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Investigation of the pH Induced Conformational Rearrangement of Influenza Hemagglutinin
Sunday, February 28, 2016 functionality on bacterial attachment. Altogether, by integrating biophysical and computational methods to map the FimH conformer-function relationship, this work begins to unravel how a conformational phase switch regulates bacterial adhesion and how evolutionary processes fine-tune protein dynamics to guide host colonization. 76-Plat Investigation of the pH Induced Conformational Rearrangement of Influenza Hemagglutinin Xingcheng Lin1, Jeffrey K. Noel1, Nathanial R. Eddy1, Jianpeng Ma2, Jose´ N. Onuchic1. 1 Physics and Astronomy, Rice University / CTBP, Houston, TX, USA, 2 Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA. Influenza hemaggutinin (HA), a trimeric membrane fusion glycoprotein, guides the invasion of flu viruses into its host cells through endocytosis after binding to their receptors. During this process, a reduced endosomal pH triggers a largescale structural rearrangement of HA2, the evolutionarily conserved stem domain of HA. This structural change is responsible for the fusion of the viral and the host membranes, facilitating the subsequent delivery of viral genome. Although the end states structures of the HA2 transition have been crystallized, the molecular details of this transition is unknown. To map out the overall picture, here we employ a combination of both coarse-grained and explicit solvent molecular dynamics simulations with the aim of understanding the underlying mechanism of membrane fusion. Our simulations suggest that the overall transition is separated into a fast phase and a slow phase. At the fast phase, HA2 experiences a fast order-disorder transition at a junction domain (Loop3-4) and reaches an asymmetric configuration. In the subsequent slow phase HA2 starts two transition pathways. Also, the results reveal that a reduction in pH destabilizes HA2 by protonating some key residues near its fusion peptides. Finally, a timescale competition among different domains of HA2 is shown to be necessary for HA function. 77-Plat Atomic Structure of a Non-Enveloped dsRNA Virus Reveals pH Sensing for Cell Entry Xing Zhang. CNSI, UCLA, Los Angeles, CA, USA. Mechanism to monitor and response to environmental changes by nonenveloped dsRNA viruses during cell entry is largely unclear, which is in contrast to that by envelope viruses. In particular, how these viruses sense pH change inside endosomes and disrupt host membrane to gain entry into cytoplasm are not understood. To gain insight into cell entry by nonenveloped dsRNA viruses, we determined the atomic structure of the bluetongue virus (BTV) at pH 8.5, a non-enveloped dsRNA virus that undergoes a two-stage endosomal process of cell entry. Our structure reveals that the receptor-binding protein VP2 possesses a conserved zinc-finger motif which may be sensitive to low pH, and that the membrane penetration protein VP5 has three domains: dagger, unfurling and anchoring. The core of the anchoring domain not only contains a b-meander motif with a histidine cluster for low-pH sensing, but also has features potentially for membrane interactions: aromatic clusters, a hydrophobic surface and a WHXL motif. The cryoEM structure of BTV at low pH (5.5 & 3.4) reveals that the VP2 detaches from virus particles, and that the VP5 undergoes dramatic conformational change with the dagger and unfurling domains of VP5 trimer project outwards to form a long filament structure while the anchoring domain remains attached on virus inner capsid. Consistently, biochemical and structure-based mutagenesis studies support these mechanistic interpretations.
Platform: Skeletal Muscle Mechanics, Structure, and Regulation 78-Plat The Force Producing ADP State of Myosin Bound to Actin Rasmus R. Schroeder1, Sarah F. Wulf1, Virginie Ropars2, Setsuko Fujita-Becker1, Marco Oster1, Goetz Hofhaus1, Leonardo G. Trabuco3, Olena Pylypenko2, H. Lee Sweeney4, Anne Houdusse2. 1 Cryo Electron Microscopy, Universitaet Heidelberg, Heidelberg, Germany, 2 Institut Curie, Paris, France, 3Universitaet Heidelberg, Heidelberg, Germany, 4University of Florida, Gainesville, FL, USA. Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly ˚ bound, while myosin is strongly bound to actin. We have generated an 8A
13a
cryo-electron microscopy reconstruction of this state for myosin V, and used Molecular Dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The new structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This is accomplished by a new conformation of the beta sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5 rotation of the myosin lever arm, coupled to a beta sheet rearrangement. Thus the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release from myosin that is essential for processive myosins, such as myosin V. 79-Plat Ribbons, Not Subfilaments Michael K. Reedy, Robert J. Perz-Edwards. Cell Biology, Duke Univ Med Ctr, Durham, NC, USA. Sixty years ago Francis Crick proposed a stable protein structure called the alpha-helical coiled-coil. Muscle contains two classic examples of coiledcoils, tropomyosin and the long rod-domain of myosin. In situ, tropomyosin wraps around actin and is therefore a coiled-coiled-coil, but almost nothing is known about how myosin rods pack together to form the thick filament. The longest-standing theory for myosin has been that the rods from 2-4 molecules would wrap around each other, and thus also be coiled-coiled-coils, forming ~4-nm diameter sub-filaments. Various numbers of these hypothetical subfilaments would then pack together to give the varied symmetries observed in thick filaments from different species. Numerous low-resolution EM studies, either analysis of 2D projections or 3D reconstructions of thick filaments, have supported the idea that myosin rods might be packed into ~4-nm diameter sub-filaments. We present a 0.5-nm resolution 3D cryo-EM structure of the thick filament from Lethocerus flight muscle in which the entire rod domain of myosin can be traced. When filtered to lower resolution and viewed in projection, our reconstruction also shows an approximate 12-fold symmetry that suggests sub-filaments; however, at full resolution it is clear that the myosin rods do not wrap around each other. We see no evidence of sub-filaments. Instead, 3-4 rods lie approximately parallel to one another forming a continuous, flat ribbon-like structure. The heads of each myosin molecule project from one side of the ribbon with the C-terminus ending at the other side. Adjacent molecules within the ribbon are axially separated by 43.5 nm. Twelve ribbons are then packed together, with adjacent ribbons staggered by 14.5 nm (= 43.5/3) axially, to give the long-observed 4-fold symmetric thick filament with crowns of myosin heads separated by 14.5 nm. 80-Plat Stress-Sensing Mobilizes Myosin Motors in the Thick Filaments of Resting Muscle Massimo Reconditi1, Elisabetta Brunello2, Marco Caremani1, Luca Fusi3, Marco Linari1, Theyencheri Narayanan4, Gabriella Piazzesi1, Malcom Irving3, Vincenzo Lombardi1. 1 Biology, University of Florence, Firenze, Italy, 2Biology, King’s College London, London, United Kingdom, 3King’s College London, London, United Kingdom, 4ESRF, Grenoble, France. We recently described a novel mechanism mediated by thick filament stress that controls load-dependent recruitment of myosin motors during muscle contraction (Linari et al. Nature in press). Here we show by time-resolved X-ray diffraction with a 5-m camera at beamline ID02 (ESRF) that this mechanism is also present in resting muscle. Force steps of 0.25 times the maximum active isometric force were imposed on resting mouse EDL muscles. Sarcomere length (SL), measured by X-ray diffraction with a 30-m camera, increased from SL 2.4 to 2.9 mm during the force step, and the intensities of the M2, M4 and M5 reflections associated with axial perturbations of myosin motors in the filament region containing myosin binding protein-C (MyBP-C) decreased by ca. 60%. These changes are linked to SL rather than filament stress, because similar reductions were produced by slowly increasing SL to 2.9 mm at low resting force. The spacing and fine structure of the M3 reflection were almost constant during the force step, but its intensity (IM3) decreased by ca. 50%, as did that of the first myosin layer line (IML1), indicating loss of the helical arrangement of the myosin motors associated with the thick filament OFF state. The spacing of the M6 reflection (SM6) increased by 0.4% during the step, indicating increased strain in the thick filament backbone. Much smaller changes in IM3, IML1 and SM6 were observed when SL was increased slowly from 2.4 to 2.9 mm at low force. Thus thick filament stress directly triggers the rapid release of myosin motors from the helical OFFstate in the absence of calcium, but MyBP-C links between the filaments do not mediate the effect. Supported by MIUR-PRIN and FIRB-Futuro in Ricerca (Italy), MRC (UK).
Platform: Skeletal Muscle Mechanics, Structure, and Regulation 78-Plat The Force Producing ADP State of Myosin Bound to Actin Rasmus R. Schroeder1, Sarah F. Wulf1, Virginie Ropars2, Setsuko Fujita-Becker1, Marco Oster1, Goetz Hofhaus1, Leonardo G. Trabuco3, Olena Pylypenko2, H. Lee Sweeney4, Anne Houdusse2. 1 Cryo Electron Microscopy, Universitaet Heidelberg, Heidelberg, Germany, 2 Institut Curie, Paris, France, 3Universitaet Heidelberg, Heidelberg, Germany, 4University of Florida, Gainesville, FL, USA. Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly ˚ bound, while myosin is strongly bound to actin. We have generated an 8A
13a
cryo-electron microscopy reconstruction of this state for myosin V, and used Molecular Dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The new structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This is accomplished by a new conformation of the beta sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5 rotation of the myosin lever arm, coupled to a beta sheet rearrangement. Thus the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release from myosin that is essential for processive myosins, such as myosin V. 79-Plat Ribbons, Not Subfilaments Michael K. Reedy, Robert J. Perz-Edwards. Cell Biology, Duke Univ Med Ctr, Durham, NC, USA. Sixty years ago Francis Crick proposed a stable protein structure called the alpha-helical coiled-coil. Muscle contains two classic examples of coiledcoils, tropomyosin and the long rod-domain of myosin. In situ, tropomyosin wraps around actin and is therefore a coiled-coiled-coil, but almost nothing is known about how myosin rods pack together to form the thick filament. The longest-standing theory for myosin has been that the rods from 2-4 molecules would wrap around each other, and thus also be coiled-coiled-coils, forming ~4-nm diameter sub-filaments. Various numbers of these hypothetical subfilaments would then pack together to give the varied symmetries observed in thick filaments from different species. Numerous low-resolution EM studies, either analysis of 2D projections or 3D reconstructions of thick filaments, have supported the idea that myosin rods might be packed into ~4-nm diameter sub-filaments. We present a 0.5-nm resolution 3D cryo-EM structure of the thick filament from Lethocerus flight muscle in which the entire rod domain of myosin can be traced. When filtered to lower resolution and viewed in projection, our reconstruction also shows an approximate 12-fold symmetry that suggests sub-filaments; however, at full resolution it is clear that the myosin rods do not wrap around each other. We see no evidence of sub-filaments. Instead, 3-4 rods lie approximately parallel to one another forming a continuous, flat ribbon-like structure. The heads of each myosin molecule project from one side of the ribbon with the C-terminus ending at the other side. Adjacent molecules within the ribbon are axially separated by 43.5 nm. Twelve ribbons are then packed together, with adjacent ribbons staggered by 14.5 nm (= 43.5/3) axially, to give the long-observed 4-fold symmetric thick filament with crowns of myosin heads separated by 14.5 nm. 80-Plat Stress-Sensing Mobilizes Myosin Motors in the Thick Filaments of Resting Muscle Massimo Reconditi1, Elisabetta Brunello2, Marco Caremani1, Luca Fusi3, Marco Linari1, Theyencheri Narayanan4, Gabriella Piazzesi1, Malcom Irving3, Vincenzo Lombardi1. 1 Biology, University of Florence, Firenze, Italy, 2Biology, King’s College London, London, United Kingdom, 3King’s College London, London, United Kingdom, 4ESRF, Grenoble, France. We recently described a novel mechanism mediated by thick filament stress that controls load-dependent recruitment of myosin motors during muscle contraction (Linari et al. Nature in press). Here we show by time-resolved X-ray diffraction with a 5-m camera at beamline ID02 (ESRF) that this mechanism is also present in resting muscle. Force steps of 0.25 times the maximum active isometric force were imposed on resting mouse EDL muscles. Sarcomere length (SL), measured by X-ray diffraction with a 30-m camera, increased from SL 2.4 to 2.9 mm during the force step, and the intensities of the M2, M4 and M5 reflections associated with axial perturbations of myosin motors in the filament region containing myosin binding protein-C (MyBP-C) decreased by ca. 60%. These changes are linked to SL rather than filament stress, because similar reductions were produced by slowly increasing SL to 2.9 mm at low resting force. The spacing and fine structure of the M3 reflection were almost constant during the force step, but its intensity (IM3) decreased by ca. 50%, as did that of the first myosin layer line (IML1), indicating loss of the helical arrangement of the myosin motors associated with the thick filament OFF state. The spacing of the M6 reflection (SM6) increased by 0.4% during the step, indicating increased strain in the thick filament backbone. Much smaller changes in IM3, IML1 and SM6 were observed when SL was increased slowly from 2.4 to 2.9 mm at low force. Thus thick filament stress directly triggers the rapid release of myosin motors from the helical OFFstate in the absence of calcium, but MyBP-C links between the filaments do not mediate the effect. Supported by MIUR-PRIN and FIRB-Futuro in Ricerca (Italy), MRC (UK).