Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine

AbstractThe hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.

Download Full-text

Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

Nature Communications ◽

10.1038/s41467-021-23702-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hauke S. Hillen ◽

Elena Lavdovskaia ◽

Franziska Nadler ◽

Elisa Hanitsch ◽

Andreas Linden ◽

...

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Structural Basis ◽

Peptidyl Transferase ◽

Mitochondrial Ribosomes ◽

Mitochondrial Ribosome ◽

In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

Download Full-text

Structural basis for DEAH-helicase activation by G-patch proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913880117 ◽

2020 ◽

Vol 117 (13) ◽

pp. 7159-7170 ◽

Cited By ~ 2

Author(s):

Michael K. Studer ◽

Lazar Ivanović ◽

Marco E. Weber ◽

Sabrina Marti ◽

Stefanie Jonas

Keyword(s):

Conformational Changes ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Atp Hydrolysis ◽

Protein Complexes ◽

Adenosine Diphosphate ◽

Structural Basis ◽

Rna Helicases ◽

Catalytic Core ◽

Terminal Domains

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA–protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch–mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

Download Full-text

Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05702-11 ◽

2011 ◽

Vol 56 (2) ◽

pp. 603-612 ◽

Cited By ~ 192

Author(s):

Katherine S. Long ◽

Birte Vester

Keyword(s):

Binding Site ◽

Ribosomal Proteins ◽

Resistance Mechanism ◽

Ribosomal Subunit ◽

Resistance Mechanisms ◽

Drug Binding ◽

23S Rrna ◽

Detailed Knowledge ◽

Linezolid Resistance ◽

Almost All

ABSTRACTLinezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms.

Download Full-text

Structural Basis of ATP Hydrolysis and Intersubunit Signaling in the AAA+ ATPase p97

Structure ◽

10.1016/j.str.2015.10.026 ◽

2016 ◽

Vol 24 (1) ◽

pp. 127-139 ◽

Cited By ~ 35

Author(s):

Petra Hänzelmann ◽

Hermann Schindelin

Keyword(s):

Atp Hydrolysis ◽

Structural Basis ◽

Aaa Atpase

Download Full-text

The CryoEM Structure of the Ribosome Maturation Factor Rea1

10.1101/455634 ◽

2018 ◽

Author(s):

Piotr Sosnowski ◽

Linas Urnavicius ◽

Andreas Boland ◽

Robert Fagiewicz ◽

Johan Busselez ◽

...

Keyword(s):

Amino Acid ◽

Atpase Activity ◽

Conformational Changes ◽

Ribosomal Subunit ◽

Force Generation ◽

Docking Site ◽

Aaa Atpase ◽

Helical Bundle ◽

Maturation Factor ◽

Assembly Factors

AbstractThe biogenesis of the 60S ribosomal subunit is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualize the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide induced conformational changes that create a docking site for the substrate binding MIDAS domain of Rea1 on the AAA+ ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

Download Full-text

Structural basis for dynamic regulation of the human 26S proteasome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614614113 ◽

2016 ◽

Vol 113 (46) ◽

pp. 12991-12996 ◽

Cited By ~ 96

Author(s):

Shuobing Chen ◽

Jiayi Wu ◽

Ying Lu ◽

Yong-Bei Ma ◽

Byung-Hoon Lee ◽

...

Keyword(s):

Conformational Changes ◽

Ground State ◽

Atp Hydrolysis ◽

26S Proteasome ◽

Structural Basis ◽

Core Particle ◽

Dynamic Regulation ◽

Aaa Atpase ◽

The Core ◽

Aaa Atpases

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

Download Full-text

Binding Site of Macrolide Antibiotics on the Ribosome: New Resistance Mutation Identifies a Specific Interaction of Ketolides with rRNA

Journal of Bacteriology ◽

10.1128/jb.183.23.6898-6907.2001 ◽

2001 ◽

Vol 183 (23) ◽

pp. 6898-6907 ◽

Cited By ~ 85

Author(s):

Georgina Garza-Ramos ◽

Liqun Xiong ◽

Ping Zhong ◽

Alexander Mankin

Keyword(s):

Binding Site ◽

Specific Interaction ◽

Resistance Mutation ◽

Ribosomal Subunit ◽

Drug Binding ◽

Structural Basis ◽

23S Rrna ◽

Macrolide Antibiotics ◽

Crystallographic Structure ◽

Large Ribosomal Subunit

ABSTRACT Macrolides represent a clinically important class of antibiotics that block protein synthesis by interacting with the large ribosomal subunit. The macrolide binding site is composed primarily of rRNA. However, the mode of interaction of macrolides with rRNA and the exact location of the drug binding site have yet to be described. A new class of macrolide antibiotics, known as ketolides, show improved activity against organisms that have developed resistance to previously used macrolides. The biochemical reasons for increased potency of ketolides remain unknown. Here we describe the first mutation that confers resistance to ketolide antibiotics while leaving cells sensitive to other types of macrolides. A transition of U to C at position 2609 of 23S rRNA rendered E. coli cells resistant to two different types of ketolides, telithromycin and ABT-773, but increased slightly the sensitivity to erythromycin, azithromycin, and a cladinose-containing derivative of telithromycin. Ribosomes isolated from the mutant cells had reduced affinity for ketolides, while their affinity for erythromycin was not diminished. Possible direct interaction of ketolides with position 2609 in 23S rRNA was further confirmed by RNA footprinting. The newly isolated ketolide-resistance mutation, as well as 23S rRNA positions shown previously to be involved in interaction with macrolide antibiotics, have been modeled in the crystallographic structure of the large ribosomal subunit. The location of the macrolide binding site in the nascent peptide exit tunnel at some distance from the peptidyl transferase center agrees with the proposed model of macrolide inhibitory action and explains the dominant nature of macrolide resistance mutations. Spatial separation of the rRNA residues involved in universal contacts with macrolides from those believed to participate in structure-specific interactions with ketolides provides the structural basis for the improved activity of the broader spectrum group of macrolide antibiotics.

Download Full-text

Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics

10.1101/2021.06.18.448924 ◽

2021 ◽

Author(s):

Caillan Crowe-McAuliffe ◽

Victoriia Murina ◽

Marje Kasari ◽

Hiraku Takada ◽

Kathryn Jane Turnbull ◽

...

Keyword(s):

Binding Sites ◽

Ribosomal Subunit ◽

Drug Binding ◽

Structural Basis ◽

Translation Elongation ◽

Drug Binding Site ◽

Nascent Chain ◽

70S Ribosome ◽

Context Specific ◽

Peptidyltransferase Center

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF) that confer resistance to oxazolidinone, such as linezolid, and phenicol antibiotics, such as chloramphenicol. PoxtA/OptrA are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria. These target protection proteins are thought to confer resistance by binding to the ribosome and dislodging the antibiotics from their binding sites. However, a structural basis for their mechanism of action has been lacking. Here we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9-3.1A, as well as the complete E. faecalis 70S ribosome at 2.2-2.5 A. The structures reveal that PoxtA binds within the ribosomal E-site with its antibiotic resistance domain (ARD) extending towards the peptidyltransferase center (PTC) on the large ribosomal subunit. At its closest point, the ARD of PoxtA is still located >15A from the linezolid and chloramphenicol binding sites, suggesting that drug release is elicited indirectly. Instead, we observe that the ARD of PoxtA perturbs the CCA-end of the P-site tRNA causing it to shift by ~4A out of the PTC, which correlates with a register shift of one amino acid for the attached nascent polypeptide chain. Given that linezolid and chloramphenicol are context-specific translation elongation inhibitors, we postulate that PoxtA/OptrA confer resistance to oxazolidinones and phenicols indirectly by perturbing the P-site tRNA and thereby altering the conformation of the attached nascent chain to disrupt the drug binding site.

Download Full-text

Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation

The Journal of Cell Biology ◽

10.1083/jcb.201205021 ◽

2012 ◽

Vol 199 (5) ◽

pp. 771-782 ◽

Cited By ~ 40

Author(s):

Lisa Kappel ◽

Mathias Loibl ◽

Gertrude Zisser ◽

Isabella Klein ◽

Gernot Fruhmann ◽

...

Keyword(s):

Atpase Activity ◽

Ribosome Biogenesis ◽

Atp Hydrolysis ◽

Large Subunit ◽

Exact Role ◽

Aaa Atpase ◽

Energy Consuming

Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.

Download Full-text

Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone

Nature Communications ◽

10.1038/s41467-019-13743-9 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Carol Cho ◽

Juwon Jang ◽

Yujin Kang ◽

Hiroki Watanabe ◽

Takayuki Uchihashi ◽

...

Keyword(s):

High Speed ◽

Atp Hydrolysis ◽

Structural Features ◽

Structural Basis ◽

Dependent Manner ◽

Histone Chaperones ◽

Nucleosome Assembly ◽

Chromatin Dynamics ◽

Aaa Atpase

AbstractThe fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3–H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3–H4 loading by utilizing ATP.

Download Full-text