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26 Janelia Publications

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    05/16/22 | In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes
    Zhen Chen , Garrett A. Greenan , Momoko Shiozaki , Yanxin Liu , Will M. Skinner , Xiaowei Zhao , Shumei Zhao , Rui Yan , Caiying Guo , Zhiheng Yu , Polina V. Lishko , David A. Agard , Ronald D. Vale
    bioRxiv. 2022 May 16:. doi: 10.1101/2022.05.15.492011

    The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm axonemes, providing the highest resolution structural information to date. Our subtomogram averages reveal mammalian sperm- specific protein complexes within the outer microtubule doublets, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm- specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

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    04/15/22 | Allosteric interactions prime androgen receptor dimerization and activation.
    Wasmuth EV, Broeck AV, LaClair JR, Hoover EA, Lawrence KE, Paknejad N, Pappas K, Matthies D, Wang B, Feng W, Watson PA, Zinder JC, Karthaus WR, de la Cruz MJ, Hite RK, Manova-Todorova K, Yu Z, Weintraub ST, Klinge S, Sawyers CL
    Molecular Cell. 2022 Apr 15:. doi: 10.1016/j.molcel.2022.03.035

    The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.

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    02/21/22 | Allosteric interactions prime androgen receptor dimerization and activation.
    Elizabeth V. Wasmuth , Arnaud Vanden Broeck , Justin R. LaClair , Elizabeth A. Hoover , Kayla E. Lawrence , Navid Paknejad , Kyrie Pappas , Doreen Matthies , Biran Wang , Weiran Feng , Philip A. Watson , John C. Zinder , Wouter R. Karthaus , M. Jason de la Cruz , Richard K. Hite , Katia Manova-Todorova , Zhiheng Yu , Susan T. Weintraub , Sebastian Klinge , Charles L. Sawyers
    bioRxiv. 2022 Feb 21:. doi: 10.1101/2022.02.20.481229

    The androgen receptor (AR) is a steroid receptor and master transcription factor that governs gene expression programs required for luminal development of prostate epithelium, formation of muscle tissue and maintenance of the male phenotype. AR misregulation is a hallmark of multiple malignancies, including prostate cancer, where AR hyperactivation and expansion of its transcriptome occur in part through AR gene amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR’s individual domains and its protein cofactors cooperate to bind DNA have remained elusive. Using a combination of reconstitution biochemistry and single particle cryo-electron microscopy (EM), we have isolated three conformational states of AR bound to DNA. We observe that AR forms a non-obligate dimer, with the buried dimer interface utilized by related ancestral nuclear receptors repurposed to facilitate cooperative DNA binding. We identify surfaces bridging AR’s domains responsible for allosteric communication, that are compromised in partial androgen insensitivity syndrome (PAIS), and are reinforced by AR’s oncoprotein cofactor, ERG, and DNA binding site motifs. Finally, we present evidence that this plastic dimer interface for transcriptional activation may have been adopted by AR at the expense of DNA binding. Our work highlights how fine-tuning of AR’s cooperative interactions translate to consequences in development and disease.

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    12/21/21 | Structure and RNA template requirements of RNA-DEPENDENT RNA POLYMERASE 2.
    Fukudome A, Singh J, Mishra V, Reddem E, Martinez-Marquez F, Wenzel S, Yan R, Shiozaki M, Yu Z, Wang JC, Takagi Y, Pikaard CS
    Proceedings of the National Academy of Sciences of the U.S.A.. 2021 Dec 21;118(51):. doi: 10.1073/pnas.2115899118

    RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In , RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

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    09/30/21 | The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson’s disease
    Aurelie de Rus Jacquet , Jenna L. Tancredi , Andrew L. Lemire , Michael C. DeSantis , Wei-Ping Li , Erin K. O’Shea
    eLife. 2021 Sep 30:. doi:

    Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinson’s disease (PD), dopaminergic neurons are progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remains largely unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes generated from patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes, and we identify the abnormal accumulation of key PD-related proteins within multi vesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs and that LRRK2 G2019S EVs are abnormally enriched in neurites and fail to provide full neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties may participate in the progression of PD.

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    05/13/21 | CryoEM structure of the antibacterial target PBP1b at 3.3 Å resolution.
    Caveney NA, Workman SD, Yan R, Atkinson CE, Yu Z, Strynadka NC
    Nature Communications. 2021 May 13;12(1):2775. doi: 10.1038/s41467-021-23063-6

    The pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of β-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.

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    12/02/20 | Cryo-EM structure of the inhibited (10S) form of myosin II.
    Yang S, Tiwari P, Lee KH, Sato O, Ikebe M, Padrón R, Craig R
    Nature. 2020 Dec 02;588(7838):521-25. doi: 10.1038/s41586-020-3007-0

    Myosin II is the motor protein that enables muscle cells to contract and nonmuscle cells to move and change shape. The molecule has two identical heads attached to an elongated tail, and can exist in two conformations: 10S and 6S, named for their sedimentation coefficients. The 6S conformation has an extended tail and assembles into polymeric filaments, which pull on actin filaments to generate force and motion. In 10S myosin, the tail is folded into three segments and the heads bend back and interact with each other and the tail, creating a compact conformation in which ATPase activity, actin activation and filament assembly are all highly inhibited. This switched-off structure appears to function as a key energy-conserving storage molecule in muscle and nonmuscle cells, which can be activated to form functional filaments as needed-but the mechanism of its inhibition is not understood. Here we have solved the structure of smooth muscle 10S myosin by cryo-electron microscopy with sufficient resolution to enable improved understanding of the function of the head and tail regions of the molecule and of the key intramolecular contacts that cause inhibition. Our results suggest an atomic model for the off state of myosin II, for its activation and unfolding by phosphorylation, and for understanding the clustering of disease-causing mutations near sites of intramolecular interaction.

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    09/30/20 | Structural insight into the ATP-driven exporter of virulent peptide toxins.
    Zeytuni N, Dickey SW, Hu J, Chou HT, Worrall LJ, Alexander JA, Carlson ML, Nosella M, Duong F, Yu Z, Otto M, Strynadka NC
    Science Advances. 2020 Sep 30;6(40):. doi: 10.1126/sciadv.abb8219

    is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an "open" conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

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    11/27/19 | Cryo-EM structure of the human FLCN-FNIP2-Rag-Ragulator complex.
    Shen K, Rogala KB, Chou H, Huang RK, Yu Z, Sabatini DM
    Cell. 2019 Nov 27;179(6):1319-29. doi: 10.1016/j.cell.2019.10.036
    08/01/19 | T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly.
    Hu J, Worrall LJ, Vuckovic M, Hong C, Deng W, Atkinson CE, Brett Finlay B, Yu Z, Strynadka NC
    Nature Microbiology. 2019 Aug;4(11):2010-19. doi: 10.1038/s41564-019-0545-z

    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 ‘export apparatus’ 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.

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