@article {64782, title = {Cryo-EM structure of the homohexameric T3SS ATPase-central stalk complex reveals rotary ATPase-like asymmetry.}, journal = {Nature Communications}, volume = {10}, year = {2019}, month = {2019 Feb 07}, pages = {626}, abstract = {

Many Gram-negative bacteria, including causative agents of dysentery, plague, and typhoid fever, rely on a type III secretion system - a multi-membrane spanning syringe-like apparatus - for their pathogenicity. The cytosolic ATPase complex of this injectisome is proposed to play an important role in energizing secretion events and substrate recognition. We present the 3.3 {\r A} resolution cryo-EM structure of the enteropathogenic Escherichia coli ATPase EscN in complex with its central stalk EscO. The structure shows an asymmetric pore with different functional states captured in its six catalytic sites, details directly supporting a rotary catalytic mechanism analogous to that of the heterohexameric F/V-ATPases despite its homohexameric nature. Situated at the C-terminal opening of the EscN pore is one molecule of EscO, with primary interaction mediated through an electrostatic interface. The EscN-EscO structure provides significant atomic insights into how the ATPase contributes to type III secretion, including torque generation and binding of chaperone/substrate complexes.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-08477-7}, author = {Majewski, Dorothy D and Worrall, Liam J and Hong, Chuan and Atkinson, Claire E and Vuckovic, Marija and Watanabe, Nobuhiko and Yu, Zhiheng and Strynadka, Natalie C J} } @article {65260, title = {Structure of an endosomal signaling GPCR-G protein-β-arrestin megacomplex.}, journal = {Nature Structural and Molecular Biology}, volume = {26}, year = {2019}, month = {2019 Dec 26}, pages = {1123-1131}, abstract = {

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-β-arr {\textquoteright}megaplex{\textquoteright}. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (βVR). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.

}, issn = {1545-9985}, doi = {10.1038/s41594-019-0330-y}, author = {Nguyen, Anthony H and Thomsen, Alex R B and Cahill, Thomas J and Huang, Rick and Huang, Li-Yin and Marcink, Tara and Clarke, Oliver B and Heissel, S{\o}ren and Masoudi, Ali and Ben-Hail, Danya and Samaan, Fadi and Dandey, Venkata P and Tan, Yong Zi and Hong, Chuan and Mahoney, Jacob P and Triest, Sarah and Little, John and Chen, Xin and Sunahara, Roger and Steyaert, Jan and Molina, Henrik and Yu, Zhiheng and des Georges, Amedee and Lefkowitz, Robert J} } @article {65109, title = {T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly.}, journal = {Nature Microbiology}, volume = {4}, year = {2019}, month = {2019 Aug}, pages = {2010-19}, abstract = {

The bacterial injectisome is a syringe-shaped macromolecular nanomachine utilized by many pathogenic Gram-negative bacteria, including the causative agents of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. Bacterial proteins destined for self-assembly and host-cell targeting are translocated by the injectisome in a process known as type III secretion (T3S). The core structure is the ~4 MDa needle complex (NC), built on a foundation of three highly oligomerized ring-forming proteins that create a hollow scaffold spanning the bacterial inner membrane (IM) (24-mer ring-forming proteins PrgH and PrgK in the\ Salmonella entericaserovar Typhimurium\ Salmonella\ pathogenicity island 1 (SPI-1) type III secretion system (T3SS)) and outer membrane (OM) (15-mer InvG, a member of the broadly conserved secretin pore family). An internalized helical needle projects from the NC and bacterium, ultimately forming a continuous passage to the host, for delivery of virulence effectors. Here, we have captured snapshots of the entire prototypical SPI-1 NC in four distinct needle assembly states, including near-atomic resolution, and local reconstructions in the absence and presence of the needle. These structures reveal the precise localization and molecular interactions of the internalized SpaPQR {\textquoteleft}export apparatus{\textquoteright} complex, which is intimately encapsulated and stabilized within the IM rings in the manner of a nanodisc, and to which the PrgJ rod directly binds and functions as an initiator and anchor of needle polymerization. We also describe the molecular details of the extensive and continuous coupling interface between the OM secretin and IM rings, which is remarkably facilitated by a localized 16-mer stoichiometry in the periplasmic-most coupling domain of the otherwise 15-mer InvG oligomer.

}, doi = {10.1038/s41564-019-0545-z}, url = {http://www.nature.com/articles/s41564-019-0545-zhttp://www.nature.com/articles/s41564-019-0545-z.pdfhttp://www.nature.com/articles/s41564-019-0545-z.pdfhttp://www.nature.com/articles/s41564-019-0545-z}, author = {Hu, Jinhong and Worrall, Liam J. and Vuckovic, Marija and Hong, Chuan and Deng, Wanyin and Atkinson, Claire E. and Brett Finlay, B. and Yu, Zhiheng and Strynadka, Natalie C. J.} } @article {49123, title = {Architecture of the human GATOR1 and GATOR1-Rag GTPases complexes.}, journal = {Nature}, volume = {556}, year = {2018}, month = {2018 Mar 28}, pages = {64-9}, abstract = {

Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.

}, issn = {1476-4687}, doi = {10.1038/nature26158}, author = {Shen, Kuang and Huang, Rick K and Brignole, Edward J and Condon, Kendall J and Valenstein, Max L and Chantranupong, Lynne and Bomaliyamu, Aimaiti and Choe, Abigail and Hong, Chuan and Yu, Zhiheng and Sabatini, David M} } @article {49315, title = {Cryo-EM structure of an essential Plasmodium vivax invasion complex.}, journal = {Nature}, volume = {559}, year = {2018}, month = {2018 Jul}, pages = {135-139}, abstract = {

Plasmodium vivax is the most widely distributed malaria parasite that infects humans. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1). TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 {\r A} resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

}, issn = {1476-4687}, doi = {10.1038/s41586-018-0249-1}, author = {Gruszczyk, Jakub and Huang, Rick K and Chan, Li-Jin and Menant, S{\'e}bastien and Hong, Chuan and Murphy, James M and Mok, Yee-Foong and Griffin, Michael D W and Pearson, Richard D and Wong, Wilson and Cowman, Alan F and Yu, Zhiheng and Tham, Wai-Hong} } @article {49574, title = {Structure of Plasmodium falciparum Rh5-CyRPA-Ripr invasion complex.}, journal = {Nature}, volume = {565}, year = {2018}, month = {2018 Dec 12}, pages = {118-21}, abstract = {

Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5-CyRPA-Ripr complex with the erythrocyte receptor basigin, which is an essential step for entry into human erythrocytes. Here we show that the Rh5-CyRPA-Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5-CyRPA-Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4-6 of the β-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5-CyRPA and CyRPA-Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5-basigin with the cryo-electron microscopy structure of Rh5-CyRPA-Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.

}, issn = {1476-4687}, doi = {10.1038/s41586-018-0779-6}, author = {Wong, Wilson and Huang, Rick and Menant, S{\'e}bastien and Hong, Chuan and Sandow, Jarrod J and Birkinshaw, Richard W and Healer, Julie and Hodder, Anthony N and Kanjee, Usheer and Tonkin, Christopher J and Heckmann, Denise and Soroka, Vladislav and S{\o}gaard, Teit Max Moscote and J{\o}rgensen, Thomas and Duraisingh, Manoj T and Czabotar, Peter E and de Jongh, Willem A and Tham, Wai-Hong and Webb, Andrew I and Yu, Zhiheng and Cowman, Alan F} } @article {48713, title = {Near-atomic resolution cryoelectron microscopy structure of the 30-fold homooligomeric SpoIIIAG channel essential to spore formation in Bacillus subtilis.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, year = {2017}, month = {2017 Aug 07}, abstract = {

Bacterial sporulation allows starving cells to differentiate into metabolically dormant spores that can survive extreme conditions. Following asymmetric division, the mother cell engulfs the forespore, surrounding it with two bilayer membranes. During the engulfment process, an essential channel, the so-called feeding tube apparatus, is thought to cross both membranes to create a direct conduit between the mother cell and the forespore. At least nine proteins are required to create this channel, including SpoIIQ and SpoIIIAA-AH. Here, we present the near-atomic resolution structure of one of these proteins, SpoIIIAG, determined by single-particle cryo-EM. A 3D reconstruction revealed that SpoIIIAG assembles into a large and stable 30-fold symmetric complex with a unique mushroom-like architecture. The complex is collectively composed of three distinctive circular structures: a 60-stranded vertical β-barrel that forms a large inner channel encircled by two concentric rings, one β-mediated and the other formed by repeats of a ring-building motif (RBM) common to the architecture of various dual membrane secretion systems of distinct function. Our near-atomic resolution structure clearly shows that SpoIIIAG exhibits a unique and dramatic adaptation of the RBM fold with a unique β-triangle insertion that assembles into the prominent channel, the dimensions of which suggest the potential passage of large macromolecules between the mother cell and forespore during the feeding process. Indeed, mutation of residues located at key interfaces between monomers of this RBM resulted in severe defects both in vivo and in vitro, providing additional support for this unprecedented structure.

}, issn = {1091-6490}, doi = {10.1073/pnas.1704310114}, author = {Zeytuni, Natalie and Hong, Chuan and Flanagan, Kelly A and Worrall, Liam J and Theiltges, Kate A and Vuckovic, Marija and Huang, Rick K and Massoni, Shawn C and Camp, Amy H and Yu, Zhiheng and Strynadka, Natalie C} } @article {48945, title = {Structural basis of bacterial transcription activation.}, journal = {Science (New York, N.Y.)}, volume = {358}, year = {2017}, month = {2017 Nov 17}, pages = {947-951}, abstract = {

In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3{\textquoteright},5{\textquoteright}-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ(70)-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.

}, issn = {1095-9203}, doi = {10.1126/science.aao1923}, author = {Liu, Bin and Hong, Chuan and Huang, Rick K and Yu, Zhiheng and Steitz, Thomas A} } @article {48317, title = {A near atomic structure of the active human apoptosome.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 Oct 04}, pages = {e17755}, abstract = {

In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy. The resulting model gives insights into cytochrome c binding, nucleotide exchange and conformational changes that drive assembly. During activation an acentric disk is formed on the central hub of the apoptosome. This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy. On average, Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub, when an odd number of zymogens are bound. This suggests a stoichiometry of one or at most, two pc-9 dimers per active apoptosome. Thus, our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease.

}, issn = {2050-084X}, doi = {10.7554/eLife.17755}, author = {Cheng, Tat Cheung and Hong, Chuan and Akey, Ildik{\'o} V and Yuan, Shujun and Akey, Christopher W} }