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Architectures of Lipid Transport Systems for the Bacterial Outer Membrane
14a
Saturday, February 11, 2017
Cryo-EM Subgroup 75-Subg Architectures of Lipid Transport Systems for the Bacterial Outer Membrane Gira Bhabha1,2, Damian C. Ekiert1,2, Garrett Greenan1, Sergey Ovchinnikov3, Jeffery Cox4, Ronald D. Vale1. 1 University of California, San Francisco, San Francisco, CA, USA, 2Skirball Institute, NYU School of Medicine, New York, NY, USA, 3University of Washington, Seattle, WA, USA, 4University of California, Berkeley, Berkeley, CA, USA. All cells face the challenge of transporting hydrophobic lipids from one membrane to another through an intervening aqueous environment. Eukaryotes solve this trafficking problem using small transport vesicles that shuttle between membrane compartments. Many bacteria face a similar problem of transporting lipids between the inner and outer membrane through an aqueous periplasm, but lack vesicular transport systems. How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic periplasm not well understood. We have used cryo-EM and crystallography to elucidate the architectures of three systems, involving the mammalian cell entry (MCE) proteins, in the bacterial periplasm that have been thought to facilitate lipid transport in double-membraned bacteria The E. coli MCE protein, MlaD, forms a ring as part of a larger ABC transporter complex in the inner membrane, and employs a soluble lipid-binding protein to ferry lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the entire periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the inner and outer membranes of bacteria and some eukaryotic organelles. 76-Subg Kinase Regulation Through Dramatic Unfolding, as Told by Hsp90:Cdc37:Cdk4 Atomic Cryoem Structure Kliment A. Verba1, David A. Agard2. 1 Department of Biochemistry & Biophysics, University of California, San Francisco, San Francisco, CA, USA, 2Howard Hughes Medical Institute and the Department of Biochemistry & Biophysics, University of California, San Francisco, San Francisco, CA, USA. The Hsp90 molecular chaperone and its Cdc37 co-chaperone help stabilize and activate over half of the human kinome. However, neither the mechanism by which these chaperones assist their client kinases nor why some kinases are addicted to Hsp90 while closely related family members are independent is known. Missing has been any structural understanding of these interactions, with no full-length structures of human Hsp90, Cdc37 or either of these proteins with a kinase. Here we report a ˚ cryoEM structure of the Hsp90:Cdc37:Cdk4 kinase complex. Cdk4 3.9A is in a novel conformation, with its two lobes completely separated. Cdc37 mimics part of the kinase N-lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase b5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. Based on this novel structure and extensive previous data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions. 77-Subg Molecular Mechanisms Explained by Single Particle Cryo-Em Stefan Raunser. Structural Biochemistry, MPI of Molecular Physiology, Dortmund, Germany.
Muscular movement plays an essential role not only in our lives. Muscle contraction is initiated by the release of calcium from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors. Calcium binds to troponin, which releases tropomyosin from its blocking position allowing myosin filaments to move along actin filaments resulting in the contraction of the muscle. The underlying mechanism of the function and regulation of muscle contraction are complex but poorly understood. In my talk I will present our recent published and unpublished findings revealing important molecular details of both processes. 78-Subg The Cryo-EM Method Micro-ED Structure Determination of Type II Diabetes-Related Protein Segments Pascal Krotee1, Jose A. Rodriguez1, Michael R. Sawaya1, Duilio Cascio1, Francis E. Reyes2, Dan Shi2, Johan Hattne2, Brent L. Nannenga2, Marie E. Oskarsson3, Lin Jiang4, Gunilla T. Westermark3, Tamir Gonen2, David S. Eisenberg1. 1 HHMI, UCLA-DOE Institute, Depts. of Biol Chem and Biochem, Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA, 2Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA, 3Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden, 4Department of Neurology, Mol. Bio. Institute, Brain Res. Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA. The cryoEM method micro-electron diffraction (MicroED) utilizes 3D crystals only a few hundred nanometers thick for atomic structure determination, thus shrinking the crystal size limits set by synchrotron micro-focus beamlines. MicroED is ideal for 3D nanocrystals, such as those formed from amyloid protein segments. Amyloid proteins form fibers that are the hallmarks of a class of more than 25 amyloid diseases, ranging from Parkinson’s disease to Type II Diabetes (T2D). Although full-length amyloid proteins have so far been resistant to crystallization, select protein segments that form the spines of amyloid fibrils do form such tiny 3D crystals. Gaining knowledge of the atomic structures of these protein segments is critical to our understanding of amyloid fibril structures that contribute to disease onset and progression. Here, we use MicroED to determine the structures of two 11-residue segments from Islet ˚ and 2.0 A ˚ resolution, respectively. Amyloid Polypeptide (IAPP) at 1.4 A Amyloid fibrils of IAPP are found in approximately 90% of T2D patients and the formation of these fibrils is tightly linked to pancreatic b-cell death and insulin dependence. The two segments overlap in sequence by 5 residues. Their structures add to the range of polymorphism observed previously in crystal structures of shorter IAPP segments. One segment, 15-25WT, forms an unusual arrangement of single, out-of-register b-sheets. The second segment, 19-29S20G, contains the familial S20G mutation that leads to early-onset T2D. It forms pairs of b-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length IAPP fibrils. Thus, this structure may serve as a model for the toxic spine of IAPP aggregates. These findings, facilitated by MicroED, give us insight into disease-relevant structures that can be utilized for structure-based design of therapeutics. 79-Subg Ligand-Dependent Structural States of a KDChannel Analyzed by Cryo-EM Roderick MacKinnon. Rockefeller University, New York, NY, USA. The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. I will describe the equilibrium ensemble of structures of Slo2.2, a neuronal Naþ-activated Kþ channel, as a function of the Naþ concentration. Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Naþ concentration. An open conformation, which correlates to functional activation, emerges from the closed ensemble in a highly Naþ-dependent manner without evidence of Naþ-dependent intermediates. Thus, channel opening is very concerted almost switch-like - analogous to a phase transition.
Saturday, February 11, 2017
Cryo-EM Subgroup 75-Subg Architectures of Lipid Transport Systems for the Bacterial Outer Membrane Gira Bhabha1,2, Damian C. Ekiert1,2, Garrett Greenan1, Sergey Ovchinnikov3, Jeffery Cox4, Ronald D. Vale1. 1 University of California, San Francisco, San Francisco, CA, USA, 2Skirball Institute, NYU School of Medicine, New York, NY, USA, 3University of Washington, Seattle, WA, USA, 4University of California, Berkeley, Berkeley, CA, USA. All cells face the challenge of transporting hydrophobic lipids from one membrane to another through an intervening aqueous environment. Eukaryotes solve this trafficking problem using small transport vesicles that shuttle between membrane compartments. Many bacteria face a similar problem of transporting lipids between the inner and outer membrane through an aqueous periplasm, but lack vesicular transport systems. How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic periplasm not well understood. We have used cryo-EM and crystallography to elucidate the architectures of three systems, involving the mammalian cell entry (MCE) proteins, in the bacterial periplasm that have been thought to facilitate lipid transport in double-membraned bacteria The E. coli MCE protein, MlaD, forms a ring as part of a larger ABC transporter complex in the inner membrane, and employs a soluble lipid-binding protein to ferry lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the entire periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the inner and outer membranes of bacteria and some eukaryotic organelles. 76-Subg Kinase Regulation Through Dramatic Unfolding, as Told by Hsp90:Cdc37:Cdk4 Atomic Cryoem Structure Kliment A. Verba1, David A. Agard2. 1 Department of Biochemistry & Biophysics, University of California, San Francisco, San Francisco, CA, USA, 2Howard Hughes Medical Institute and the Department of Biochemistry & Biophysics, University of California, San Francisco, San Francisco, CA, USA. The Hsp90 molecular chaperone and its Cdc37 co-chaperone help stabilize and activate over half of the human kinome. However, neither the mechanism by which these chaperones assist their client kinases nor why some kinases are addicted to Hsp90 while closely related family members are independent is known. Missing has been any structural understanding of these interactions, with no full-length structures of human Hsp90, Cdc37 or either of these proteins with a kinase. Here we report a ˚ cryoEM structure of the Hsp90:Cdc37:Cdk4 kinase complex. Cdk4 3.9A is in a novel conformation, with its two lobes completely separated. Cdc37 mimics part of the kinase N-lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase b5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. Based on this novel structure and extensive previous data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions. 77-Subg Molecular Mechanisms Explained by Single Particle Cryo-Em Stefan Raunser. Structural Biochemistry, MPI of Molecular Physiology, Dortmund, Germany.
Muscular movement plays an essential role not only in our lives. Muscle contraction is initiated by the release of calcium from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors. Calcium binds to troponin, which releases tropomyosin from its blocking position allowing myosin filaments to move along actin filaments resulting in the contraction of the muscle. The underlying mechanism of the function and regulation of muscle contraction are complex but poorly understood. In my talk I will present our recent published and unpublished findings revealing important molecular details of both processes. 78-Subg The Cryo-EM Method Micro-ED Structure Determination of Type II Diabetes-Related Protein Segments Pascal Krotee1, Jose A. Rodriguez1, Michael R. Sawaya1, Duilio Cascio1, Francis E. Reyes2, Dan Shi2, Johan Hattne2, Brent L. Nannenga2, Marie E. Oskarsson3, Lin Jiang4, Gunilla T. Westermark3, Tamir Gonen2, David S. Eisenberg1. 1 HHMI, UCLA-DOE Institute, Depts. of Biol Chem and Biochem, Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA, 2Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA, 3Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden, 4Department of Neurology, Mol. Bio. Institute, Brain Res. Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA. The cryoEM method micro-electron diffraction (MicroED) utilizes 3D crystals only a few hundred nanometers thick for atomic structure determination, thus shrinking the crystal size limits set by synchrotron micro-focus beamlines. MicroED is ideal for 3D nanocrystals, such as those formed from amyloid protein segments. Amyloid proteins form fibers that are the hallmarks of a class of more than 25 amyloid diseases, ranging from Parkinson’s disease to Type II Diabetes (T2D). Although full-length amyloid proteins have so far been resistant to crystallization, select protein segments that form the spines of amyloid fibrils do form such tiny 3D crystals. Gaining knowledge of the atomic structures of these protein segments is critical to our understanding of amyloid fibril structures that contribute to disease onset and progression. Here, we use MicroED to determine the structures of two 11-residue segments from Islet ˚ and 2.0 A ˚ resolution, respectively. Amyloid Polypeptide (IAPP) at 1.4 A Amyloid fibrils of IAPP are found in approximately 90% of T2D patients and the formation of these fibrils is tightly linked to pancreatic b-cell death and insulin dependence. The two segments overlap in sequence by 5 residues. Their structures add to the range of polymorphism observed previously in crystal structures of shorter IAPP segments. One segment, 15-25WT, forms an unusual arrangement of single, out-of-register b-sheets. The second segment, 19-29S20G, contains the familial S20G mutation that leads to early-onset T2D. It forms pairs of b-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length IAPP fibrils. Thus, this structure may serve as a model for the toxic spine of IAPP aggregates. These findings, facilitated by MicroED, give us insight into disease-relevant structures that can be utilized for structure-based design of therapeutics. 79-Subg Ligand-Dependent Structural States of a KDChannel Analyzed by Cryo-EM Roderick MacKinnon. Rockefeller University, New York, NY, USA. The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. I will describe the equilibrium ensemble of structures of Slo2.2, a neuronal Naþ-activated Kþ channel, as a function of the Naþ concentration. Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Naþ concentration. An open conformation, which correlates to functional activation, emerges from the closed ensemble in a highly Naþ-dependent manner without evidence of Naþ-dependent intermediates. Thus, channel opening is very concerted almost switch-like - analogous to a phase transition.