An overall structure of the T4 RNA ligase (Rnl1) with AMPcPP. Alpha-helices are colored in cyan. The beta-strands are colored in red. Loops are colored in purple. The AMPcPP molecule is shown as a stick drawing in yellow.
T4 RNA ligase (Rnl1) catalyzes the formation of phosphodiester bonds between the 5'-phosphate and the 3'-hydroxyl termini of single-stranded nucleic acids.
T4 RNA ligase is a member of a distinct subgroup of RNA ligases along with a fungal tRNA ligase (Trl1), a putative baculovirus RNA ligase and RNA ligase from the bacteriophages RM378 and TS2126. Rnl1 is also the first RNA ligase whose complete crystal structure was determined.
Rnl1 is in fact a tRNA repair enzyme used by the T4 bacteriophage to escape hosts antiviral response. Enzyme functioning requires ATP and divalent metal ions. The T4 ligase repairs the tRNALys by joining its 5'-PO4 and 3'-OH groups via series of three nucleotidyl transfer steps in a ping-pong enzymatic mechanism. First, the Lys99 of the enzyme reacts with the a phosphorus of ATP and forms a covalent intermediate: ligase-(lysyl-N)-AMP. Pyrophosphate is also produced during this step. Secondly, AMP is transferred from the intermediate to the 5'- PO4 terminus of a tRNA to form an tRNA-adenylate intermediate (AppRNA). Finally, the ligase catalyzes the attack of the 3'-OH terminus of the tRNA on the tRNA-adenylate and the two termini are joined via a phosphodiester bond, the AMP is released.
The biological role of Rnl1 is to repair a break in the anticodon loop of E.coli tRNALys and in this way to evade bacteria host antiviral defense mechanism invoked following phage infection. Bacteria have a tRNALys-specific anticodon nuclease (ACNase) which is normally kept latent by association of its core protein, PrrC, with the endonuclease EcoprrI. Upon infection, the bacteriophage expresses a T4 Stp peptide, which inhibits EcoprrI. EcoprrI dissociates from PrrC and the ACNase becomes active. The anticodon nuclease then cleaves the anticodon loop of the tRNALys which blocks phage protein synthesis and, as a consequence, stops the infection.
Bacteriophage T4 has developed way to overcome this defense mechanism using the tRNA ligase and a polynucleotide kinase (PnK) to repair the in the tRNA anticodon loop.
T4 Rnl1 and T4 polynucleotide kinase–phosphatase (PnK) together form a two-component repair system that repairs the tRNA break made by the host anticodon nuclease. First, PnK remodels the ends of the broken tRNA by converting the 2',3' cyclic phosphate to a 3'-OH, 2'-OH and by phosphorylating the 5'-OH end to form a 5'-PO4. Rnl1 then joins the 3'-OH and 5'- PO4 RNA ends to form a standard 3'–5' phosphodiester bond.
StructureStructure
Structural domains
T4 Rnl1 is a 374-amino acid polypeptide that consists of two structural domains.
The N-terminal domain is responsible for the adenylyltransferase activity of the enzyme. It includes an a helix (a1), followed by antiparallel ß-sheets (ß1–ß4). It is the central ensemble of ß strands and loops that forms the nucleotide-binding pocket.
The pocket is lined by six peptide motifs (I, Ia, III, IIIa, IV, and V). These motifs characterize the covalent nucleotidyltransferase super-family that includes DNA ligases, RNA ligases, and mRNA capping enzymes.
The N-terminal polypeptide from from 1 to 254 amino acids suffices for the ligase–adenylylation reaction of T4 Rnl1.
The C-terminal domain is an all-helical domain, specific to the Rnl1. This all-helical domain is not found in C-terminal domains of DNA ligases and RNA capping enzymes.
The C domain confers the specificity for tRNA repair. The physiological substrate for Rnl1 is a tRNA containing a single break in the anticodon loop. :Deleting the C domain abolishes the preference of the enzyme for this broken tRNA substrate, but does not abolish adenylyltransferase activity.
A conserved Arg318–Lys319 dipeptide is a possible candidate that determines the tRNA specificity.
The N- and C-terminal domains are both able to form interactions with ATP and ATP analogues. But the ß-strands of the core region in the N-terminal domain contain most of the residues involved in binding ATP. , (motif I), (motif Ia), (motif V), and (motif V) interact with the phosphate groups of ATP and ATP analogues.
Lys99 is the site of adenylation in Rnl1, [1] but in this structure this residue seems to be situated at a distance incompatible with covalent interaction with the phosphate of ATP (more than 3 Å).[2] That could suggest that the formation of a covalent bond needs some conformational changes. But we do not know if a conformational change has to occur to allow the covalent bond formation, or if the formation of this bond leads to a conformational change.
Divalent cation binding sites
Metal ions are essential for the nucleotidyltransferase catalysis by all T4 DNA and T4 RNA ligases, which use the same two-metal ions mechanism.
The enzyme binds Mg2+. The true substrate in the adenylation reaction is the ATP-Mg2+ complex, [3] but nucleotidyltransferase enzymes cannot bind ATP-Mg2 directly. They bind ATP-Mg first, then a second Mg2+ ion. Each oh these cations with one phosphoryl oxygen from AMPcPP, three water molecules and two residues (Gly269 and Asp272), which both belong to the C-terminal domain.
The enzyme binds Ca2+. are coordinated to six water molecules. They do not directly interact with the enzyme, but via water molecules interacting with Glu227, Glu159, Lys99, Glu100, and Tyr246 via hydrogen bonds. They also interact with one phosphoryl oxygen of the AMPcPP. are coordinated to four water molecules and interact with three enzyme residues (Ile211 and Asp212) via hydrogen bonds.
Calcium is very important for enzyme Rnl1 structural biology, because the enzyme crystallizes only in présence of Ca2+. This could be explained by the fact that interactions between Ca2+ and negatively charged surface of each Rnl1 allow interactions between several enzymes at crystallization interfaces.[2]
Anion binding sites
The enzyme binds Cl-.
RNA binding site
The RNA-Rnl1 complex have not be crystallized yet, because the enzyme seems to crystallize only with AMPcPP, which is incompatible with the presence of RNA in the active site. That's why we are not able to define precisely the RNA binding site. But there are some elements tending to indicate of a possible RNA biding site in the :
- The analysis of charge distribution on the protein surface shows that the Rnl1 surface is negatively charged, apart from the C-terminal domain. The positive charges present on this domain could interact with the polyanion-like RNA backbone.
- The RNA have to be close to the ATP binding site to enable the AMP transfer from Lys99. The RNA could bind at the surface of the C-terminal domain, allowing the anticodon loop to be positionned toward the ATP binding site.
- Moreover, the C-terminal helical structure matches the tRNA structure.
The Rnl1 C-terminal domain is unique in nucleotidyltransferase family, so the architecture of this domain could allow T4 RNA ligase to bind specifically tRNALysin vivo.
A chloride ion is also positionned in the active site, and could mimic the 5'-phosphate of the incoming RNA.[4][5][2]
T4 RNA ligase is also able to bind . This molecule does not allow the nucleotidyltransferase function of the enzyme, but it seems necessary to Rnl1 cristallyzation. Each AMPcPP with ten residues (Tyr37, Arg54, Lys75, Tyr98, Lys99, Glu100, Lys119, Glu159, Lys 240 and Lys242), one Ca2+, one Mg2+ and one Cl-. It also interacts with several other enzyme residues via hydrophobic interactions.
The T4 RNA ligase (Enzyme class : E.C.6.5.1.3) catalyzes the formation of phosphodiester bonds between the 5'-phosphate terminus of single-stranded nucleic acid (i) and the 3'-hydroxyl terminus of single-stranded nucleic acid (j).
In the first step, Lys99 in the conserved motif KX(D/N)G (motif I) reacts with the α-phosphate of ATP or ATP analogue (NAD or GTP) and forms a covalent bond. This step gives a covalent intermediate ligase-(lysyl-N)-AMP and a pyrophosphate. Lys99, which is responsible for this step, is the essential residue involved in the catalytic mechanism.[6]
In the second step, AMP is transferred from the covalent intermediate to the 5'-phosphate RNA, forming a tRNA-adenylate intermediate (AppRNA). Arg54, by stabilizing and orienting the RNA phosphate by hydrogen bonding, is essential for this RNA adenylation.[2]
In the third step, the 3'-hydroxyl RNA attacks the 5'-phosphate RNA. A phosphodiester bond is formed and an AMP is released.
