PNK

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Introduction

Polynucleotide kinase (Pnk) was discovered by the Richardson and Hurwitz laboratories in E. coli infected with T4 and T2 bacteriophages. Using bacteriophage T4 Pnk to label 5'DNA or RNA ends with ³²P facilitates the development of methods of nucleic acid structure, molecular cloning and nucleic acid sequencing. Although the importance of T4 Pnk in recombinant DNA research is well known, it is less widely appreciated of its contribution to the "RNA repair" pathway in vivo. During T4 infection, bacterium tries to inhibit T4 protein synthesis by inducing site-specific cleavage of tRNAs in the host cell, to which phage use Pnk and phage-encoded RNA ligase to repair the broken tRNAs. T4 Pnk catalyzes the transfer of γ phosphate from ATP to the 5'OH terminus of RNA and the hydrolytic removal of 3'PO₄ terminus from RNA in this pathway. Eukaryotic tRNA splicing requires a series of similar reactions. In fission yeast and metazoans, Pnk orthologs specific for phosphorylation of 5′ OH DNA termini function play an important role in repairing DNA damage caused by oxidation, radiation, and topoisomerase I poisons. In order to have a deeper understanding of the mechanism of action of the enzyme, we should make more efforts in structural analysis.  

Crystal structure of the kinase domain

The scientists crystallized the N-terminal kinase domain Pnk (1-181), which contains a continuous peptide from Pnk residues 1 to 152 (the N-terminal His-tag and the C-terminal segment from amino acids 153 to 181 are disordered). The kinase domain is composed of four-stranded parallel β-sheets(β2-β3-β1-β4), with three α-helices on each side. The helices α1, α5, and α6 are located on the left side of the β-sheet, while α2, α3, and α4 are located on the right side. This kinase contains a consensus Walker A-box motif (⁹GxxGxGKS¹⁶) between the first β-chain and the first α-helix. Two sulfate ions bound to Pnk, one of which binds to the P-loop, and the other binds to α2 and the loop connecting β3 and α4 to separate the NTP and phosphate receptor binding sites, respectively. Kinases can be divided into two subdomains. One is a globular pedestal supporting the two sulfates; the other bipartite module includes a lid-like structure above the sulfate-binding site. The kinase monomer contacts another monomer through the crystallographic 2-fold axis, and the surface area of the interaction interface is 2400 Ų (1200 Ų per protozoa). The molecular contacts at the interface are mainly hydrophobic.

Figure 1. Structure of the 5′-kinase domain. A stereo ribbon image is shown with the central β-sheet in green and flanking α helices in purple (Wang, L.K.; et al. 2002)

Pnk active site

The two sulfate ions play an important coordination role within the tertiary structure of Pnk. Specifically, the first sulfate has extensive contact with the backbone amide nitrogens of the P-loop and with the side chains of residues Lys15 and Ser16. Scientists speculate that this sulfate defines the NTP binding site of Pnk. Arg126 cannot be functionally replaced by lysine, highlighting the importance of bidentate interaction with the NTP substrate. Arg126 is a part of the lid subdomain covering the NTP binding site and is located within the motif ¹²²RxxxR¹²⁶. The second sulfate ion is 8.5 Å away from the first sulfate ion, and it is coordinated with the main-chain amide nitrogen and side chain Oγ of Thr86 and to both terminal guanidine nitrogens of Arg38. The Arg38 side chain is very important for the phosphorylation of 3'CMP. Mutation analysis showed that the contact of sulfate (3' phosphate) with the main chain amide of Thr86 may be indispensable, insofar as the adjacent Asp85 side chain was replaced by alanine, resulting in loss of function. Asp85 forms a bidentate hydrogen bond with the main chain amide of Arg34 and the side chain amide nitrogen of Gln64, so that Thr86-NH can be fixed in the correct position to bind the substrate. An aspartate is conserved at the equivalent position (Asp93) of CPT.

Figure 2. Pnk active site (Wang, L.K.; et al. 2002)