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TM Domain and its Interaction with the Fusion Loop Explains their Fusion Activity
78a
Sunday, February 12, 2017
by depolarization of a neuron, which in turn activates voltage-gated Ca2þ channels. The resulting Ca2þ influx then triggers the fusion of the synaptic vesicles with the plasma membrane. Synaptic vesicle fusion is mediated by a core fusion machinery SNARE complex, a small regulatory factor complexin (Cpx), and Ca2þ sensor synaptotagmin (Syt). However, it was unknown how they cooperate to trigger synaptic vesicle fusion. Combining X-ray crystallography and electrophysiological recording techniques, we determined two atomic resolution crystal structures of the synaptic vesicle fusion machinery at different states, revealing a large, specific, Ca2þ-independent interface which is essential for synchronous neurotransmitter release in mouse neuronal synapses. We propose a working model and further reveal the molecular mechanism of synchronous neurotransmitter release. 400-Pos Board B165 a-Synuclein: A Functional Role as a Regulator of SNARE-Mediated Fusion Siobhan Toal, Elizabeth Rhoades. Chemistry, University of Pennsylvania, Philadelphia, PA, USA. Fusion of vesicular and plasma membranes is mediated by SNARE proteins. In a vesicular fusion event, the t- and v-SNAREs assemble into a four-helix bundle pulling the two membranes together to cause fusion. A decrease in neurotransmitter release upon overexpression of the neuronal protein a-Synuclein (aS) has been observed in animal models, suggesting that aS may act as a regulator of neurotransmission, altering SNARE driven fusion of synaptic vesicles. Recent work in our lab has shown that aS is able to inhibit SNARE-mediated fusion in vitro, although the mechanism appears to be through binding to the lipid bilayer, not through direct interactions with SNARE proteins. Here, we investigate the possibility that aS may also modulate fusion through interactions with SNARE regulatory proteins, synaptotagmin and complexin, using an in vitro fusion assay. We find that in the presence of synaptotagmin and complexin, aS differentially alters SNARE-mediated vesicle fusion in a concentration dependent manner. At low aS concentrations, fusion is significantly enhanced in a concentration-dependent manner (i.e. increasing fusion with increasing aS). However, once a threshold concentration is exceeded, fusion is again inhibited, again in a concentration dependent manner (i.e. decreasing fusion with increasing aS). Direct evidence of protein-protein interactions was monitored using fluorescence correlation spectroscopy to measure the diffusion times of the protein components. SNARE complex formation can be observed as a function of time through an increase in the diffusion time of labeled t-SNARE protein. While aS does not appear to impact the rate of complex formation alone, substantial increases in the diffusion time of the SNARE complex in the presence of aS and complexin were observed, suggesting an interaction between the two. Taken together, our results suggest that aS may have a dual role in SNARE-mediated membrane fusion, as a chaperone of SNARE regulatory components as well as, at high enough concentrations, a fusion inhibitor. 401-Pos Board B166 Mitochondrial Fusion Proteins: A Tale of Two Membranes Andrew D. Kehr, Marisa A. Rubio, Jenny Hinshaw. NIDDK, National Institutes of Health, Bethesda, MD, USA. Dynamins are a class of GTPase enzymes responsible for the fusion, fission, and vesiculation of cellular lipid membranes throughout the cell. The dynamin-like proteins Optic Atrophy 1 (Opa1) and Mitofusin (Mfn) 1 and 2 are responsible for the fusion of the mitochondrial inner and outer membranes, respectively. Mutations in any of these proteins can lead to neuropathies including blindness and Charcot-MarieTooth, a disease characterized by progressive loss of distal muscle tissue. Currently, little is known structurally or biochemically about any of these proteins. We have developed a protocol for expressing and purifying biologically relevant and biochemically active shortened isoforms (OpaGG and Mfn1GG) in sufficient quantity to begin crystallographic studies. Both have comparable GTPase activity compared to fulllength, unstimulated Dynamin 1 when assayed at room temperature and interestingly OpaGG exists as a tetramer when assayed by size exclusion chromatography. In addition, the long, membrane-bound isoforms of Opa1 and Mfn1 have been expressed and purified in large quantities. To date, we have shown full length Mfn1 can be incorporated into proteoliposomes and in the presence of GTP forms dense tethers as seen by cryo-EM. This tethering is reversible as shown by confocal microscopy. Currently we are developing tethering assays for Opa1 and fusion assays for both Opa1 and Mfn1.
402-Pos Board B167 Structure of the Ebola Virus Envelope Protein MPER/TM Domain and its Interaction with the Fusion Loop Explains their Fusion Activity Jinwoo Lee1,2, David A. Nyenhuis1,3, Elizabeth A. Nelson1,4, David S. Cafiso1,3, Judith M. White1,4, Lukas K. Tamm1,2. 1 Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA, 2Departments of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA, 3 Department of Chemistry, University of Virginia, Charlottesville, VA, USA, 4 Department of Cell Biology, University of Virginia, Charlottesville, VA, USA. Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and releases its genetic material into the cytoplasm after membrane fusion in a late endosomal compartment. Membrane fusion is governed by the EBOV surface envelope glycoprotein (GP), which consists of subunits GP1 and GP2. GP1 binds to cellular receptors including Niemann-Pick C1 (NPC1) protein and GP2 is responsible for membrane fusion at low pH. GP1 undergoes multiple steps of proteolytic cleavage and binds to NPC1 at endosomal pH. GP2 is rearranged in a fashion that exposes the hydrophobic fusion loop (FL) of GP2, which is then inserted into the cellular target membrane, ultimately forming a sixhelix bundle structure and resulting in the formation of the fusion pore. Although major portions of the GP2 structure that have been solved in preand post-fusion states and the current model places the transmembrane (TM) and FL domains of GP2 in close proximity to each other at critical steps of membrane fusion, their structures in membrane environments and especially interactions between TM and FL have not yet been characterized. Here we present the structure of the membrane proximal external region (MPER) connected to the TM domain, i.e. the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membranemimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region, not TM region, is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry highlighting the functional importance of the MPER/TM - FL interaction in EBOV entry and fusion. 403-Pos Board B168 Leakage Induced by the Influenza Virus Haemagglutinin Depends on Target Membrane Spontaneous Curvature Sourav Haldar, Elena Mekhedov, Jane Farrington, Petr Chlanda, Paul S. Blank, Joshua Zimmerberg. Section on Integrative Biophysics, NICHD/NIH, Bethesda, MD, USA. A recent cryo-electron microscopy investigation(Chlanda et al. (2016) Nat. Microbiol. 1:16050) of hemifusion structures mediated by the influenza virus haemagglutinin posited that there exist two pathways for hemi-fusion: hemifusion-stalk and rupture-insertion. Depending on target membrane material properties, such as spontaneous curvature, one pathway will be favored over the other. A prediction of this hypothesis is that leakage of soluble content will be greater through the rupture-insertion pathway. To test this prediction, we have developed a giant unilamellar vesicle (GUV)-based dye influx assay that provides a direct measure of leakage. Our results show that leakage (influx of soluble dye in GUV) induced by influenza virus changes from ~ 80 % to ~ 40 % as the spontaneous curvature is changed from 0.02 nm1 to 0.30 nm1, supporting the hypothesis that leakage is modulated by membrane spontaneous curvature. Surprisingly, with some lipid compositions, leakage was sub-maximal, i.e. there was a variable degree of GUV filling. This result raised the possibility of a transient target membrane damage induced by the influenza virus. It was also possible that the complete fusion of a leaky virus to a GUV was responsible for the filling of the GUV. To control for this possibility, we compared leakage induced by commercially prepared virus (containing significant damaged viral membrane, as evidenced by entry of a cell impermeant nucleotidebinding dye) with lab-grown virus (with apparently minimal damaged viral membranes). 404-Pos Board B169 Viral Fusion Efficacy of Influenza Virus H3N2 Reassortment Combination to the Suppoered Lipid Layer Hunglun Hsu1, Jean Millet2, Deirdre Costello1, Gary Whittaker2, Susan Daniel1. 1 Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA, 2Department of Microbiology and Immunology, Cornell University, Ithaca, NY, USA.
Sunday, February 12, 2017
by depolarization of a neuron, which in turn activates voltage-gated Ca2þ channels. The resulting Ca2þ influx then triggers the fusion of the synaptic vesicles with the plasma membrane. Synaptic vesicle fusion is mediated by a core fusion machinery SNARE complex, a small regulatory factor complexin (Cpx), and Ca2þ sensor synaptotagmin (Syt). However, it was unknown how they cooperate to trigger synaptic vesicle fusion. Combining X-ray crystallography and electrophysiological recording techniques, we determined two atomic resolution crystal structures of the synaptic vesicle fusion machinery at different states, revealing a large, specific, Ca2þ-independent interface which is essential for synchronous neurotransmitter release in mouse neuronal synapses. We propose a working model and further reveal the molecular mechanism of synchronous neurotransmitter release. 400-Pos Board B165 a-Synuclein: A Functional Role as a Regulator of SNARE-Mediated Fusion Siobhan Toal, Elizabeth Rhoades. Chemistry, University of Pennsylvania, Philadelphia, PA, USA. Fusion of vesicular and plasma membranes is mediated by SNARE proteins. In a vesicular fusion event, the t- and v-SNAREs assemble into a four-helix bundle pulling the two membranes together to cause fusion. A decrease in neurotransmitter release upon overexpression of the neuronal protein a-Synuclein (aS) has been observed in animal models, suggesting that aS may act as a regulator of neurotransmission, altering SNARE driven fusion of synaptic vesicles. Recent work in our lab has shown that aS is able to inhibit SNARE-mediated fusion in vitro, although the mechanism appears to be through binding to the lipid bilayer, not through direct interactions with SNARE proteins. Here, we investigate the possibility that aS may also modulate fusion through interactions with SNARE regulatory proteins, synaptotagmin and complexin, using an in vitro fusion assay. We find that in the presence of synaptotagmin and complexin, aS differentially alters SNARE-mediated vesicle fusion in a concentration dependent manner. At low aS concentrations, fusion is significantly enhanced in a concentration-dependent manner (i.e. increasing fusion with increasing aS). However, once a threshold concentration is exceeded, fusion is again inhibited, again in a concentration dependent manner (i.e. decreasing fusion with increasing aS). Direct evidence of protein-protein interactions was monitored using fluorescence correlation spectroscopy to measure the diffusion times of the protein components. SNARE complex formation can be observed as a function of time through an increase in the diffusion time of labeled t-SNARE protein. While aS does not appear to impact the rate of complex formation alone, substantial increases in the diffusion time of the SNARE complex in the presence of aS and complexin were observed, suggesting an interaction between the two. Taken together, our results suggest that aS may have a dual role in SNARE-mediated membrane fusion, as a chaperone of SNARE regulatory components as well as, at high enough concentrations, a fusion inhibitor. 401-Pos Board B166 Mitochondrial Fusion Proteins: A Tale of Two Membranes Andrew D. Kehr, Marisa A. Rubio, Jenny Hinshaw. NIDDK, National Institutes of Health, Bethesda, MD, USA. Dynamins are a class of GTPase enzymes responsible for the fusion, fission, and vesiculation of cellular lipid membranes throughout the cell. The dynamin-like proteins Optic Atrophy 1 (Opa1) and Mitofusin (Mfn) 1 and 2 are responsible for the fusion of the mitochondrial inner and outer membranes, respectively. Mutations in any of these proteins can lead to neuropathies including blindness and Charcot-MarieTooth, a disease characterized by progressive loss of distal muscle tissue. Currently, little is known structurally or biochemically about any of these proteins. We have developed a protocol for expressing and purifying biologically relevant and biochemically active shortened isoforms (OpaGG and Mfn1GG) in sufficient quantity to begin crystallographic studies. Both have comparable GTPase activity compared to fulllength, unstimulated Dynamin 1 when assayed at room temperature and interestingly OpaGG exists as a tetramer when assayed by size exclusion chromatography. In addition, the long, membrane-bound isoforms of Opa1 and Mfn1 have been expressed and purified in large quantities. To date, we have shown full length Mfn1 can be incorporated into proteoliposomes and in the presence of GTP forms dense tethers as seen by cryo-EM. This tethering is reversible as shown by confocal microscopy. Currently we are developing tethering assays for Opa1 and fusion assays for both Opa1 and Mfn1.
402-Pos Board B167 Structure of the Ebola Virus Envelope Protein MPER/TM Domain and its Interaction with the Fusion Loop Explains their Fusion Activity Jinwoo Lee1,2, David A. Nyenhuis1,3, Elizabeth A. Nelson1,4, David S. Cafiso1,3, Judith M. White1,4, Lukas K. Tamm1,2. 1 Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, VA, USA, 2Departments of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA, 3 Department of Chemistry, University of Virginia, Charlottesville, VA, USA, 4 Department of Cell Biology, University of Virginia, Charlottesville, VA, USA. Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and releases its genetic material into the cytoplasm after membrane fusion in a late endosomal compartment. Membrane fusion is governed by the EBOV surface envelope glycoprotein (GP), which consists of subunits GP1 and GP2. GP1 binds to cellular receptors including Niemann-Pick C1 (NPC1) protein and GP2 is responsible for membrane fusion at low pH. GP1 undergoes multiple steps of proteolytic cleavage and binds to NPC1 at endosomal pH. GP2 is rearranged in a fashion that exposes the hydrophobic fusion loop (FL) of GP2, which is then inserted into the cellular target membrane, ultimately forming a sixhelix bundle structure and resulting in the formation of the fusion pore. Although major portions of the GP2 structure that have been solved in preand post-fusion states and the current model places the transmembrane (TM) and FL domains of GP2 in close proximity to each other at critical steps of membrane fusion, their structures in membrane environments and especially interactions between TM and FL have not yet been characterized. Here we present the structure of the membrane proximal external region (MPER) connected to the TM domain, i.e. the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membranemimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region, not TM region, is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry highlighting the functional importance of the MPER/TM - FL interaction in EBOV entry and fusion. 403-Pos Board B168 Leakage Induced by the Influenza Virus Haemagglutinin Depends on Target Membrane Spontaneous Curvature Sourav Haldar, Elena Mekhedov, Jane Farrington, Petr Chlanda, Paul S. Blank, Joshua Zimmerberg. Section on Integrative Biophysics, NICHD/NIH, Bethesda, MD, USA. A recent cryo-electron microscopy investigation(Chlanda et al. (2016) Nat. Microbiol. 1:16050) of hemifusion structures mediated by the influenza virus haemagglutinin posited that there exist two pathways for hemi-fusion: hemifusion-stalk and rupture-insertion. Depending on target membrane material properties, such as spontaneous curvature, one pathway will be favored over the other. A prediction of this hypothesis is that leakage of soluble content will be greater through the rupture-insertion pathway. To test this prediction, we have developed a giant unilamellar vesicle (GUV)-based dye influx assay that provides a direct measure of leakage. Our results show that leakage (influx of soluble dye in GUV) induced by influenza virus changes from ~ 80 % to ~ 40 % as the spontaneous curvature is changed from 0.02 nm1 to 0.30 nm1, supporting the hypothesis that leakage is modulated by membrane spontaneous curvature. Surprisingly, with some lipid compositions, leakage was sub-maximal, i.e. there was a variable degree of GUV filling. This result raised the possibility of a transient target membrane damage induced by the influenza virus. It was also possible that the complete fusion of a leaky virus to a GUV was responsible for the filling of the GUV. To control for this possibility, we compared leakage induced by commercially prepared virus (containing significant damaged viral membrane, as evidenced by entry of a cell impermeant nucleotidebinding dye) with lab-grown virus (with apparently minimal damaged viral membranes). 404-Pos Board B169 Viral Fusion Efficacy of Influenza Virus H3N2 Reassortment Combination to the Suppoered Lipid Layer Hunglun Hsu1, Jean Millet2, Deirdre Costello1, Gary Whittaker2, Susan Daniel1. 1 Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA, 2Department of Microbiology and Immunology, Cornell University, Ithaca, NY, USA.