Structure of p22 headful packaging nuclease

Abstract

Packaging of viral genomes into preformed procapsids requires the controlled and synchronized activity of an ATPase and a genome-processing nuclease, both located in the large terminase (L-terminase) subunit. In this paper, we have characterized the structure and regulation of bacteriophage P22 L-terminase (gp2). Limited proteolysis reveals a bipartite organization consisting of an N-terminal ATPase core flexibly connected to a C-terminal nuclease domain. The 2.02 Å crystal structure of P22 headful nuclease obtained by in-drop proteolysis of full-length L-terminase (FL-L-terminase) reveals a central seven-stranded β-sheet core that harbors two magnesium ions. Modeling studies with DNA suggest that the two ions are poised for two-metal ion-dependent catalysis, but the nuclease DNA binding surface is sterically hindered by a loop-helix (L(1)-α(2)) motif, which is incompatible with catalysis. Accordingly, the isolated nuclease is completely inactive in vitro, whereas it exhibits endonucleolytic activity in the context of FL-L-terminase. Deleting the autoinhibitory L(1)-α(2) motif (or just the loop L(1)) restores nuclease activity to a level comparable with FL-L-terminase. Together, these results suggest that the activity of P22 headful nuclease is regulated by intramolecular cross-talk with the N-terminal ATPase domain. This cross-talk allows for precise and controlled cleavage of DNA that is essential for genome packaging.

FIGURE 1.

Structural stability of P22 L-terminase subunit. A, schematic diagram of P22 L-terminase indicating the presence of an N-terminal ATPase domain connected to a C-terminal nuclease domain by a flexible linker. Amino acid sequences for the interdomain linker (residues 286–312) and C-terminal basic tail (residues 483–499) are also indicated, with flexible and basic residues colored in gray and blue, respectively. The site of proteolytic cleavage in the linker is indicated by a red arrow. B, time course of limited proteolysis of purified L-terminase in the presence of chymotrypsin. M.W., molecular mass. C, structural stability of FL-L-terminase against thermal denaturation monitored by measuring changes in the ellipticity intensity at 222 nm as a function of temperature. The fraction unfolded of FL-L-terminase was plotted against the temperature, revealing an apT_m of 31.3 and 42 °C, in the presence and absence of 1 mm ATP, respectively. term, terminase.

FIGURE 2.

Crystal structure of P22 l-terminase nuclease domain at 2.02 Å resolution. A, ribbon diagram of P22 headful nuclease colored by secondary structure elements with α-helices, β-strands, and loops in red, yellow, and green, respectively. The site of proteolytic cleavage (residue 289) and the beginning of the nuclease domain (residue 313) are indicated. B, topological diagram of P22 nuclease (color coded as in A) showing all secondary structure elements identified in the crystal structure. Loops L₁ and L₂ are also indicated by arrows.

FIGURE 3.

Architecture of P22 nuclease active site. A, left panel, ribbon diagram of P22 nuclease domain highlighting only active site residues and metal ions. In the right panel is a magnified view of the active site, which includes essential residues involved in catalysis, two magnesium ions (Mg_A and Mg_B) colored in purple, and several water molecules (small red spheres). The F_o − F_c electron density map (in cyan) overlaid to the magnesium sites is contoured at 7 σ above background and was computed at 2.02 Å resolution by omitting the magnesium atoms from the final model. B, comparison of five active site residues from the crystal structure of the L-terminase nuclease of P22, SPP1 (18), T4 (6), and HHV-5 (19). Acidic residues are colored in red, and nonacidic amino acids are in gray. The two metal ions are also shown in yellow.

FIGURE 4.

Modeling the DNA binding surface of P22 headful nuclease. A, model of P22 nuclease domain (in gray) bound to a DNA-RNA hybrid (in orange). The nucleic acid model was obtained by superimposing the structure of the human RNase H1·DNA-RNA complex (Protein Data Bank code 2QKB) to that of P22 nuclease and removing the atoms for the RNase H1. In this model, helix α₂ (in red) and the loop L1 (in green) adopt a conformation incompatible with DNA binding. B, magnified view of the active site highlighting the position and distance (∼7.9Å) of the two metal ions (purple sphere) symmetrically located in the vicinity of the DNA phosphate backbone.

FIGURE 5.

In vitro nuclease assay. A, time course of nuclease digestion obtained by incubating gp3-DNA with 1 μm of FL-L-terminase, FL-L-terminase + ATP, FL-L-terminase + nonameric S-terminase (referred to as gp3) with and without ATP and FL-L-terminase + dodecameric portal protein. B, time course of nuclease digestion obtained by incubating gp3-DNA with 1 μm of purified FL-nuclease, nuclease-ΔL₁, and nuclease-ΔL₁-α₂. In both panels, samples were separated on a 1.2% agarose gel followed by ethidium bromide staining. The abnormal migration of gp3-DNA in the presence of gp3 in A is caused by S-terminase tight binding to DNA and was also observed in control experiments where L-terminase was omitted (24). C and D, quantification of agarose bands in A and B. The percentage of gp3-DNA left on the gel is plotted as a function of time. The error bars are based on three independent repeats. term, terminase.