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The Drug Diazaborine Blocks Ribosome Biogenesis by Inhibiting the AAA-ATPase Drg1*

Open AccessPublished:December 26, 2013DOI:https://doi.org/10.1074/jbc.M113.536110
      The drug diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of diazaborine. Inhibitor binding into the second AAA domain of Drg1 requires ATP loading and results in inhibition of ATP hydrolysis in this site. As a consequence the physiological activity of Drg1, i.e. the release of Rlp24 from pre-60S particles, is blocked, and further progression of cytoplasmic preribosome maturation is prevented. Our results identify the first target of an inhibitor of ribosome biogenesis and provide the mechanism of inhibition of a key step in large ribosomal subunit formation.

      Introduction

      Ribosomes translate the genetic information into the amino acid sequence of proteins and are composed of one large and one small subunit containing ribosomal RNAs (rRNA) and ribosomal proteins. In eukaryotic cells, the formation of ribosomes involves >200 trans-acting factors (nonribosomal proteins), most of which are essential (for a recent introduction to ribosome biogenesis see Ref.
      • Kressler D.
      • Hurt E.
      • Bassler J.
      Driving ribosome assembly.
      ). Ribosome biogenesis starts with the transcription of precursors of ribosomal RNA (pre-rRNA) in the nucleolus. Joining of ribosomal and nonribosomal proteins with pre-rRNA results in formation of precursor particles for the large and small subunits. A complex maturation pathway driven by transiently joining nonribosomal proteins leads to export-competent particles that are transported through the nuclear pore complex into the cytoplasm where final maturation steps take place. These include release and recycling of shuttling maturation and export factors and incorporation of late joining ribosomal proteins (for review, see Ref.
      • Panse V.G.
      • Johnson A.W.
      Maturation of eukaryotic ribosomes: acquisition of functionality.
      ). We and others demonstrated recently that the AAA
      The abbreviations used are:
      AAA
      ATPases associated with diverse cellular activities
      AMP-PNP
      adenosine 5′-(β,γ-imino)triphosphate
      DSF
      differential scanning fluorometry
      res
      diazaborine-resistant
      TAP
      tandem affinity purification
      YPD
      yeast extract peptone dextrose.
      protein Drg1 is a key factor in cytoplasmic pre-60S maturation in yeast (
      • Kappel L.
      • Loibl M.
      • Zisser G.
      • Klein I.
      • Fruhmann G.
      • Gruber C.
      • Unterweger S.
      • Rechberger G.
      • Pertschy B.
      • Bergler H.
      Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation.
      ,
      • Lo K.Y.
      • Li Z.
      • Bussiere C.
      • Bresson S.
      • Marcotte E.M.
      • Johnson A.W.
      Defining the pathway of cytoplasmic maturation of the 60S ribosomal subunit.
      ,
      • Pertschy B.
      • Saveanu C.
      • Zisser G.
      • Lebreton A.
      • Tengg M.
      • Jacquier A.
      • Liebminger E.
      • Nobis B.
      • Kappel L.
      • van der Klei I.
      • Högenauer G.
      • Fromont-Racine M.
      • Bergler H.
      Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1.
      ,
      • Bassler J.
      • Klein I.
      • Schmidt C.
      • Kallas M.
      • Thomson E.
      • Wagner M.A.
      • Bradatsch B.
      • Rechberger G.
      • Strohmaier H.
      • Hurt E.
      • Bergler H.
      The conserved Bud20 zinc finger protein is a new component of the ribosomal 60S subunit export machinery.
      ). Drg1 is composed of an N domain and the two ATPase domains D1 and D2 and forms hexamers in the presence of ATP (
      • Thorsness P.E.
      • White K.H.
      • Ong W.C.
      AFG2, an essential gene in yeast, encodes a new member of the Sec18p, Pas1p, Cdc48p, TBP-1 family of putative ATPases.
      ,
      • Wendler F.
      • Bergler H.
      • Prutej K.
      • Jungwirth H.
      • Zisser G.
      • Kuchler K.
      • Högenauer G.
      Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3.
      ,
      • Zakalskiy A.
      • Högenauer G.
      • Ishikawa T.
      • Wehrschütz-Sigl E.
      • Wendler F.
      • Teis D.
      • Zisser G.
      • Steven A.C.
      • Bergler H.
      Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae: decoupling of intracellular function from ATPase activity and hexamerization.
      ). The protein is recruited to cytoplasmic pre-60S particles by direct interaction with the shuttling protein Rlp24 (
      • Kappel L.
      • Loibl M.
      • Zisser G.
      • Klein I.
      • Fruhmann G.
      • Gruber C.
      • Unterweger S.
      • Rechberger G.
      • Pertschy B.
      • Bergler H.
      Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation.
      ). The C-terminal domain of Rlp24 then stimulates ATP hydrolysis in both AAA domains of Drg1. ATP hydrolysis in D2 is essential and triggers the release of Rlp24 from pre-60S particles whereas ATP hydrolysis in D1 is required for the subsequent dissociation from Rlp24. The release of Rlp24 by Drg1 represents a critical event in pre-60S maturation and is a prerequisite for downstream maturation steps like the release of other shuttling factors and association of late joining cytoplasmic proteins (
      • Kappel L.
      • Loibl M.
      • Zisser G.
      • Klein I.
      • Fruhmann G.
      • Gruber C.
      • Unterweger S.
      • Rechberger G.
      • Pertschy B.
      • Bergler H.
      Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation.
      ).
      We showed previously that the drug diazaborine blocks ribosome biogenesis in yeast (
      • Pertschy B.
      • Zisser G.
      • Schein H.
      • Köffel R.
      • Rauch G.
      • Grillitsch K.
      • Morgenstern C.
      • Durchschlag M.
      • Högenauer G.
      • Bergler H.
      Diazaborine treatment of yeast cells inhibits maturation of the 60S ribosomal subunit.
      ). Diazaborine is a heterocyclic boron-containing compound with strong antimicrobial activity (
      • Grassberger M.A.
      • Turnowsky F.
      • Hildebrandt J.
      Preparation and antibacterial activities of new 1,2,3-diazaborine derivatives and analogues.
      ). In Gram-negative bacteria the drug blocks fatty acid biosynthesis by inhibiting the enoyl-ACP reductase FabI (
      • Bergler H.
      • Wallner P.
      • Ebeling A.
      • Leitinger B.
      • Fuchsbichler S.
      • Aschauer H.
      • Kollenz G.
      • Högenauer G.
      • Turnowsky F.
      Protein EnvM is the NADH-dependent enoyl-ACP reductase (FabI) of Escherichia coli.
      ,
      • Kater M.M.
      • Koningstein G.M.
      • Nijkamp H.J.
      • Stuitje A.R.
      The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes.
      ). In yeast, fatty acid biosynthesis is not affected by diazaborine (
      • Zakalskiy A.
      • Högenauer G.
      • Ishikawa T.
      • Wehrschütz-Sigl E.
      • Wendler F.
      • Teis D.
      • Zisser G.
      • Steven A.C.
      • Bergler H.
      Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae: decoupling of intracellular function from ATPase activity and hexamerization.
      ). Instead, the drug specifically interferes with large ribosomal subunit formation (
      • Pertschy B.
      • Zisser G.
      • Schein H.
      • Köffel R.
      • Rauch G.
      • Grillitsch K.
      • Morgenstern C.
      • Durchschlag M.
      • Högenauer G.
      • Bergler H.
      Diazaborine treatment of yeast cells inhibits maturation of the 60S ribosomal subunit.
      ). Resistance to diazaborine in yeast is mediated by certain allelic forms of DRG1 (
      • Wendler F.
      • Bergler H.
      • Prutej K.
      • Jungwirth H.
      • Zisser G.
      • Kuchler K.
      • Högenauer G.
      Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3.
      ). However, the exact relationship between Drg1 and the drug remained elusive, because the basal ATPase activity of Drg1 was not inhibited by diazaborine in vitro, and direct binding of radiolabeled inhibitor could not be demonstrated using equilibrium dialysis (
      • Zakalskiy A.
      • Högenauer G.
      • Ishikawa T.
      • Wehrschütz-Sigl E.
      • Wendler F.
      • Teis D.
      • Zisser G.
      • Steven A.C.
      • Bergler H.
      Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae: decoupling of intracellular function from ATPase activity and hexamerization.
      ).
      We show here that diazaborine binds to Drg1 and specifically blocks ATP hydrolysis in the D2 domain, thereby preventing Rlp24 release from pre-60S particles. Thus, our results identify the target and describe the mechanism of action of the first specific inhibitor of the ribosome biogenesis pathway.

      DISCUSSION

      Ribosome biogenesis in eukaryotic cells involves the coordinated activity of >200 trans-acting factors. Because most of these factors are essential, ribosome biogenesis provides a promising target for antimicrobial and anti-tumor chemotherapy. In this study, we investigated the mechanism of action of the first specific inhibitor of this pathway, the heterocyclic boron-containing compound diazaborine. Our data demonstrate that the AAA-ATPase Drg1 is the direct target of diazaborine. Drg1 is only found in eukaryotic cells and was previously shown to be a key factor of pre-60S ribosome maturation in yeast. Accordingly, treatment with the drug leads to the same phenotypic consequences as those caused by the functional inactivation of Drg1, i.e. a failure to release Rlp24 from pre-60S particles and a block in downstream maturation (Fig. 1, compare with Ref.
      • Kappel L.
      • Loibl M.
      • Zisser G.
      • Klein I.
      • Fruhmann G.
      • Gruber C.
      • Unterweger S.
      • Rechberger G.
      • Pertschy B.
      • Bergler H.
      Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation.
      ).
      What is the mechanism of inhibition of the drug? Our work shows that diazaborine specifically binds into the second AAA domain of Drg1 and thereby inhibits ATP hydrolysis in this site. However, the drug does not act as a competitive inhibitor for ATP. Instead, ATP binding to D2 is a prerequisite for diazaborine to recognize Drg1. Nucleotide binding is also required for inhibitor interaction with the enoyl-ACP reductase (FabI), which is the target of diazaborine in Gram-negative bacteria. For FabI, it was shown that the NAD+ cofactor has to bind to the enzyme to enable interaction with the inhibitor (
      • Bergler H.
      • Wallner P.
      • Ebeling A.
      • Leitinger B.
      • Fuchsbichler S.
      • Aschauer H.
      • Kollenz G.
      • Högenauer G.
      • Turnowsky F.
      Protein EnvM is the NADH-dependent enoyl-ACP reductase (FabI) of Escherichia coli.
      ,
      • Kater M.M.
      • Koningstein G.M.
      • Nijkamp H.J.
      • Stuitje A.R.
      The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes.
      ). Structural studies showed that in the active site of FabI, the boron atom of diazaborine forms a covalent bond with the 2′-OH of the nicotinamide ribose moiety of the NAD+ cofactor (
      • Baldock C.
      • Rafferty J.B.
      • Sedelnikova S.E.
      • Bithell S.
      • Stuitje A.R.
      • Slabas A.R.
      • Rice D.W.
      Crystallization of Escherichia coli enoyl reductase and its complex with diazaborine.
      ,
      • Levy C.W.
      • Baldock C.
      • Wallace A.J.
      • Sedelnikova S.
      • Viner R.C.
      • Clough J.M.
      • Stuitje A.R.
      • Slabas A.R.
      • Rice D.W.
      • Rafferty J.B.
      A study of the structure-activity relationship for diazaborine inhibition of Escherichia coli enoyl-ACP reductase.
      ). It is however currently unknown whether or not binding of diazaborine to Drg1 also involves the formation of a covalent bond between ATP and the inhibitor. Our homology model of Drg1 shows that all up to now identified amino acid exchanges causing resistance to diazaborine are located in close proximity to the nucleotide in the D2 domain of the protein (Fig. 4C). This observation supports the possibility of a close contact between ATP and diazaborine within the D2 domain of Drg1. Therefore, formation of covalent intermediates or at least tight binding between a nucleotide cofactor and the inhibitor could be a general principle for the mode of action of diazaborine. Our structural model also offers an explanation for the resistance mechanism: The mutations resulting in the resistant phenotype in the drg1-1, drg1-3, and drg1-4 alleles introduce amino acid residues with bulkier side chains than those present in the wild-type and therefore may reduce the accessibility of the binding pocket for the drug. As diazaborine is unable to bind to the Drg1-1 protein, the resistance mechanism may involve the occlusion of diazaborine from its binding site near the D2 nucleotide binding pocket of Drg1.
      Taken together, our work shows that ribosome biogenesis in general and particularly energy-consuming enzymes within this pathway may represent promising targets for the development of novel inhibitors. These enzymes include additional AAA-ATPases, like Rix7 and Rea1, but also a number of RNA helicases and GTPases (
      • Kressler D.
      • Hurt E.
      • Bassler J.
      Driving ribosome assembly.
      ). Furthermore, AAA-ATPases have emerged as interesting new targets for low molecular weight inhibitors in recent years. For example, significant efforts were undertaken to identify inhibitors for p97/VCP (
      • Bursavich M.G.
      • Parker D.P.
      • Willardsen J.A.
      • Gao Z.H.
      • Davis T.
      • Ostanin K.
      • Robinson R.
      • Peterson A.
      • Cimbora D.M.
      • Zhu J.F.
      • Richards B.
      2-Anilino-4-aryl-1,3-thiazole inhibitors of valosin-containing protein (VCP or p97).
      ,
      • Chou T.F.
      • Deshaies R.J.
      Development of p97 AAA-ATPase inhibitors.
      ,
      • Magnaghi P.
      • D'Alessio R.
      • Valsasina B.
      • Avanzi N.
      • Rizzi S.
      • Asa D.
      • Gasparri F.
      • Cozzi L.
      • Cucchi U.
      • Orrenius C.
      • Polucci P.
      • Ballinari D.
      • Perrera C.
      • Leone A.
      • Cervi G.
      • Casale E.
      • Xiao Y.
      • Wong C.
      • Anderson D.J.
      • Galvani A.
      • Donati D.
      • O'Brien T.
      • Jackson P.K.
      • Isacchi A.
      Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death.
      ,
      • Wang Q.
      • Shinkre B.A.
      • Lee J.G.
      • Weniger M.A.
      • Liu Y.
      • Chen W.
      • Wiestner A.
      • Trenkle W.C.
      • Ye Y.
      The ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group.
      ), which is closely related to Drg1 but involved in different cellular pathways. The mechanistic insights gained from our study will therefore be helpful for the rational design of novel inhibitors of other AAA-ATPases.

      Acknowledgments

      We thank Kerstin Klein and Anke Loregger for expert help during early stages of this work; Micheline Fromont-Racine, Bernard L. Trumpower, Juan P. Ballesta, Arlen W. Johnson, Ed Hurt, and Sabine Rospert for generously supplying reagents; and Tamsyn Stanborough for critically reading the manuscript.

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