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

Abstract

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 ...

T4 DNA ligase structure reveals a prototypical ATP-dependent ligase with a unique mode of sliding clamp interaction

“…Glu344 (motif IV ) interacts closely with Lys159 to form an ion pair. Glu344, Glu217 and intervening Asp161 (motif I ) are likely to coordinate metal ions essential for activating the catalytic Lys159 residue ( 61 ) and stabilizing the negatively charged intermediate of the nick sealing reaction. We observed electron density for several water molecules or putative magnesium ions surrounded by these residues (Figure 4A ).…”

Structural insight into DNA joining: from conserved mechanisms to diverse scaffolds

“…This single metal ion is coordinated with octahedral geometry by six ligands which include the 3′OH terminus of the nick and one of the non-bridging phosphate oxygens of the 5′ terminus as well as water-mediated interactions to residues in motif I, III and IV (Figure 3C ). The position of this Mn directly corresponds to the site of the catalytic metal ion in the ATP-bound pre-step 1 complexes the of RNA ligases and Ecl-Lig ND-ligase ( 20 , 21 ). The direct Mn contact to the DNA 5′ phosphate Pmar-Lig (PreS3-Mn) replaces the interaction with the AMP α-phosphate seen in the pre-Step 1 complexes, which is consistent with an exchange of location of the chemical transformation as the metal ion is now able to stabilize the pentacoordinate transition state of the 5′phosphate as well as decreasing the p K a of the 3′OH nucleophile.…”

Structures of ATP-bound DNA ligase D in a closed domain conformation reveal a network of amino acid and metal contacts to the ATP phosphates

“…The superfamily is defined by (i) the formation of a lysyl-NMP intermediate en route to the nucleotidylation of nucleic acid 5Ј ends and (ii) a conserved NTase catalytic domain and a set of NTase motifs that forms the NMP-binding pocket. Six distinct families of ATP-dependent RNA ligase (Rnl) have been characterized, each consisting of an NTase domain fused to a structurally unique flanking domain that defines the Rnl family (32)(33)(34)(35)(36)(37)(38)(39). By contrast, ATPdependent DNA ligases, NAD ϩ -dependent DNA ligases, and GTP-dependent mRNA capping enzymes share a core structure composed of a proximal NTase domain fused to a distal OB-fold domain (1).…”

“…Recent studies have highlighted a conserved mechanism of metal-dependent lysine adenylylation by ATP-dependent RNA ligases and NAD ϩ -dependent DNA ligases, whereby an octahedral catalytic metal-(H 2 O) 5 complex stabilizes the transition state of the ATP or NAD ϩ ␣ phosphate (32,33,35,38,39). Three side chains of NTase motifs I, III, and IV bind the pentahydrated metal cofactor via waters.…”

“…Three side chains of NTase motifs I, III, and IV bind the pentahydrated metal cofactor via waters. Structures of the Michaelis complex of RNA ligases from three different families reveal a two-metal mechanism of lysine adenylylation whereby a second octahedral noncatalytic metal complex bridges the ATP ␤ and ␥ phosphates and orients the PP i leaving group for in-line attackbythelysinenucleophile (32,33,39).Bycontrast,NAD ϩ -dependent DNA ligase employs a one-metal mechanism of lysine adenylylation and relies on direct enzymic contacts to orient the nicotinamide mononucleotide leaving group (33).…”