RNA ligase Rnl2 complexed with AMP
Bacteriophage T4 (virus)
Polynucleotide ligases are ubiquitous enzymes that rectify breaks in nucleic acids by joining 3'OH and 5'PO4 termini to form a phosphodiester. Although these enzymes are classified to two families, DNA ligases and RNA ligases, both undergo the three pathways shown below.
The Rnl2 of bacteriophage T4, which requires duplex RNAs for substrate, has a substrate specificity only for the two terminal ribonucleotides on the 3'OH side of the nick. Although the replacement of these two nucleotides to 2'-deoxynucleotide causes enzyme activity reduction, the RNA recognition mechanism remains poorly understood.
The Rnl2 consists of two domains. One is the N-terminal adenylyltransferase domain, which is similar to that of DNA ligase. The other is the C-terminal domain (C domain), which has no apparent sequence similarity to the OB-fold domain found at equivalent position of all DNA ligases. The unique C domain is implicated as critical for step 2 of the ligation pathway. The elucidation of structural transformation in each ligation step seems to clear mechanism of the substrate specificity.
The structure shown here is the Rnl2 of bacteriophage T4 complexed with AMP, which binds to the Lys35 in the N-terminal domain. The complex is the product of the first ligation step (Figure 1).
Figure 1: The structure of Rnl2 of bacteriophage T4 bound to AMP. The AMP binding residue Lys35 is shown in sticks, the AMP molecule is in balls and sticks, and the others are in cartoon expression, respectively. The carboxy terminal and amino terminal are indicated with by the letter C and N, respectively.
The C domain consists of four alpha helices. It is indispensable for the second step, in which AMP is transfered from ligase to polynucleotide chain, and contacts neither binding polynucleotide chain nor the N-terminal domain but adjacent binding polynucleotide chain. Although this domain contacts with neither substrate polynucleotide chain nor N-terminal domain, it contacts with the polynucleotide chain bound to adjacent Rnl2.
The N-terminal adenylyltransferase domain serves the AMP binding site (adenylate binding pocket) in the first step, and the favorable conformation of the nick 3'OH for nucleophilic attack on the 5' phosphate.
At the first step (figure 1), a ATP fits to the adenylate binding pocket, which consists of six motifs, and then the alpha phosphate of the ATP binds to Lys35 of N-terminal domain with release of pyrophosphate. For this step, a magnesium ion and active site core residues (Arg55, Glu99, Phe119, Glu204, and Lys227) of binding pocket are essential.
Figure 2: The structure of the N-terminal domain of the product of first step (this PDB entry). The AMP is displayed in balls and sticks, the Lys35 residue bound to the AMP is in white sticks, the residues at the active site core are in pink sticks, and the others in grey cartoon expression, respectively. Some residues which exist in the front of active site are omitted.
At the second step, the essential residue Arg55 recognises the nick of polynucleotide chain, and transfers the AMP to the nick 5' phosphate.
Figure 3: The structure of the N-terminal domain of the product of second step (complex A of xPSSS:2HVR). The essential residue Arg55 is displayed in pink sticks, other residues in N-terminal domain is in grey cartoon, the template nucleotide chain is in blue sticks, 5' phosphate side chain of joining target is orange sticks, 3' OH side chain is in yellow sticks except two nucleotides beside the nick. The 3'-deoxyribocytidine monophosphate (3'dCMP) faced the 3'OH side of nick is in violet sticks, and the adjacent 2'-methylribocytidine monophosphate (2'OMeCMP) is in purple sticks, respectively. Comparing to figure 2, AMP leaves from Lys35 and approaches to 5' phosphate side nucleotide. Some residues which exist in the front of active site are omitted.
At the third step, phosphate binding residues leave and the pyrophosphate consists of the AMP and the nick 5' phosphate rotate to serve the favorable conformation of the nick 3'OH for nucleophilic attack on the 5' phosphate.
Figure 4: The structure of the N-terminal domain complexed with nucleotide chains. Enlarged the area of the nucleotide nick. The color and expression method are same as the figure 3. The phosphate anhydride bond between the AMP and the adjacent dCMP, which is the nucleophilic target of 3'OH, is displayed in the more thick stick. a) The product of step 2 (complex A of xPSSS:2HVR) b) The substrate of step 3 (complex B of xPSSS:2HVR) Compared to a), the b) indicates that the Arg55 and Lys227 leave from dCMP and AMP, respectively, and then the phosphate of AMP rotates to the opposite direction of 3'dCMP and makes a favorite conformation to join nucleotide chains.
If the sugar of the nick 3'OH terminal is replaced by 2'-deoxyribose, the activity of this step is significantly reduced. The result suggests that 2'-deoxyribose which adopts 2'-endo puckered conformation. The result suggests that 2'-deoxyribose with 2'-endo pucker conformation displaces the nick 3'OH to AMP leaving group so that it is not well disposed for nucleophilic attack on the nick 5' phosphate. Whereas the sugar of the nick 3'OH terminal is replaced by 3'-deoxyribose (xPSSS:2HVS) had no effect. These results seem to be due to the 3'-deoxyribose's 3'-endo pucker conformation, which does not induce conformational inhibition shown in 2'-deoxyribose.
Figure 5: (Right) The structure of a ribose molecule takeing 3'-endo pucker structure. (Left) The structure of a 2'-deoxyribose molecule taking 2'-endo pucker structure. Both are displayed in stick expression. In ribose, the 3'OH directs to the out of franose plane (equatorial position), whereas in deoxyribose, it directs to the up of franose plane (axial position).
Protein Data Bank (PDB)
author: Takahiro Kudou