Structure-function analysis of the 3' phosphatase component of T4 polynucleotide kinase/phosphatase

Abstract

T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity.

Fig. 1. Primary and secondary structure of the phosphatase domain of T4 Pnkp

The amino acid sequence of T4 Pnkp from amino acids 159 to 301 is aligned with that of the phosphatase domain of the ORF86 polypeptide encoded by Autographa californica nucleopolyhedrovirus (AcNPV). Positions of side chain identity/similarity are denoted by ^ below the aligned sequences. The secondary structure elements of T4 Pnkp are shown above the amino acid sequence with β strands as arrows and α helices as bars. The amino acids of Pnkp that were mutated previously to alanine are denoted by + (nonessential for activity) or ● (essential or important for 3′ phosphatase activity). The amino acids in Pnkp that were mutated singly to alanine in the present study are indicated by !.

Fig. 2. Pnkp mutants

Aliquots (5 μg) of the nickel-agarose preparations of full-length wild-type (WT) Pnkp and the indicated 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.

Fig. 3. Velocity sedimentation of phosphatase-defective Pnkp-Ala mutants

Sedimentation analysis was performed as described under Methods. The distributions of Pnkp (either WT, D187A, S211A, K258A or D277A as indicated) and the marker proteins catalase, BSA, and cytochrome c in each gradient were analyzed by SDS-PAGE. Scans of the Coomassie-blue stained gels are shown.

Fig. 4. Pnkp phosphatase active site

(A) Stereo view of the superimposed phosphatase active sites from Pnkp protomers A, B, C, and D (with the carbon atoms colored cyan for chain A, beige for chain B, yellow for chain C, and magenta for chain D) and the active site from the symmetrical Pnkp homotetramer structure reported previously (with carbon atoms in green). The Mg²⁺ of protomer B is depicted as a magenta sphere. The sulfate of protomer B is shown poised between Arg279 and Arg246. (B) Refined 2Fo-Fc electron density map of protomer D contoured at 1σ. The Mg²⁺ (cyan sphere) and sulfate ligands are shown. Waters are depicted as red spheres. The images were prepared in Pymol (DeLano, 2002).

Fig. 5. Active site similarity between Pnkp phosphatase and CTD serine phosphatase

Stereo view of the phosphatase active site from Pnkp protomer D (green) superimposed on the BeF₃-modified active site of CTD phosphatase Scp1 (beige). The aspartyl-BeF₃ adduct is depicted with the beryllium in yellow and the fluorines colored red (reflecting their mimicry of the phosphate of the aspartyl-phosphate intermediate). The Mg²⁺ of Scp1 is depicted as a cyan sphere and the associated waters as red spheres. Atomic interactions in the Scp1 active site are indicated by dashed lines. Pnkp side chains are labeled.

Fig. 6. The phosphatase homodimer interface

A stereo view is shown with one phosphatase protomer colored beige and the other colored gray. Amino acids that line the homodimer interface are shown in stick representation; residues from the gray promoter are labeled in italics and those from the beige protomer are in regular font style. Hydrogen-bonding and ionic interactions of these side chains are indicated by dashed lines. Landmark active site residues and the Mg and sulfate ligands are shown for the beige protomer only.

Fig. 7. Sedimentation analysis of phosphatase dimer interface mutants

Sedimentation analysis was performed as described under Methods. The distributions of Pnkp Ala-Ala mutants and the marker proteins catalase, BSA, and cytochrome c in each gradient were analyzed by SDS-PAGE. Scans of the Coomassie-blue stained gels are shown.