Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme

Abstract

T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exemplifies a family of bifunctional enzymes with 5'-kinase and 3'-phosphatase activities that play key roles in RNA and DNA repair. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. The 2.0 A crystal structure of the isolated kinase domain highlights a tunnel-like active site through the heart of the enzyme, with an entrance on the 5' OH acceptor side that can accommodate a single-stranded polynucleotide. The active site is composed of essential side chains that coordinate the beta phosphate of the NTP donor and the 3' phosphate of the 5' OH acceptor, plus a putative general acid that activates the 5' OH. The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.

Fig. 1. Functional domains of T4 Pnk. (A) Domain organization of T4 Pnk. The N-terminal 5′-kinase and C-terminal 3′-phosphatase domains are depicted as horizontal bars. The Walker A-box motif near the N-terminus of the kinase domain is in black. Basic and acidic side chains essential for the kinase (K15, S16, D35, R38, D85 and R126) and phosphatase (D165, D167, R176, R213, D254 and D278) activities are highlighted. (B) Aliquots (3 µg) of the Ni–agarose preparations of full-length wild-type (WT) Pnk and the indicated deletion mutants were analyzed by SDS–PAGE. Polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (in kDa) of marker proteins are indicated on the left. (C) Phosphate release from 3′ dTMP is plotted as a function of input WT (closed cirles), NΔ138 (open circles), NΔ148 (squares) and NΔ158 (triangles) protein. (D) Pnk(149–301) [NΔ148] was sedimented in a glycerol gradient together with BSA, ovalbumin and cytochrome c. Aliquots (20 µl) of the odd numbered fractions were analyzed by SDS–PAGE. A Coomassie Blue-stained gel is shown. The direction of sedimentation is from the right (top of gradient) to the left (bottom).

Fig. 2. 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. Two enzyme-bound sulfates coordinated at the active site tunnel are shown as a stick models. Coordination of one sulfate within the oxyanion hole formed by the P-loop is depicted by dashed lines.

Fig. 3. Active site tunnel of Pnk. Space-filling images of the protein in CPK coloring highlight a see-through tunnel in T4 Pnk. (Left) A view looking into the tunnel from the NTP phosphate-donor side of the active site. (Right) A view from the phosphate-acceptor side. The sulfates corresponding to the β phosphate of the NTP and the 3′ phosphate of the terminal nucleotide of the 5′ OH acceptor are shown in the tunnel.

Fig. 4. Structural similarity between Pnk and CPT. The secondary structures of T4 Pnk (pnk) and Streptomyces venezuelae CPT (cpt) are shown above and below their respective amino acid sequences. The crystal structures were aligned by DALI. Gaps in the alignment are indicated by dashes. The essential side chains of Pnk are highlighted in shaded boxes, which include the equivalent positions of CPT when conserved.

Fig. 5. Crystallographic dimer interfaces in Pnk and CPT. The backbone traces of the Pnk and CPT dimers are shown with one protomer in purple and the other protomer in green. The dimers were superimposed by a least-squares fit in program O and the images were offset vertically in SETOR.

Fig. 6. Pnk active site. Stereo view highlighting the interactions of two sulfates bound at the active site. The P-loop main-chain amides and side chains Lys15, Ser16 and Arg126 coordinate a sulfate corresponding to the β phosphate of the NTP substrate. A second sulfate corresponding to the 3′ phosphate of the 3′ NMP substrate is coordinated to the NH and Oγ of Thr86 and the side chain of Arg38. Contacts are denoted by dashed lines.

Fig. 7. A model of T4 Pnk quaternary structure. We speculate that native Pnk homotetramer is assembled via high-affinity interactions between the C-terminal phosphatase domains (P) to form Pnk homodimers that are then converted into tetramers via lower-affinity interactions between the N-terminal kinase domains (K). The tetramer is depicted in a square planar arrangement for the sake of clarity.