, XylE, PepTso, GalP and GlpT spontaneously insert into liposomes, without the aid of an insertase such as SecYEG. This indicates that the innate hydrophobicity of these transporters is sufficient for insertion into a bilayer. Additionally, spontaneously inserted LacY is able to transport a fluorescent substrate across a Droplet Interface Bilayer, a good indication that it is folded correctly once inserted. This spontaneous insertion is highly influenced by the lipid composition of the liposomes. All the transporters tested to date prefer a lipid headgroup composition at least 50 % phosphatidylglycerol (PG), whereas bilayers containing phosphatidylcholine (PC) headgroups have the lowest spontaneous insertion yield. On-going work will investigate whether the two helical domains of MFS transporters fold independently or cooperatively when expressed as two separate polypeptides. Preliminary results indicate that they are able to fold independently. 1010-Pos Board B78 Folding Membrane Proteins by Contacts Inferred from Non-Membrane Proteins and Near-Atomic Level Refinement Sheng Wang1,2, Zongan Wang3,4, John Jumper3,4, Karl F. Freed3,4, Tobin R. Sosnick5,6, Jinbo Xu1,7. 1 Toyota Technological Institute at Chicago, Chicago, IL, USA, 2Department of Human Genetics, University of Chicago, Chicago, IL, USA, 3Department of Chemistry, University of Chicago, Chicago, IL, USA, 4James Franck Institute, University of Chicago, Chicago, IL, USA, 5Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA, 6Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 7Computation Institute, University of Chicago, Chicago, IL, USA. Computational prediction of membrane protein structure is very challenging due to lack of sufficient solved structures as templates or training data. Recently direct evolutionary coupling (EC) analysis has shed some light on protein contact prediction and accordingly contact-assisted folding. Limited by information within a single protein family, EC analysis is effective only on some very large-sized families. We have developed a deep transfer learning method to predict contacts for membrane proteins not in a large family, by making use of information in thousands of non-membrane protein families. Deep learning can learn complex patterns from large datasets and has recently revolutionized object and speech recognition and the GO game. Our deep learning method learns contact patterns and the complex relationship between contacts and protein features (amino acid property, residue conservation level and residue coevolution strength) from non-membrane proteins, but can predict membrane protein contacts very well. Tested on 398 non-redundant membrane proteins, the average top L/10 long-range contact prediction accuracy of our method is 0.78, much better than the representative EC method CCMpred (0.52), the CASP11 winner MetaPSICOV (0.61) and the methods trained by only membrane proteins (0.60). When some membrane proteins are added into the training data, we can further improve accuracy by ~4%. Using only our predicted contacts, we can correctly fold 160 test membrane proteins with ˚ while homology modeling, CCMpred and MetaPSICOV contacts RMSD <8A can fold only 10, 60 and 87 proteins, respectively. We further refine the 3D