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Structures of RNA Complexes with the Escherichia coli RNA Pyrophosphohydrolase RppH Unveil the Basis for Specific 5′-End-dependent mRNA Decay*

  • Nikita Vasilyev
    Affiliations
    Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York 10016
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  • Alexander Serganov
    Correspondence
    To whom correspondence should be addressed:Dept. of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 550 First Ave., New York, NY 10016. Tel.: 212-263-4446
    Affiliations
    Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York 10016
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  • Author Footnotes
    * This work was supported by New York University start-up funds (to A. S.).
Open AccessPublished:February 05, 2015DOI:https://doi.org/10.1074/jbc.M114.634824
      Background: RppH initiates mRNA degradation in bacteria by removing pyrophosphate from the triphosphorylated 5′ terminus.
      Results: X-ray structures of E. coli RppH have revealed molecular features important for RNA recognition, discrimination against mononucleotides, and catalysis.
      Conclusion: RppH balances specificity and promiscuity.
      Significance: Our findings explain the broad influence of RppH on E. coli mRNA decay and suggest a catalytic mechanism for Nudix hydrolases.
      5′-End-dependent RNA degradation impacts virulence, stress responses, and DNA repair in bacteria by controlling the decay of hundreds of mRNAs. The RNA pyrophosphohydrolase RppH, a member of the Nudix hydrolase superfamily, triggers this degradation pathway by removing pyrophosphate from the triphosphorylated RNA 5′ terminus. Here, we report the x-ray structures of Escherichia coli RppH (EcRppH) in apo- and RNA-bound forms. These structures show distinct conformations of EcRppH·RNA complexes on the catalytic pathway and suggest a common catalytic mechanism for Nudix hydrolases. EcRppH interacts with RNA by a bipartite mechanism involving specific recognition of the 5′-terminal triphosphate and the second nucleotide, thus enabling discrimination against mononucleotides as substrates. The structures also reveal the molecular basis for the preference of the enzyme for RNA substrates bearing guanine in the second position by identifying a protein cleft in which guanine interacts with EcRppH side chains via cation-π contacts and hydrogen bonds. These interactions explain the modest specificity of EcRppH at the 5′ terminus and distinguish the enzyme from the highly selective RppH present in Bacillus subtilis. The divergent means by which RNA is recognized by these two functionally and structurally analogous enzymes have important implications for mRNA decay and the regulation of protein biosynthesis in bacteria.

      Introduction

      Messenger RNA degradation is essential for all living cells and is especially critical for bacteria, which live in a rapidly changing environment and require quick changes in protein synthesis to maximize their competitive advantage. An important mRNA decay pathway in bacteria involves endonucleolytic cleavage followed by 3′ to 5′-exonucleolytic digestion (
      • Belasco J.G.
      All things must pass: contrasts and commonalities in eukaryotic and bacterial mRNA decay.
      ). Recent studies have demonstrated that bacterial mRNA degradation can also be initiated at the 5′-end (
      • Mathy N.
      • Bénard L.
      • Pellegrini O.
      • Daou R.
      • Wen T.
      • Condon C.
      5′-to-3′ exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation and 5′ stability of mRNA.
      ,
      • Even S.
      • Pellegrini O.
      • Zig L.
      • Labas V.
      • Vinh J.
      • Bréchemmier-Baey D.
      • Putzer H.
      Ribonucleases J1 and J2: two novel endoribonucleases in B. subtilis with functional homology to E. coli RNase E.
      ). This degradation route requires initial conversion of the triphosphorylated 5′ terminus to a monophosphate by the RNA pyrophosphohydrolase RppH (
      • Celesnik H.
      • Deana A.
      • Belasco J.G.
      Initiation of RNA decay in Escherichia coli by 5′ pyrophosphate removal.
      ,
      • Deana A.
      • Celesnik H.
      • Belasco J.G.
      The bacterial enzyme RppH triggers messenger RNA degradation by 5′ pyrophosphate removal.
      ), followed by 5′ to 3′-exonucleolytic digestion by RNase J in Bacillus subtilis (
      • Richards J.
      • Liu Q.
      • Pellegrini O.
      • Celesnik H.
      • Yao S.
      • Bechhofer D.H.
      • Condon C.
      • Belasco J.G.
      An RNA pyrophosphohydrolase triggers 5′-exonucleolytic degradation of mRNA in Bacillus subtilis.
      ) or internal cleavage by the 5′-monophosphate-dependent endonuclease RNase E in Escherichia coli (
      • Celesnik H.
      • Deana A.
      • Belasco J.G.
      Initiation of RNA decay in Escherichia coli by 5′ pyrophosphate removal.
      ,
      • Deana A.
      • Celesnik H.
      • Belasco J.G.
      The bacterial enzyme RppH triggers messenger RNA degradation by 5′ pyrophosphate removal.
      ). The protective influence of a triphosphate on the 5′-end of bacterial mRNA resembles that of a eukaryotic cap structure; therefore, pyrophosphate removal by RppH activity is both structurally and functionally analogous to mRNA decapping by eukaryotic enzymes. Similarity between the RppH-mediated reaction and eukaryotic decapping is further highlighted by commonalities in the cleavage mechanism. RppH and some eukaryotic decapping enzymes, such as Dcp2, belong to the superfamily of Nudix hydrolyses that share the metal-coordinating Nudix box GX5EX7REXXEEXG, the motif essential for the chemistry of the reaction (
      • McLennan A.G.
      The Nudix hydrolase superfamily.
      ). Despite the different mechanisms and proteins involved in the 5′-end-dependent mRNA decay pathway in E. coli and B. subtilis, RppH is common to both species. Nevertheless, E. coli RppH (EcRppH)
      The abbreviations used are: EcRppH
      E. coli RppH
      BsRppH
      B. subtilis RppH
      BdRppH
      B. bacteriovorus RppH
      Ap4A
      P1,P4-di(adenosine-5′) tetraphosphate.
      and B. subtilis RppH (BsRppH) have low (∼20%) sequence identity (
      • Richards J.
      • Liu Q.
      • Pellegrini O.
      • Celesnik H.
      • Yao S.
      • Bechhofer D.H.
      • Condon C.
      • Belasco J.G.
      An RNA pyrophosphohydrolase triggers 5′-exonucleolytic degradation of mRNA in Bacillus subtilis.
      ) and share little similarity outside of the Nudix box. How these two rather distinct RppHs bind substrate RNA and perform an analogous catalytic reaction remains puzzling.
      Most Nudix hydrolases are active on mononucleotide substrates, and only a few, such as RppH, can act on polynucleotide substrates (
      • McLennan A.G.
      The Nudix hydrolase superfamily.
      ,
      • McLennan A.G.
      Substrate ambiguity among the nudix hydrolases: biologically significant, evolutionary remnant, or both?.
      ). RppH appears to be a ubiquitous bacterial enzyme and one of the most widespread Nudix hydrolases affecting several critical cellular processes. The loss of RppH activity causes diminished virulence (
      • Badger J.L.
      • Wass C.A.
      • Kim K.S.
      Identification of Escherichia coli K1 genes contributing to human brain microvascular endothelial cell invasion by differential fluorescence induction.
      ,
      • Bessman M.J.
      • Walsh J.D.
      • Dunn C.A.
      • Swaminathan J.
      • Weldon J.E.
      • Shen J.
      The gene ygdP, associated with the invasiveness of Escherichia coli K1, designates a Nudix hydrolase, Orf176, active on adenosine (5′)-pentaphospho-(5′)-adenosine (Ap5A).
      ), sensitivity to stress (
      • Lundin A.
      • Nilsson C.
      • Gerhard M.
      • Andersson D.I.
      • Krabbe M.
      • Engstrand L.
      The NudA protein in the gastric pathogen Helicobacter pylori is an ubiquitous and constitutively expressed dinucleoside polyphosphate hydrolase.
      ), abnormal mutagenic repair of DNA breaks (
      • Al Mamun A.A.
      • Lombardo M.J.
      • Shee C.
      • Lisewski A.M.
      • Gonzalez C.
      • Lin D.
      • Nehring R.B.
      • Saint-Ruf C.
      • Gibson J.L.
      • Frisch R.L.
      • Lichtarge O.
      • Hastings P.J.
      • Rosenberg S.M.
      Identity and function of a large gene network underlying mutagenic repair of DNA breaks.
      ), and other deficiencies, even though the protein destabilizes only a fraction of mRNAs (hundreds in E. coli) (
      • Deana A.
      • Celesnik H.
      • Belasco J.G.
      The bacterial enzyme RppH triggers messenger RNA degradation by 5′ pyrophosphate removal.
      ,
      • Luciano D.J.
      • Hui M.P.
      • Deana A.
      • Foley P.L.
      • Belasco K.J.
      • Belasco J.G.
      Differential control of the rate of 5′-end-dependent mRNA degradation in Escherichia coli.
      ). Such unexpected selectivity of RppH-dependent degradation can be explained, at least in part, by the recently discovered 5′-terminal requirements of these enzymes. BsRppH needs two but prefers three or more unpaired 5′-terminal nucleotides for activity and strictly requires guanine at the second position of its substrates (Fig. 1A) (
      • Hsieh P.K.
      • Richards J.
      • Liu Q.
      • Belasco J.G.
      Specificity of RppH-dependent RNA degradation in Bacillus subtilis.
      ,
      • Piton J.
      • Larue V.
      • Thillier Y.
      • Dorléans A.
      • Pellegrini O.
      • Li de la Sierra-Gallay I.
      • Vasseur J.J.
      • Debart F.
      • Tisné C.
      • Condon C.
      Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
      ), suggesting that it triggers the decay of a distinct set of mRNAs. This sequence preference does not correlate with the nucleotide frequency at the 5′-end of B. subtilis transcripts (
      • Hsieh P.K.
      • Richards J.
      • Liu Q.
      • Belasco J.G.
      Specificity of RppH-dependent RNA degradation in Bacillus subtilis.
      ,
      • Piton J.
      • Larue V.
      • Thillier Y.
      • Dorléans A.
      • Pellegrini O.
      • Li de la Sierra-Gallay I.
      • Vasseur J.J.
      • Debart F.
      • Tisné C.
      • Condon C.
      Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
      ). Thus, RppH can be considered a master regulator of protein biosynthesis that selectively controls mRNA translation through different rates of the 5′-end-dependent mRNA decay.
      Figure thumbnail gr1
      FIGURE 15′-End-dependent mRNA degradation pathways triggered by RppH. A and B, schematics depict sequence- and length-specific removal of pyrophosphate from triphosphorylated 5′ mRNA termini in B. subtilis (A) and E. coli (B). Nucleotide preferences at the first three RNA positions (
      • Luciano D.J.
      • Hui M.P.
      • Deana A.
      • Foley P.L.
      • Belasco K.J.
      • Belasco J.G.
      Differential control of the rate of 5′-end-dependent mRNA degradation in Escherichia coli.
      ,
      • Hsieh P.K.
      • Richards J.
      • Liu Q.
      • Belasco J.G.
      Specificity of RppH-dependent RNA degradation in Bacillus subtilis.
      ,
      • Foley P.L.
      • Hsieh P.K.
      • Luciano D.
      • Belasco J.G.
      Specificity and evolutionary conservation of Escherichia coli RppH.
      ) are shown in green. PPi, pyrophosphate; Pi, orthophosphate.
      Although vitally important for all organisms, mRNA degradation, especially RppH-mediated 5′-end-dependent mRNA decay in microbes, is among the least understood mechanisms of gene regulation. The biological significance of RppH prompted determination of the NMR structure of free EcRppH (
      • Bi Y.
      • Li H.
      • Fan S.
      • Xia B.
      • Jin C.
      1H, 13C and 15N resonance assignments of RNA pyrophosphohydrolase RppH from Escherichia coli.
      ), x-ray structures of Bdellovibrio bacteriovorus RppH (BdRppH) in the free form and bound to GTP (
      • Messing S.A.
      • Gabelli S.B.
      • Liu Q.
      • Celesnik H.
      • Belasco J.G.
      • Piñeiro S.A.
      • Amzel L.M.
      Structure and biological function of the RNA pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus.
      ), and x-ray structures of BsRppH bound to GTP and RNA (
      • Piton J.
      • Larue V.
      • Thillier Y.
      • Dorléans A.
      • Pellegrini O.
      • Li de la Sierra-Gallay I.
      • Vasseur J.J.
      • Debart F.
      • Tisné C.
      • Condon C.
      Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
      ). However, these RppH structures do not reveal whether EcRppH retains the same specificity as BsRppH or how EcRppH binds mRNA because the putative RNA-binding regions of EcRppH and BsRppH are strikingly different. Moreover, the determined structures do not feature RNA·RppH complexes in catalytically active conformations and do not provide sufficient details to unambiguously explain why BsRppH cleaves off two orthophosphates sequentially, although EcRppH normally removes pyrophosphate in one step (although it too sometimes catalyzes consecutive cleavage reactions) (
      • Deana A.
      • Celesnik H.
      • Belasco J.G.
      The bacterial enzyme RppH triggers messenger RNA degradation by 5′ pyrophosphate removal.
      ,
      • Richards J.
      • Liu Q.
      • Pellegrini O.
      • Celesnik H.
      • Yao S.
      • Bechhofer D.H.
      • Condon C.
      • Belasco J.G.
      An RNA pyrophosphohydrolase triggers 5′-exonucleolytic degradation of mRNA in Bacillus subtilis.
      ,
      • Piton J.
      • Larue V.
      • Thillier Y.
      • Dorléans A.
      • Pellegrini O.
      • Li de la Sierra-Gallay I.
      • Vasseur J.J.
      • Debart F.
      • Tisné C.
      • Condon C.
      Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
      ).
      To reveal the molecular basis of mRNA recognition and catalysis by EcRppH, we have identified minimal RNA substrates, determined x-ray structures of EcRppH in the apo- and RNA-bound forms, and conducted mutational studies. Our structural and biochemical data demonstrate novel structural principles of low specificity mRNA recognition that are characteristic of innumerable proteobacterial RppHs and distinct from those used by more specific RNA pyrophosphohydrolases like BsRppH. Our results also suggest a common catalytic mechanism for many hydrolases of the Nudix superfamily and therefore will have an impact on numerous biological systems.

      DISCUSSION

      To understand the molecular basis of the bacterial reaction functionally analogous to decapping of eukaryotic mRNAs, we determined the crystal structure of EcRppH in the apo-form and bound to RNA and Mg2+ cations. Although crystallization of low specificity RNA·protein complexes often yields poor electron density maps because of the mobility of the loosely bound RNA and the dissociation of the weak complexes under harsh crystallization conditions, we developed crystal treatment procedures that allowed us to visualize the first two nucleotides of EcRppH-bound RNA almost in their entirety. The EcRppH·RNA structure is more complete than the previously reported BsRppH·RNA structure, which mapped only the 5′-terminal triphosphate moiety, the second nucleotide, and two Mg2+ cations (
      • Piton J.
      • Larue V.
      • Thillier Y.
      • Dorléans A.
      • Pellegrini O.
      • Li de la Sierra-Gallay I.
      • Vasseur J.J.
      • Debart F.
      • Tisné C.
      • Condon C.
      Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
      ).
      The EcRppH·RNA structures reveal that EcRppH binds in a bipartite manner to the 5′-phosphates and to a nucleobase at the second position, as well as to adjoined sugar-phosphate moieties (Figs. 7A and 8A). As in the BsRppH·RNA complex (Fig. 8C) (
      • Piton J.
      • Larue V.
      • Thillier Y.
      • Dorléans A.
      • Pellegrini O.
      • Li de la Sierra-Gallay I.
      • Vasseur J.J.
      • Debart F.
      • Tisné C.
      • Condon C.
      Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
      ), the first and third nucleobases are not specifically recognized but must be unpaired to place the second nucleobase into the RNA-binding site (
      • Piton J.
      • Larue V.
      • Thillier Y.
      • Dorléans A.
      • Pellegrini O.
      • Li de la Sierra-Gallay I.
      • Vasseur J.J.
      • Debart F.
      • Tisné C.
      • Condon C.
      Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
      ,
      • Foley P.L.
      • Hsieh P.K.
      • Luciano D.
      • Belasco J.G.
      Specificity and evolutionary conservation of Escherichia coli RppH.
      ). Recognition of the second nucleotide by EcRppH and BsRppH allows discrimination between mRNAs and mononucleotides and prevents hydrolysis of cellular NTPs by RppH. Too short to simultaneously reach both the triphosphate and nucleobase two binding sites, NTPs are expected to be poor substrates, as confirmed by the very slow cleavage of the γ phosphate of ATP. This conclusion raises questions about the specificity of Bdellovibrio RppH in vivo because GTP binds to BdRppH at a position equivalent to where the first nucleotide of RNA binds to EcRppH and BsRppH (Fig. 8, A–C) (
      • Messing S.A.
      • Gabelli S.B.
      • Liu Q.
      • Celesnik H.
      • Belasco J.G.
      • Piñeiro S.A.
      • Amzel L.M.
      Structure and biological function of the RNA pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus.
      ). Although the triphosphate of GTP reaches the catalytic center of BdRppH, it was not hydrolyzed in the crystals, suggesting that the published structure of the BdRppH·3Mg·GTP complex may not be representative of how the enzyme functions with an RNA substrate.
      Figure thumbnail gr7
      FIGURE 7Recognition of the second RNA nucleotide by bacterial RppHs. Gray shading depicts the surface of the RNA-binding site visible through a cross-section made above a base plane. A, edge-specific interactions with guanine in the semi-open cleft of EcRppH. B, specific recognition of guanine in the cavity of BsRppH. The preferences of each enzyme for the second RNA nucleotide are shown below the structures.
      Figure thumbnail gr8
      FIGURE 8Comparison of catalytic sites in ternary complexes of EcRppH, BdRppH, and BsRppH with Mg2+ ions and triphosphorylated substrates. RNAs are in stick representation. A–C, surface views of the ligand-binding sites in EcRppH (A), Bd(RppH) (B), and BsRppH (C) colored according to surface potential. Red and blue colors correspond to negatively and positively charged areas, respectively. Mg2+ cations are shown as magenta spheres. D–F, zoomed-in views of catalytic sites in EcRppH (D), BdRppH (E), and BsRppH (F) complex structures. Water molecules (red color) and Mg2+ cations (in colors of corresponding proteins) are shown by spheres. Hydrogen and coordination bonds are depicted by dashed lines.
      Our structural studies uncovered markedly dissimilar modes of RNA binding by EcRppH and BsRppH despite their structural and functional similarities. Although both prefer guanine at the second position of their RNA substrates, the enzymes bind the second nucleobase differently, in either a deep cavity (BsRppH) or a cleft (EcRppH), each lined by a distinct set of nonequivalent amino acids (Fig. 7). The most notable sequence-specific contact in the EcRppH·RNA complex is two hydrogen bonds from Lys-140 to the Hoogsteen edge of guanine. These interactions would be expected to play a large part in determining the specificity of mRNA recognition by EcRppH because only guanine in an anti conformation can accept two hydrogen bonds from the lysine side chain, whereas adenine and uracil can accept only one hydrogen bond and cytosine cannot accept a hydrogen bond. Our structural prediction is corroborated by the reduced selectivity of the K140M mutant reported in the accompanying article (
      • Foley P.L.
      • Hsieh P.K.
      • Luciano D.
      • Belasco J.G.
      Specificity and evolutionary conservation of Escherichia coli RppH.
      ). An additional hydrogen bond between Ser-32 and N2 of guanine might not contribute much to the specificity of EcRppH because only one of two alternative conformations of this residue allows it to contact the RNA. Nevertheless, mutations of Ser-32 can enhance selectivity, suggesting that this residue contributes to the promiscuity of EcRppH (
      • Foley P.L.
      • Hsieh P.K.
      • Luciano D.
      • Belasco J.G.
      Specificity and evolutionary conservation of Escherichia coli RppH.
      ).
      Although the structures with adenosine and pyrimidines in the second position have not been determined yet, our structures with Gua in this position suggest that, in contrast to many other RNA·protein complexes, direct hydrogen bonding alone cannot explain the preference of EcRppH for guanosine. Most likely, hydrophobic and cation-π interactions also contribute to the specificity of RNA binding. Compared with pyrimidines, purines have a larger surface area that promotes stronger interactions with the pedestal formed by the hydrophobic side chains of Val-137 and Phe-139. Purines also form stronger cation-π interactions with the guanidinium group of Arg-27 according to the calculated strength of cation-π interactions in DNA·protein complexes (G > A> T > C) (
      • Wintjens R.
      • Liévin J.
      • Rooman M.
      • Buisine E.
      Contribution of cation-π interactions to the stability of protein-DNA complexes.
      ). Thus, loss of cation-π interactions explains the reduced ability of the R27A mutant to discriminate between purines and pyrimidines (
      • Foley P.L.
      • Hsieh P.K.
      • Luciano D.
      • Belasco J.G.
      Specificity and evolutionary conservation of Escherichia coli RppH.
      ). Although cation-π interactions with ligands are not common among Nudix hydrolases, they may be involved in adenine binding by the plant Ap4A hydrolase (
      • Fletcher J.I.
      • Swarbrick J.D.
      • Maksel D.
      • Gayler K.R.
      • Gooley P.R.
      The structure of Ap4A hydrolase complexed with ATP-MgFx reveals the basis of substrate binding.
      ).
      The structures illustrate that multiple features of EcRppH that favor binding of purines are compatible with weaker interactions with pyrimidines. Therefore, an unusual combination of low specificity interactions and an open RNA-binding site defines the relaxed specificity of EcRppH. In contrast to EcRppH, BsRppH binds guanine in a tight pocket where the base is surrounded by amino acid side chains that recognize the base through a set of direct hydrogen bonds incompatible with other nucleobases (Fig. 7). Structural comparison reveals that the only sequence-specific contact with nucleobase 2 common to both RNA·protein complexes has remarkably resulted from convergent evolution that positioned nonequivalent lysines to recognize the Hoogsteen edge of guanine.
      Our structures of the EcRppH·RNA complexes present two important pieces of information that encourage rethinking the catalytic mechanism of Nudix proteins. The EcRppH·2Mg·ppcpAGU structure demonstrates that Glu-56 makes a hydrogen bond with water molecule Wat-1. The EcRppH·3Mg·ppcpAGU structure reveals that Wat-1 is flanked by two Mg2+ cations and is ideally positioned for the catalytic reaction. These structures hint at the direct involvement of Glu-56 in catalysis, namely in activating Wat-1 for in-line nucleophilic attack on the β phosphorus atom. This hypothesis is supported by the severe effect of the E56A mutation on activity. In addition, in several structures of Nudix hydrolases, the counterpart of Glu-56 is positioned in proximity to a water molecule analogous to Wat-1 (
      • Bailey S.
      • Sedelnikova S.E.
      • Blackburn G.M.
      • Abdelghany H.M.
      • Baker P.J.
      • McLennan A.G.
      • Rafferty J.B.
      The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms.
      ,
      • Kang L.W.
      • Gabelli S.B.
      • Cunningham J.E.
      • O'Handley S.F.
      • Amzel L.M.
      Structure and mechanism of MT-ADPRase, a nudix hydrolase from Mycobacterium tuberculosis.
      ). Glu-56 is one of the most conserved residues of the Nudix motif and is thought to be essential for coordinating metal cations (
      • Mildvan A.S.
      • Xia Z.
      • Azurmendi H.F.
      • Saraswat V.
      • Legler P.M.
      • Massiah M.A.
      • Gabelli S.B.
      • Bianchet M.A.
      • Kang L.W.
      • Amzel L.M.
      Structures and mechanisms of Nudix hydrolases.
      ). However, in the EcRppH·3Mg·ppcpAGU and several other structures (
      • Bailey S.
      • Sedelnikova S.E.
      • Blackburn G.M.
      • Abdelghany H.M.
      • Baker P.J.
      • McLennan A.G.
      • Rafferty J.B.
      The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms.
      ,
      • Kang L.W.
      • Gabelli S.B.
      • Cunningham J.E.
      • O'Handley S.F.
      • Amzel L.M.
      Structure and mechanism of MT-ADPRase, a nudix hydrolase from Mycobacterium tuberculosis.
      • Scarsdale J.N.
      • Peculis B.A.
      • Wright H.T.
      Crystal structures of U8 snoRNA decapping nudix hydrolase, X29, and its metal and cap complexes.
      ), this residue does not coordinate divalent cations. Thus Glu-56 may be more important for activating a water molecule for catalysis than for binding Mg2+ cations involved in catalysis.
      Previous studies proposed that another glutamate of the Nudix motif (Glu-53 in EcRppH) sometimes acts as a catalytic residue in Nudix hydrolases (
      • Mildvan A.S.
      • Xia Z.
      • Azurmendi H.F.
      • Saraswat V.
      • Legler P.M.
      • Massiah M.A.
      • Gabelli S.B.
      • Bianchet M.A.
      • Kang L.W.
      • Amzel L.M.
      Structures and mechanisms of Nudix hydrolases.
      ). However, this residue coordinates two Mg2+ cations in the EcRppH·RNA complex, BdRppH (
      • Messing S.A.
      • Gabelli S.B.
      • Liu Q.
      • Celesnik H.
      • Belasco J.G.
      • Piñeiro S.A.
      • Amzel L.M.
      Structure and biological function of the RNA pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus.
      ), and other Nudix enzyme structures (
      • Bailey S.
      • Sedelnikova S.E.
      • Blackburn G.M.
      • Abdelghany H.M.
      • Baker P.J.
      • McLennan A.G.
      • Rafferty J.B.
      The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms.
      ,
      • Kang L.W.
      • Gabelli S.B.
      • Cunningham J.E.
      • O'Handley S.F.
      • Amzel L.M.
      Structure and mechanism of MT-ADPRase, a nudix hydrolase from Mycobacterium tuberculosis.
      ) and therefore may have difficulty deprotonating the nucleophilic water molecule. To alleviate this contradiction, it was suggested that Glu-53 could function as a catalytic residue in Nudix hydrolases if coordinated to only a single Mg2+ cation (
      • Mildvan A.S.
      • Xia Z.
      • Azurmendi H.F.
      • Saraswat V.
      • Legler P.M.
      • Massiah M.A.
      • Gabelli S.B.
      • Bianchet M.A.
      • Kang L.W.
      • Amzel L.M.
      Structures and mechanisms of Nudix hydrolases.
      ). Our structural data and the residual activity of the Glu-53 mutants of EcRppH and BdRppH (
      • Messing S.A.
      • Gabelli S.B.
      • Liu Q.
      • Celesnik H.
      • Belasco J.G.
      • Piñeiro S.A.
      • Amzel L.M.
      Structure and biological function of the RNA pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus.
      ) do not support the idea that Glu-53 is the sole catalytic residue of these RNA pyrophosphohydrolases. Nevertheless, our data do not exclude the possibility that Glu-53 is responsible for the residual activity observed for the E56A mutant at high EcRppH concentrations.
      The E57A mutation abolished EcRppH activity entirely, in agreement with the severe effects of Glu-57 mutations on the activity of other Nudix hydrolases, such as MutT (
      • Mildvan A.S.
      • Xia Z.
      • Azurmendi H.F.
      • Saraswat V.
      • Legler P.M.
      • Massiah M.A.
      • Gabelli S.B.
      • Bianchet M.A.
      • Kang L.W.
      • Amzel L.M.
      Structures and mechanisms of Nudix hydrolases.
      ). Our structures do not reveal any water molecule that could be activated for nucleophilic attack by this residue, but its side chain does undergo a conformational change upon Mg2+ binding. Because Glu-57 coordinates two key Mg2+ cations, this amino acid residue is essential for properly positioning the enzyme-bound metal cations.
      The crystal structures of the EcRppH·RNA complexes revealed a conformational change in the side chain of Glu-120 that allows it to coordinate Mg1 and Mg2. Despite the seemingly strategic position of this side chain, which “covers” Mg1 and Mg2 and stabilizes their enzyme-bound positions, Glu-120 has a relatively low impact on catalytic activity, emphasizing the fact that it assists catalysis but does not play an essential role in the reaction mechanism. This glutamate residue not only is absent from many Nudix hydrolases but also is not conserved among RppHs. For example, it is replaced by His in BdRppH.
      Mutational analysis showed that replacement of positively charged Arg-8 or Arg-52 significantly reduces EcRppH activity. This effect is easily explained for Arg-52, a conserved residue that orients the side chain of Glu-53 to facilitate its function. The strong reduction in activity observed for the R8A mutant was less expected because Arg-8 is not conserved among RppHs. This residue contacts the γ and β phosphates and likely stabilizes the leaving group. Although Arg-8 is not conserved, BdRppH and BsRppH contain lysines that are situated in proximity to the 5′-terminal phosphates and that may have a function similar to that of Arg-8 in EcRppH (Fig. 8, D–F).
      The catalytic mechanism proposed for EcRppH may also pertain to BdRppH (
      • Messing S.A.
      • Gabelli S.B.
      • Liu Q.
      • Celesnik H.
      • Belasco J.G.
      • Piñeiro S.A.
      • Amzel L.M.
      Structure and biological function of the RNA pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus.
      ) and BsRppH (
      • Piton J.
      • Larue V.
      • Thillier Y.
      • Dorléans A.
      • Pellegrini O.
      • Li de la Sierra-Gallay I.
      • Vasseur J.J.
      • Debart F.
      • Tisné C.
      • Condon C.
      Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
      ), which have similarly organized catalytic centers despite the presence of different numbers of bound Mg2+ cations (Fig. 8, D–F). In all of these complexes, Glu-56 adopts the same conformation and therefore has the potential to deprotonate the catalytic water molecule Wat-1. The EcRppH and BdRppH structures show that Wat-1 is aligned for nucleophilic attack on the β phosphorus atom and hydrolysis between the α and β phosphates. Although the BsRppH structure does not reveal Wat-1, such a water molecule would attack the γ phosphorus atom and hydrolyze the bond between the γ and β phosphates. Thus, the preferential initial cleavage between the γ and β phosphates reported for BsRppH can be explained by the differential binding of the triphosphate moiety in the catalytic site so that the γ phosphate in the BsRppH·RNA complex is positioned in the location of the β phosphate in the EcRppH·RNA complex. As suggested in the earlier model of the BsRppH catalysis (
      • Piton J.
      • Larue V.
      • Thillier Y.
      • Dorléans A.
      • Pellegrini O.
      • Li de la Sierra-Gallay I.
      • Vasseur J.J.
      • Debart F.
      • Tisné C.
      • Condon C.
      Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.
      ), the flexibility of the first nucleotide may facilitate movement of the β phosphate closer to the metal cations for the second round of catalysis after the γ phosphate has left.
      Species ranging from bacteria to mammals can contain over 20 Nudix enzymes involved in distinct cellular activities. Therefore, our mechanistic study to gain insights on the catalytic mechanism of EcRppH will have an impact on many prokaryotic and eukaryotic systems. At the moment, there is no catalytic mechanism accepted as universal for Nudix hydrolases, and several amino acid residues have been proposed to play a catalytic role in different enzymes. However, the Nudix motif is the only set of residues common to all Nudix hydrolases, and because an important catalytic residue is expected to be preserved in evolution, it is likely that such a residue would reside in this motif in the majority of Nudix enzymes. The difficulty in understanding Nudix catalysis likely stems from the abundance of noncatalytic enzyme structures that have been reported and the strong effects on catalysis of various residues that form the catalytic site. The structure of the E. coli ADP-ribose pyrophosphatase (
      • Gabelli S.B.
      • Bianchet M.A.
      • Ohnishi Y.
      • Ichikawa Y.
      • Bessman M.J.
      • Amzel L.M.
      Mechanism of the Escherichia coli ADP-ribose pyrophosphatase, a Nudix hydrolase.
      ) indirectly supports our hypothesis that Glu-56 directly participates in catalysis by the majority of Nudix hydrolases. This enzyme has an extra glutamate that can activate the nucleophilic water molecule. However, this glutamate is not conserved, and many Nudix proteins do not have a specialized non-Nudix amino acid residue that could play a similar catalytic role.
      An intriguing observation in our study is a slightly different position of the Gua2 nucleobase in the binding cleft in two EcRpH·RNA complexes. Surprisingly, specific hydrogen bonding and deep positioning of this nucleobase in the cleft were observed in the catalytically inactive complex although shallow positioning and loss of specific hydrogen bonds were seen in the catalytically relevant complex. We also noticed that placement of the α phosphate in the catalytically relevant conformation appears to be structurally incompatible with deep binding of the Gua2 nucleobase in the cleft. To explain two distinct means of the Gua2 nucleobase binding, we hypothesize that EcRppH initially forms base-specific interactions with the nucleotide at the second position of RNA, although the triphosphate moiety and Mg2+ cations are not fully bound to the enzyme. Weak initial binding of the triphosphate moiety may constitute a specific adaptation of RppH to reject nucleotides and other phosphate-containing noncognate substrates so that productive interactions are formed only with RNA. Following initial binding, movement of the triphosphate to the catalytic conformation would then result in tighter binding, hydrolysis, and sliding of the second nucleobase partway out of the cleft, thereby facilitating release of the monophosphorylated RNA product from the protein.
      The crystal structures of the EcRppH·RNA complexes reveal how a Nudix hydrolase that does not possess an established RNA-binding fold has nevertheless evolved for RNA binding and catalysis. The bipartite mRNA recognition mechanism involving the 5′-terminal phosphates and the second RNA nucleotide distinguishes EcRppH from Argonaute (
      • Ma J.B.
      • Yuan Y.R.
      • Meister G.
      • Pei Y.
      • Tuschl T.
      • Patel D.J.
      Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein.
      ), RIG-I (
      • Wang Y.
      • Ludwig J.
      • Schuberth C.
      • Goldeck M.
      • Schlee M.
      • Li H.
      • Juranek S.
      • Sheng G.
      • Micura R.
      • Tuschl T.
      • Hartmann G.
      • Patel D.J.
      Structural and functional insights into 5′-ppp RNA pattern recognition by the innate immune receptor RIG-I.
      ,
      • Lu C.
      • Xu H.
      • Ranjith-Kumar C.T.
      • Brooks M.T.
      • Hou T.Y.
      • Hu F.
      • Herr A.B.
      • Strong R.K.
      • Kao C.C.
      • Li P.
      The structural basis of 5′ triphosphate double-stranded RNA recognition by RIG-I C-terminal domain.
      ), and other proteins (
      • Leung D.W.
      • Amarasinghe G.K.
      Structural insights into RNA recognition and activation of RIG-I-like receptors.
      ,
      • Abbas Y.M.
      • Pichlmair A.
      • Górna M.W.
      • Superti-Furga G.
      • Nagar B.
      Structural basis for viral 5′-PPP-RNA recognition by human IFIT proteins.
      ) that recognize RNA ends. The unique combination of molecular features revealed by the EcRppH·RNA complexes expands the known repertoire of principles that govern RNA binding and demonstrates that functionally and structurally analogous enzymes can employ very different structural principles for the recognition of similar substrates.

      Acknowledgments

      We thank the Belasco laboratory for fruitful discussions and sharing unpublished results. We thank the staff of beamlines X-25 (National Synchrotron Light Source, Brookhaven National Laboratory).

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