scholarly journals Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

2021 ◽  
Author(s):  
Koichiro E. Kishi ◽  
Yoon Seok Kim ◽  
Masahiro Fukuda ◽  
Tsukasa Kusakizako ◽  
Elina Thadhani ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transmembrane Helix ◽  
Action Spectrum ◽  
Light Sensitivity ◽  
Structural Basis ◽  
Ion Pump ◽  
Cryo Electron Microscopy ◽  
Architectural Features ◽  
Computational Analyses ◽  
Insight Into

ChRmine, a recently-discovered bacteriorhodopsin-like cation-conducting channelrhodopsin, exhibits puzzling properties (unusually-large photocurrents, exceptional red-shift in action spectrum, and extreme light-sensitivity) that have opened up new opportunities in optogenetics. ChRmine and its homologs function as light-gated ion channels, but by primary sequence more closely resemble ion pump rhodopsins; the molecular mechanisms for passive channel conduction in this family of proteins, as well as the unusual properties of ChRmine itself, have remained mysterious. Here we present the cryo-electron microscopy structure of ChRmine at 2.0 Å resolution. The structure reveals striking architectural features never seen before in channelrhodopsins including trimeric assembly, a short transmembrane-helix 3 unwound in the middle of the membrane, a prominently-twisting extracellular-loop 1, remarkably-large intracellular cavities and extracellular vestibule, and an unprecedented hydrophilic pore that extends through the center of the trimer, separate from the three individual monomer pores. Electrophysiological, spectroscopic, and computational analyses provide insight into conduction and gating of light-gated channels with these distinct design features, and point the way toward structure-guided creation of novel channelrhodopsins for optogenetic applications in biology.

scholarly journals Pathogenic siderophore ABC importer YbtPQ adopts a surprising fold of exporter

2020 ◽  
Vol 6 (6) ◽  
pp. eaay7997 ◽  
Author(s):  
Zhiming Wang ◽  
Wenxin Hu ◽  
Hongjin Zheng
Keyword(s):  
Bound States ◽  
Molecular Mechanisms ◽  
Transmembrane Helix ◽  
Human Pathogens ◽  
Binding Domains ◽  
Type Iv ◽  
Cryo Electron Microscopy ◽  
Open Conformation ◽  
Multiple Structures ◽  
First Time

To fight for essential metal ions, human pathogens secrete virulence-associated siderophores and retake the metal-chelated siderophores through a subfamily of adenosine triphosphate (ATP)–binding cassette (ABC) importer, whose molecular mechanisms are completely unknown. We have determined multiple structures of the yersiniabactin importer YbtPQ from uropathogenic Escherichia coli (UPEC) at inward-open conformation in both apo and substrate-bound states by cryo–electron microscopy. YbtPQ does not adopt any known fold of ABC importers but surprisingly adopts the fold of type IV ABC exporters. To our knowledge, it is the first time an exporter fold of ABC importer has been reported. We have also observed two unique features in YbtPQ: unwinding of a transmembrane helix in YbtP upon substrate release and tightly associated nucleotide-binding domains without bound nucleotides. Together, our study suggests that siderophore ABC importers have a distinct transport mechanism and should be classified as a separate subfamily of ABC importers.

scholarly journals The cryo-EM structure of a 12-subunit variant of RNA polymerase I reveals dissociation of the A49-A34.5 heterodimer and rearrangement of subunit A12.2

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Lucas Tafur ◽  
Yashar Sadian ◽  
Jonas Hanske ◽  
Rene Wetzel ◽  
Felix Weis ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Reversible Binding ◽  
Cryo Electron Microscopy ◽  
Nucleotide Analog ◽  
Pol I ◽  
Polymerase I ◽  
Insight Into

RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-electron microscopy (EM) structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-EM structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 Å resolution that provide additional insight into the functional interplay between the Pol I-specific transcription-like factors A49-A34.5 and A12.2. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our structural and biochemical data suggest a mechanism where reversible binding of the A49-A34.5 heterodimer could contribute to the regulation of Pol I transcription initiation and elongation.

Cryo-EM structures of the human sodium-potassium pump revealing the gating mechanism on the cytoplasmic side

2021 ◽  
Author(s):  
Yingying Guo ◽  
Yuanyuan Zhang ◽  
Renhong Yan ◽  
Bangdong Huang ◽  
Fangfei Ye ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Structural Basis ◽  
Extracellular Potassium ◽  
Ion Pump ◽  
Sodium Potassium ◽  
Working Cycle ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Membrane Bound ◽  
Cytoplasmic Side

Abstract Na+/K+-ATPase (NKA) is a membrane-bound ion pump that generates electrochemical gradient of sodium ion and potassium ion across the plasma membrane via hydrolyzing ATP. During each so-called Post-Albers cycle, NKA exchanges three cytoplasmic sodium ions for two extracellular potassium ions through alternating E1 and E2 states. Hitherto, there are several steps remained unknown during the complete working cycle of NKA. Here, we report cryo-electron microscopy (cryo-EM) structures of recombinant over-expressed human NKA in three distinct states at 3.1–3.4 Å resolution, representing the E1·3Na state, in which the cytosolic gate is open, and the E1·3Na·ATP state preceding ATP hydrolysis and a basic E2·[2K] state. These structures reveal the ATP-dependent Na+-binding site remodeling for the close of the cytoplasmic gate, filling a gap in the structural elucidation of the Post-Albers cycle of NKA and providing structural basis for understanding the cytoplasmic Na+ entrance pathway.

scholarly journals Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

2020 ◽  
Author(s):  
Gong Qin ◽  
Kim Robinson ◽  
Xu Chenrui ◽  
Zhang Jiawen ◽  
Boo Zhao Zhi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Inner Core ◽  
Inflammatory Diseases ◽  
Structural Features ◽  
Structural Basis ◽  
Card Domain ◽  
Domain Interactions ◽  
Cultured Human Cells ◽  
Insight Into

AbstractNod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related NLR proteins, NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, the molecular mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to trigger the assembly of distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core composed of oligomerized CARD domains and the outer layer consisting of FIINDUPA rings. Biochemically, oligomerized NLRP1-CARD is sufficient to drive ASC speck formation in cultured human cells via filament formation-a process that is greatly enhanced by NLRP1-FIINDUPA, which forms ring-like oligomers in vitro. In addition, we report the cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments at 3.7 Å, which uncovers unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide unique structural insight into the mechanisms of activation for human NLRP1 and CARD8, uncovering an unexpected level of specificity in inflammasome signaling mediated by heterotypic CARD domain interactions.

scholarly journals Cryo-EM structures of undocked innexin-6 hemichannels in phospholipids

2020 ◽  
Vol 6 (7) ◽  
pp. eaax3157 ◽  
Author(s):  
Batuujin Burendei ◽  
Ruriko Shinozaki ◽  
Masakatsu Watanabe ◽  
Tohru Terada ◽  
Kazutoshi Tani ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Structural Basis ◽  
Wild Type ◽  
Amino Terminal ◽  
Functional Assays ◽  
Cryo Electron Microscopy ◽  
Dynamics Simulations ◽  
Open States ◽  
Insight Into ◽  
Junction Proteins

Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo–electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.

scholarly journals Cryo-EM structures of NPC1L1 reveal mechanisms of cholesterol transport and ezetimibe inhibition

2020 ◽  
Vol 6 (25) ◽  
pp. eabb1989 ◽  
Author(s):  
Ching-Shin Huang ◽  
Xinchao Yu ◽  
Preston Fordstrom ◽  
Kaylee Choi ◽  
Ben C. Chung ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Molecular Target ◽  
The Other ◽  
Cholesterol Transport ◽  
Central Cavity ◽  
Cryo Electron Microscopy ◽  
Lowering Therapy ◽  
Therapeutic Development ◽  
Niemann Pick ◽  
Insight Into

The intestinal absorption of cholesterol is mediated by a multipass membrane protein, Niemann-Pick C1-Like 1 (NPC1L1), the molecular target of a cholesterol lowering therapy ezetimibe. While ezetimibe gained Food and Drug Administration approval in 2002, its mechanism of action has remained unclear. Here, we present two cryo–electron microscopy structures of NPC1L1, one in its apo form and the other complexed with ezetimibe. The apo form represents an open state in which the N-terminal domain (NTD) interacts loosely with the rest of NPC1L1, leaving the NTD central cavity accessible for cholesterol loading. The ezetimibe-bound form signifies a closed state in which the NTD rotates ~60°, creating a continuous tunnel enabling cholesterol movement into the plasma membrane. Ezetimibe blocks cholesterol transport by occluding the tunnel instead of competing with cholesterol binding. These findings provide insight into the molecular mechanisms of NPC1L1-mediated cholesterol transport and ezetimibe inhibition, paving the way for more effective therapeutic development.

scholarly journals Structures of dimeric human NPC1L1 provide insight into mechanisms for cholesterol absorption

2021 ◽  
Vol 7 (34) ◽  
pp. eabh3997
Author(s):  
Tao Long ◽  
Yang Liu ◽  
Yu Qin ◽  
Russell A. DeBose-Boyd ◽  
Xiaochun Li
Keyword(s):  
Low Density Lipoprotein ◽  
Transmembrane Helix ◽  
Dietary Cholesterol ◽  
Density Lipoprotein ◽  
Cholesterol Absorption ◽  
Oligomeric State ◽  
Cryo Electron Microscopy ◽  
Niemann Pick ◽  
Circulating Levels ◽  
Insight Into

Polytopic Niemann-Pick C1-like 1 (NPC1L1) plays a major role in intestinal absorption of biliary cholesterol, vitamin E (VE), and vitamin K (VK). The drug ezetimibe inhibits NPC1L1-mediated absorption of cholesterol, lowering of circulating levels of low-density lipoprotein cholesterol. Here, we report cryo–electron microscopy structures of human NPC1L1 (hNPC1L1) bound to either cholesterol or a lipid resembling VE. These findings, together with functional assays, reveal that the same intramolecular channel in hNPC1L1 mediates transport of VE and cholesterol. hNPC1L1 exists primarily as a homodimer; dimerization is mediated by aromatic residues within a region of transmembrane helix 2 that exhibits a horizonal orientation in the membrane. Mutation of tryptophan-347 lies in this region disrupts dimerization and the resultant monomeric NPC1L1 exhibits reduced efficiency of cholesterol uptake. These findings identify the oligomeric state of hNPC1L1 as a target for therapies that inhibit uptake of dietary cholesterol and reduce the incidence of cardiovascular disease.

scholarly journals The cryo-electron microscopy structure of the human CDK-activating kinase

2020 ◽  
Author(s):  
Basil J. Greber ◽  
Juan M. Perez-Bertoldi ◽  
Kif Lim ◽  
Anthony T. Iavarone ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transcription Initiation ◽  
Control Cell ◽  
Regulatory Elements ◽  
Structural Basis ◽  
Control Of Gene Expression ◽  
Cryo Electron Microscopy ◽  
Insight Into ◽  
Cdk Activating Kinase

AbstractThe human CDK-activating kinase (CAK), a complex composed of cyclin dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II subunit Rpb1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell-cycle progression. Here, we have determined the three-dimensional structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and aid in the rational design of therapeutic compounds.SignificanceControl of gene expression and the cell cycle is critical for appropriate cell growth and timely cell division. Failure of the mechanisms regulating these processes can result in proliferative diseases. A molecular complex termed the CDK activating kinase (CAK) impinges on both of these regulatory networks in human cells and is thus a possible drug target for treatment of cancer. Here, we use cryo-electron microscopy to describe the detailed molecular structure of the human CAK, revealing its architecture and the interactions between its regulatory elements. Additionally, we have obtained the structure of the CAK in complex with a small-molecule inhibitor.

scholarly journals Structural basis for the therapeutic advantage of dual and triple agonists at the human GIP, GLP-1 or GCG receptors

2021 ◽  
Author(s):  
Fenghui Zhao ◽  
Qingtong Zhou ◽  
Zhaotong Cong ◽  
Kaini Hang ◽  
Xinyu Zou ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Atomic Level ◽  
Structural Basis ◽  
Therapeutic Advantage ◽  
Cryo Electron Microscopy ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Diabetes And Obesity ◽  
Functional Versatility

Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dualagonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their superior efficacies over monoagonist such as semaglutide, we determined cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility and therapeutic supremacy of tirzepatide and peptide 20.

Structural insights into immunoglobulin M

Science ◽  
2020 ◽  
Vol 367 (6481) ◽  
pp. 1014-1017 ◽  
Author(s):  
Yaxin Li ◽  
Guopeng Wang ◽  
Ningning Li ◽  
Yuxin Wang ◽  
Qinyu Zhu ◽  
...  
Keyword(s):  
Mucosal Immunity ◽  
Molecular Mechanisms ◽  
Immunoglobulin M ◽  
Structural Basis ◽  
Polymeric Immunoglobulin Receptor ◽  
Immunoglobulin Receptor ◽  
J Chain ◽  
Cryo Electron Microscopy ◽  
Large Conformational Change ◽  
Structural Insights

Immunoglobulin M (IgM) plays a pivotal role in both humoral and mucosal immunity. Its assembly and transport depend on the joining chain (J-chain) and the polymeric immunoglobulin receptor (pIgR), but the underlying molecular mechanisms of these processes are unclear. We report a cryo–electron microscopy structure of the Fc region of human IgM in complex with the J-chain and pIgR ectodomain. The IgM-Fc pentamer is formed asymmetrically, resembling a hexagon with a missing triangle. The tailpieces of IgM-Fc pack into an amyloid-like structure to stabilize the pentamer. The J-chain caps the tailpiece assembly and bridges the interaction between IgM-Fc and the polymeric immunoglobulin receptor, which undergoes a large conformational change to engage the IgM-J complex. These results provide a structural basis for the function of IgM.

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