The adenylyltransferase domain of bacterial Pnkp defines a unique RNA ligase family

Smith, Paul; Wang, Li Kai; Nair, Pravin A.; Shuman, Stewart

doi:10.1073/pnas.1116827109

Cited by 27 publications

(29 citation statements)

References 24 publications

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“…1B;Shuman 2006a,b, 2007). The ligase domain reacts with ATP to form a covalent LIG-AMP adduct, but it is unable per se to seal RNA strands (Martins and Shuman 2005;Smith et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system

Wang

Das

Smith

et al. 2012

RNA

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Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 59-kinase, a central 29,39 phosphatase, and a C-terminal ligase. Here we report the crystal structure of the kinase domain of Clostridium thermocellum Pnkp bound to ATPdMg 2+ (substrate complex) and ADPdMg 2+ (product complex). The protein consists of a core P-loop phosphotransferase fold embellished by a distinctive homodimerization module composed of secondary structure elements derived from the N and C termini of the kinase domain. ATP is bound within a crescent-shaped groove formed by the P-loop ( 15 GSSGSGKST 23 ) and an overlying helix-loop-helix ''lid.'' The a and b phosphates are engaged by a network of hydrogen bonds from Thr23 and the P-loop main-chain amides; the g phosphate is anchored by the lid residues Arg120 and Arg123. The P-loop lysine (Lys21) and the catalytic Mg 2+ bridge the ATP b and g phosphates. The P-loop serine (Ser22) is the sole enzymic constituent of the octahedral metal coordination complex. Structure-guided mutational analysis underscored the essential contributions of Lys21 and Ser22 in the ATP donor site and Asp38 and Arg41 in the phosphoacceptor site. Our studies suggest a catalytic mechanism whereby Asp38 (as general base) activates the polynucleotide 59-OH for its nucleophilic attack on the g phosphorus and Lys21 and Mg 2+ stabilize the transition state.

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“…1B;Shuman 2006a,b, 2007). The ligase domain reacts with ATP to form a covalent LIG-AMP adduct, but it is unable per se to seal RNA strands (Martins and Shuman 2005;Smith et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system

Wang

Das

Smith

et al. 2012

RNA

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“…There are presently four structurally characterized RNA ligase families, exemplified by the following: bacteriophage T4 RNA ligase 1 (Rnl1 family) (El Omari et al 2006); T4 RNA ligase 2 and Trypanosome REL1 (Rnl2 family) (Nandakumar et al 2006); Pyrococccus abyssi RNA ligase (Rnl3 family) (Brooks et al 2008); and Clostridium thermocellum RNA ligase (Rnl4 family) (Smith et al 2012;Wang et al 2012). The physiology and biochemistry of the Rnl families suggest a division of labor in RNA repair, whereby Rnl1 and Rnl4 ligases are tailored to seal single-strand breaks in the loop of RNA stem-loops (Amitsur et al 1987;Wang et al 2007;Nandakumar et al 2008;Zhang et al 2012), whereas Rnl2 ligases are designed to seal 3 ′ -OH/5 ′ -PO 4 nicks in duplex RNAs and/ or RNA:DNA hybrids (Blanc et al 1999;Chauleau and Shuman 2013).…”

Section: Introductionmentioning

confidence: 99%

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Unciuleac

Shuman

2015

RNA

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The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3 ′ -OH/5 ′ -PO 4 duplexes in which the 3 ′ -OH strand is RNA. It does so via the "classic" ligase pathway, entailing reaction with ATP to form a covalent NgrRnl-AMP intermediate, transfer of AMP to the nick 5 ′ -PO 4 , and attack of the RNA 3 ′ -OH on the adenylylated nick to form a 3 ′ -5 ′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3 ′ -OH/5 ′ -PO 4 nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new "Rnl5 family" of nick-sealing ligases with a signature domain organization.

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“…Several crystal structures of ligases absent metals provided scant support for either explanation. In these structures, the motif I lysine nucleophile is located next to a motif IV glutamate or aspartate side chain (2,(4)(5)(6). The lysine and the motif IV carboxylate form an ion pair, the anticipated effect of which is to increase the pK a of lysine by virtue of surrounding negative charge.…”

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Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD ⁺ -dependent polynucleotide ligases

Unciuleac

Goldgur

Shuman

2017

Proc. Natl. Acad. Sci. U.S.A.

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Polynucleotide ligases comprise a ubiquitous superfamily of nucleic acid repair enzymes that join 3′-OH and 5′-PO 4 ). In step 3, ligase catalyzes attack by a DNA or RNA 3′-OH on the polynucleotide-adenylate to seal the two ends via a phosphodiester bond and release AMP. All steps in the ligase pathway require a divalent cation cofactor.The autoadenylylation reaction of polynucleotide ligases is performed by a nucleotidyltransferase (NTase) domain that is conserved in ATP-dependent DNA and RNA ligases and NAD + -dependent DNA ligases (1, 2). The NTase domain includes defining peptide motifs that form the nucleotide-binding pocket. Motif I (KxDG) contains the lysine that becomes covalently attached to the AMP. As Robert Lehman pointed out in 1974 (3), it is unclear how lysine (with a predicted pK a value of ∼10.5) loses its proton at physiological pH to attain the unprotonated state required for attack on the α phosphorus of ATP or NAD + . In principle, a ligase might use a general base to deprotonate the lysine. Alternatively, the pK a could be driven down by positive charge potential of protein amino acids surrounding lysine-Nζ. Several crystal structures of ligases absent metals provided scant support for either explanation. In these structures, the motif I lysine nucleophile is located next to a motif IV glutamate or aspartate side chain (2, 4-6). The lysine and the motif IV carboxylate form an ion pair, the anticipated effect of which is to increase the pK a of lysine by virtue of surrounding negative charge. It is unlikely that a glutamate or aspartate anion could serve as a general base to abstract a proton from the lysine cation. A potential solution to the problem would be if a divalent cation abuts the lysine-Nζ and drives down its pK a .A metal-driven mechanism was revealed by the recent crystal structure of Naegleria gruberi RNA ligase (NgrRnl) as a step 1 Michaelis complex with ATP and manganese (its preferred metal cofactor) (7). The key to capturing the Michaelis-like complex was the replacement of the lysine nucleophile by an isosteric methionine. The 1.9-Å structure contained ATP and two manganese ions in the active site. The "catalytic" metal was coordinated with octahedral geometry to five waters that were, in turn, coordinated by the carboxylate side chains of conserved residues in motifs I, III, and IV. The sixth ligand site in the catalytic metal complex was occupied by an ATP α phosphate oxygen, indicative of a role for the metal in stabilizing the transition state of the autoadenylylation reaction. A key insight, fortified by superposition of the Michaelis complex on the structure of the covalent NgrRnl-(Lys-Nζ)-AMP intermediate, concerned the role of the catalytic metal complex in stabilizing the unprotonated state of the lysine nucleophile before catalysis, via local positive charge and atomic contact of Lys-Nζ to one of the metal-bound waters (7).The NgrRnl Michaelis complex revealed a second metal, coordinated octahedrally to four waters and to ATP β and γ phosphate oxygens. The metal ...

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The adenylyltransferase domain of bacterial Pnkp defines a unique RNA ligase family

Cited by 27 publications

References 24 publications

Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system

Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD ⁺ -dependent polynucleotide ligases

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The adenylyltransferase domain of bacterial Pnkp defines a unique RNA ligase family

Cited by 27 publications

References 24 publications

Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system

Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD + -dependent polynucleotide ligases

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Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD ⁺ -dependent polynucleotide ligases