Trans-interaction of Arg-493 with Glu-343 or Asn-468 in the nucleotide-binding site of T7 DNA helicase. A, the crystal structure of the helicase domain of T7 gp4 (Protein Data Bank accession code 1E0J) drawn in PyMOL. The structure reveals a ring-shaped molecule with a central core. The structure shows the location of a central β-hairpin (green), helicase motif Ia (cyan), and motif IV (blue) and their rearrangement in respect to the NTP bound or empty state of the nucleotide-binding pocket. B, a magnified view of the nucleotide bound state of the nucleotide-binding site shows the arrangement of the indicated amino acids in response to AMPPNP binding. C, a magnified view of the arrangement of the indicated amino acids in an empty site, devoid of a nucleotide in the nucleotide-binding site. Note that in the NTP bound state, Glu-343 interacts with the γ-phosphate. However, in the empty state Glu-343 interacts with Arg-493. Arg-493 is otherwise paired with Asn-468.

dTTP hydrolysis activity in the absence and presence of ssDNA. dTTP hydrolysis assays were performed in 10-μl reactions containing the indicated concentrations of [α-³²P]dTTP, 100 nm of either wild-type or altered gp4, and 1 nm circular M13 ssDNA where indicated. After incubation at 37 °C for 30 min, EDTA was added to stop the reaction. The reaction was spotted on TLC plates coated with polyethyleneimine and dTDP was separated from dTTP in a buffer containing 0.5 m LiCl₂ and 0.5 m formic acid. The quantity of dTTP hydrolysis was calculated by measuring the intensities corresponding to [α-³²P]dTDP and unhydrolyzed [α-³²P]dTTP. Reaction kinetics was derived with the help of Michaelis-Menten equation in GraphPad Prism software and presented in Table 2. A, dTTP hydrolysis in the absence of ssDNA. B, dTTP hydrolysis in the presence of 1 nm M13 ssDNA. The values in the y axis are in log₁₀ scale. Note that 1 nm M13 ssDNA could stimulate the rate of dTTP hydrolysis by wild-type gp4 up to 100-fold. C, effect of M13 ssDNA on dTTP hydrolysis catalyzed by gp4-R493A/N468A compared with wild-type gp4. The assays were carried out as described above, except that different concentrations of M13 ssDNA (0–5 nm) were added to the reactions containing 200 μm [α-³²P]dTTP. The rate of dTTP hydrolysis by gp4-R493A/N468A (0.35 μm/s) and wild-type gp4 (0.22 μm/s) in the absence of M13 ssDNA were normalized to 1. The rates of dTTP hydrolysis in the presence of different concentrations of the M13 ssDNA were converted relative to the corresponding protein activity in the absence of DNA. Please note that in all cases less than 30% of the dTTP is hydrolyzed in 30 min for each of the concentrations examined.

Effect of nucleotides on the binding of gp4 to ssDNA. Binding of T7 gp4 to ssDNA was measured in a nitrocellulose filter assay. Reactions contained 1 nm 5′-³²P-labeled 50-mer oligonucleotide, 1 mm β,γ-methylene dTTP, 10 mm MgCl₂, 40 mm Tris-HCl (pH 7.5), 10 mm DTT, 50 mm potassium glutamate, and the indicated concentrations of either wild-type or altered gp4. After incubation at 37 °C for 30 min, the reaction mixture was filtered through a nitrocellulose membrane atop a charged ζ probe membrane. Protein-bound DNA on the nitrocellulose membrane and unbound free DNA on the ζ probe membrane were measured by radioactive intensity in a PhosphorImager. The percentage of oligonucleotide bound by each of the proteins is plotted against the corresponding protein concentration. The dissociation constant (K_D) calculated for each of the proteins using GraphPad Prism software is presented in Table 3. A, the graph shows the ability of wild-type and variants of gp4 to bind the 50-mer ssDNA in the presence of β,γ-methylene dTTP. B, the graph shows the ability of wild-type and variants of gp4 to bind 1 nm 50-mer ssDNA in the presence of dTDP. Note that wild-type gp4 and gp4-R493A/N468A are unable to bind ssDNA in this reaction. The error bars represent the S.D. obtained from three independent experiments.

Unwinding of duplex DNA by T7 DNA helicase. The DNA used to measure the unwinding activity of gp4 was a short duplex DNA with a 5′- and 3′-ssDNA tail at one end of the molecule. The latter strand has a ³²P label at its 5′ terminus. The DNA substrate was prepared by annealing a radioactive labeled 75-mer with an unlabeled 95-mer, generating a duplex region of 56 bp. Unwinding assays were carried out in a reaction containing 100 nm DNA substrate, 100 nm gp4, and 5 mm dTTP as described under “Experimental Procedures.” After incubation at 37 °C the reaction was stopped after 10 min. The unwound ssDNA was separated from the dsDNA substrate in a 10% non-denaturing polyacrylamide gel. The identity of each of the gp4 variants is indicated. The control reaction does not contain gp4. The separation of unwound ssDNA from the dsDNA substrate can be seen from the gel picture.

Oligomerization of gp4. A, oligomerization reactions contained 2 μm wild-type or altered proteins, 0.01–1 mm β,γ-methylene dTTP, and 1 μm 50-mer oligonucleotide and incubated at 37 °C for 20 min as described under “Experimental Procedures.” Glutaralderhyde was added to 0.033% to stabilize the oligomeric forms. The proteins were analyzed by electrophoresis on a 10% non-denaturing polyacrylamide gel. The state of the oligomerization of the proteins was determined after staining the gel with Coomassie Blue. The formation of hexamers, higher order oligomers, and lower order oligomers are shown in the gel picture. The identity of the protein is also indicated in the gel picture. B, the gel picture shows the oligomerization ability of wild-type and altered proteins in the presence of different concentrations of dTDP (0.01–1 mm). The experiments were carried out as described in A except that β,γ-methylene dTTP was replaced with dTDP. C, fraction of hexamers and higher order oligomers formed by each of the proteins in the presence of different concentrations of β,γ-methylene dTTP or dTDP are quantitated by the software AlphaEase FC (AlphaImager 3400). The data were fit to the Hill equation and provided an apparent K_d for β,γ-methylene dTTP or dTDP-dependent hexamer formation. The error bars represent the S.D. of the result from three independent experiments.

Role of glutamate switch in the NTP-dependent ssDNA binding by T7 DNA helicase. Interaction of Arg-493 (glutamate switch) with either Glu-343 (catalytic glutamate) or Asn-468 (central β-hairpin) from the adjacent subunit depends on the nature of nucleotide in the nucleotide-binding site. The model is derived from our experimental results and the crystal structures of the helicase domain of T7 gp4 (PDB code 1E0J as presented in Fig. 1). Arrangement of the three indicated amino acid residues in response to the presence of either dTTP or dTDP in the nucleotide-binding site are depicted for wild-type gp4, gp4-E343Q, gp4-E343D, and gp4-E343N. Note that when Arg-493 interacts with Asn-468 of the neighboring subunit, the helicase binds ssDNA in its central core. In wild-type gp4, Glu-343 interacts with the γ-phosphate of dTTP. Arg-493 interacts with Asn-468 and the helicase binds ssDNA. In the presence of dTDP, Arg-493 interacts with Glu-343. An interruption in the Arg-493 and Asn-468 interaction makes the wild-type gp4 defective in ssDNA binding. In gp4-E343Q, Gln-343 neither interacts with the γ-phosphate of dTTP nor Arg-493. Consequently Arg-493 interacts with Asn-468 at the central core and facilitates the altered helicase, gp4-E343Q to bind ssDNA irrespective of the dTTP or dTDP present in the nucleotide-binding site. In gp4-E343D, Asp-343 can interact with the γ-phosphate of dTTP and Arg-493 interacts with Asn-468 at the central core of the helicase ring and facilitates ssDNA binding, but in the presence of dTDP, Arg-493 can interact with either Asp-343 or Asn-468. The choice of Arg-493 to interact with Asp-343 compromises the interaction of Arg-493 with Asn-468 in the central core leads to moderate ssDNA binding by gp4-E343D. In gp4-E343N, Asn-343 cannot bind the γ-phosphate of dTTP but can interact with Arg-493. Thus irrespective of the NTP present in the nucleotide-binding site, Arg-493 can interact with either Asn-343 or Asn-468 leading to the observed DNA binding.