The small terminase, gp16, of bacteriophage T4 is a regulator of the DNA packaging motor

Abstract

Tailed bacteriophages and herpes viruses use powerful molecular motors to translocate DNA into a preassembled prohead and compact the DNA to near crystalline density. The phage T4 motor, a pentamer of 70-kDa large terminase, gp17, is the fastest and most powerful motor reported to date. gp17 has an ATPase activity that powers DNA translocation and a nuclease activity that cuts concatemeric DNA and generates the termini of viral genome. An 18-kDa small terminase, gp16, is also essential, but its role in DNA packaging is poorly understood. gp16 forms oligomers, most likely octamers, exhibits no enzymatic activities, but stimulates the gp17-ATPase activity, and inhibits the nuclease activity. Extensive mutational and biochemical analyses show that gp16 contains three domains, a central oligomerization domain, and N- and C-terminal domains that are essential for ATPase stimulation. Stimulation occurs not by nucleotide exchange or enhanced ATP binding but by triggering hydrolysis of gp17-bound ATP, a mechanism reminiscent of GTPase-activating proteins. gp16 does not have an arginine finger but its interaction with gp17 seems to position a gp17 arginine finger into the catalytic pocket. gp16 inhibits DNA translocation when gp17 is associated with the prohead. gp16 restricts gp17-nuclease such that the putative packaging initiation cut is made but random cutting is inhibited. These results suggest that the phage T4 packaging machine consists of a motor (gp17) and a regulator (gp16). The gp16 regulator is essential to coordinate the gp17 motor ATPase, translocase, and nuclease activities, otherwise it could be suicidal to the virus.

FIGURE 1.

Domain organization of phage T4 small terminase, gp16. A, domain end points of the large terminase, gp17, were determined by the x-ray structures of ATPase and nuclease domains and the full-length gp17 (7, 8). The functional motifs were determined by mutational and biochemical studies (, –, data not shown). The domain organization of the small terminase, gp16, is predicted by this study. Numbers represent the number of amino acids in the respective coding sequence. B, T4 family gp16 sequences with various degree of similarity to the T4 sequence were selected for multiple sequence alignment by ClustalW and secondary structure predictions by JPRED (accession numbers: T4, NP049775; 44RR2.8t, NP932507RB69, RB43, AAX78740;NP861868; RB49, NP891723; KVP40, NP899600; S-PM2, CAF34160 sequences were obtained from Phage Tulane website. Pairwise alignment scores of each gp16 with the T4 gp16 are shown in parentheses after the name of each phage. Amino acids highlighted in cyan represent the endpoints for N-terminal truncations. Amino acids highlighted in yellow represent the endpoints of C-terminal truncations. CCM-1 heptad residues are shown in orange. Heptad-1 and heptad-2 residues in T4 gp16 sequence are indicated by blue and pink underlines, respectively. Conserved arginines, aspartic acids, glutamine, and tyrosine that are mutated are highlighted in red. Linker regions predicted by DLP-SVM (34) and DISOPRED2 (35) are boxed in green. The sequences corresponding to N- (amino acids 1–35), central (amino acids 36–115), and C-domains (amino acids 116–164) are underlined by horizontal blue, orange, and green bars, respectively. Consensus sequence is shown below the secondary structure predictions (H residues correspond to α-helix, E residues to β-strand, and − residues to loop).

FIGURE 2.

The gp16 C-domain truncation mutants show partial loss of gp17-ATPase stimulation. A, schematic of the gp16 polypeptide showing domain organization (N-domain in blue, central domain in orange, and C-domain in green), predicted helices (h1–h6), and endpoints of C-terminal truncation clones (e.g. T139 refers to truncation at the threonine 139 residue). Note that the secondary structure prediction of gp16 sequence, unlike that of the aligned T4 family gp16 sequences (Fig. 1), shows six helices (h1–h6). B, SDS-polyacrylamide gel (12%) showing the purity of mutant proteins. C, 4–20% native non-denaturing gradient gel showing the oligomeric state of mutants. Both the gels were stained with Coomassie Blue R. The WT gp16 migrates as two oligomeric species (labeled as O₁ and O₂). All the C-terminal truncation mutants except M107 in which the truncation extended into the CCM-1 motif produced oligomers. M107 (lane 7), like the CCM-1 double mutant L103R-I110H (lane 2) (30), produced monomeric gp16 (labeled as M). D, gp17-ATPase stimulation activity of gp16 mutants expressed as percentage of the WT gp16 activity. The amount of P_i produced was quantified by phosphorimaging and ImageQuant 5.2 software. Values represent average of duplicates from two independent experiments. E, autoradiogram showing the gp17-ATPase stimulation activity of various mutants. Each ATPase assay was done in duplicate as per the procedure described under “Experimental Procedures.”

FIGURE 3.

Both the N- and C-domains of gp16 are important for gp17-ATPase stimulation. A, schematic of the gp16 polypeptide showing the endpoints of the truncation clones (see Fig. 2 legend for description of the schematic). The truncations are of two types: (i) truncations in N-terminal domain (L9-D164, I12-D164, and S36-D164); and (ii) truncations in both N- and C-terminal domains (I12-T111, I12-V115, E22-I128, E27-T94, and S36-V115). The S36-V115 mutant also contained a T96L mutation that increases the length of CCM-1 from two to three heptads but is phenotypically similar to the WT (30). B, SDS-polyacrylamide gel (12%) showing the purity of mutant proteins. C, 4–20% native non-denaturing gel showing the oligomeric state of gp16 mutants. Both the gels were stained with Coomassie Blue R. All the mutants, like the WT gp16, produced oligomers. D, gp17-ATPase stimulation activity of gp16 mutants expressed as percentage of the WT gp16. The amount of P_i produced was quantified by phosphorimaging and ImageQuant 5.2 software. Panels B and C are composites from different experiments, and the values in the table represent average of duplicates from two independent experiments.

FIGURE 4.

gp16 has a nucleotide binding site that is inactivated in mutants. The ability of WT and gp16 mutants to bind ATP or ADP was determined by UV cross-linking in the presence of [γ-³²P]8N₃ATP (A) or [γ-³²P]8N₃ADP (B). Each sample was cross-linked in duplicate and electrophoresed in adjacent lanes. See “Experimental Procedures” for details.

FIGURE 5.

None of the conserved arginines or the glutamine is essential for gp17-ATPase stimulation. A, schematic of the gp16 clones showing the positions of the mutated residues (see Fig. 2 legend for description of the schematic). B, Coomassie Blue R-stained SDS-polyacrylamide gel (12%) showing the purity of the mutant proteins. C, gp17-ATPase stimulation activity of mutants expressed as the percentage of WT gp16. D, autoradiogram showing the gp17-ATPase stimulation activity of various mutants. See legend to Fig. 2 and “Experimental Procedures” for more details.

FIGURE 6.

gp16 does not show nucleotide exchange activity. WT gp17 (1 μm) and WT gp16 (8.5 μm) either alone (lanes 1 and 2) or together (lane 3) were incubated with 5 μm [α-³²P]8N₃ADP for 5 min prior to UV cross-linking. For lane 4, gp16 was preincubated with [α-³²P] 8N₃ADP for 5 min before the addition of gp17. For lane 5, gp17 was preincubated with [α³²P] 8N₃ADP for 5 min before the addition of gp16. Panel A, Coomassie Blue R stained SDS-polyacrylamide gel, Panel B, phosphorimage of panel A. Panel C, histogram showing the quantification of cross-linking data from panel B.

FIGURE 7.

gp16 stimulates the hydrolysis of gp17-bound ATP. Vertical columns: gp17-K577 (columns A and C) or gp17-ATPase domain (column B) (1.4 μm) was incubated either alone or in the presence of increasing concentration of gp16 (1 μm-18 μm) and 3 μm of [γ-³²P]8N₃ATP (columns A and B) or 5 μm of [γ-³²P]2N₃ATP (column C). Following the ATPase assay, 1-μl aliquots of the samples were removed, and thin layer chromatography was performed. The rest was exposed to UV light for 5 min at 4 °C and subjected to SDS-PAGE and phosphorimaging (see “Experimental Procedures” for details of assays). Horizontal columns: 1: Coomassie Blue R-stained SDS-polyacrylamide gel; 2: phosphorimage of column 1; 3: Autoradiogram of samples following thin layer chromatography; 4: quantification of cross-linking and ATP hydrolysis data from columns 2 and 3, respectively. The positions of gp17, gp17-ATPase domain, and gp16 bands are marked with arrows.

FIGURE 8.

gp16 interaction to prohead-assembled gp17 inhibits DNA translocation. Defined in vitro DNA translocation was performed in the presence of gp17 alone (1.5 μm; 1.8 × 10¹³ molecules) or in the presence of gp17 plus increasing concentrations of gp16 (67–333 nm; 0.5–3 × 10¹¹ gp16 oligomers). The reaction mixture contained 6 × 10⁹ proheads and 6 × 10⁹ 48-kb phage λ DNA molecules (see “Experimental Procedures” for assay details). The amount of packaged DNA was quantified using the control lane C where 30 ng (10% of the total DNA) of phage λ DNA was loaded, and the quantified data are shown in the histogram below.

FIGURE 9.

gp16 regulates gp17-nuclease. Four sets of reactions were carried out with four different concentrations of gp17 (A, 0.2 μm; B, 0.4 μm; C, 0.8 μm; D, 1.0 μm). In each set, the amount of gp17 was kept constant and incubated either alone (lanes 2) or in the presence of increasing concentrations of gp16 (lanes 3–7). The ratio of gp16 (oligomer):17 (monomer) is shown at the bottom of lanes. Lane 1 corresponds to control sample containing no gp16 or gp17. See “Experimental Procedures” for assay details.