Structural and biophysical characterization of the proteins interacting with the herpes simplex virus 1 origin of replication

Abstract

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8DeltaC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8DeltaC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (Kd approximately 6 nM) followed by a second binding event (Kd approximately 0.8 nM). It is also shown that the stoichiometry of the ternary UL9ct-ICP8DeltaC-dsDNA15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.

FIGURE 1.

A, solution scattering curves (dots with error bars) for the UL9ct monomer (lower) and the UL9ct-ICP8ΔC complex (upper) together with the calculated curves for the models shown in B and E. B, molecular envelope of the UL9ct monomer as determined from solution scattering experiments with approximate dimensions. The two views are rotated with respect to each other by a 90° rotation about the vertical axis. C, envelope of the UL9ct dimer, which forms over time. D and E, two possible models of the UL9ct-ICP8ΔC binary complex derived from the x-ray scattering data using the known structure of ICP8ΔC and the envelope of UL9ct. The UL9ct envelope (spheres about the dummy residues) is shown in green, and a similar representation of ICP8ΔC is shown in magenta. The two ICP8ΔC domains (the smaller C-terminal helical domain (26) is seen as a protrusion on the right) were allowed to move independently in each case, constrained by a linker of 10 residues. The expected ssDNA-binding cleft (27) is indicated by a yellow line, indicating that ssDNA binding would be obstructed if UL9ct is bound. The red circles show the approximate positions of the surface lysines on ICP8ΔC that are modified by the cross-linker (supplemental material) suggesting a preference for the structure shown in E.

FIGURE 2.

A, scattering curve for the UL9ct-dsDNA complex (dots with errors bars) with MONSA (blue solid curve) and SASREF fits (red dashed curve). B, ab initio MONSA envelope for the 2:1 UL9ct-dsDNA_15-mer complex in two orientations related to each other by a vertical rotation of 90°. The protein is colored green and the DNA colored brown.

FIGURE 3.

Sedimentation velocity analysis. A, distribution of sedimentation coefficients c(s). B, distribution of molecular weights c(M_r) for the 4:1 molar mixture of UL9ct and the dsDNA_15-mer. The analysis shows three species as follows: UL9ct (37.8 kDa, s_20,w = 1.77 S, D_20,w = 4.06 × 10⁻⁷ cm² s⁻¹; % of mixture = 13.8), 2UL9ct:DNA (78.3 kDa, s_20,w = 2.80 S. D_20,w = 3.22 × 10⁻⁷ cm² s⁻¹, % of mixture = 78.8)- and a higher molecular weight species (108.6 kDa; s_20,w = 3.57S, D_20,w = 2.85 × 10 −⁷ cm² s⁻¹, % of mixture = 7.4), which can be interpreted as a 3:1 UL9ct-DNA complex.

FIGURE 4.

Isothermal titration microcalorimetric analysis of the Box I-containing dsDNA_15-mer to UL9ct. A, trace of the calorimetric titration of 30 × 2.5-μl aliquots of 463 μm DNA injected into 1.8 ml of 15.8 μm UL9ct. B, solid line in the lower plot represents the best fit to the data given by the parameters in Table 2.

FIGURE 5.

A, analytical gel filtration of the ternary complex using the fluorescein-labeled dsDNA_15-mer. The chromatogram in mauve is for a 1:1 ICP8ΔC:DNA mixture where only the DNA is observed, whereas the chromatogram in blue is for a 1:1:1 UL9ct:ICP8ΔC:DNA mixture. The labels are as follows: a, ternary ICP8ΔC-UL9ct-DNA complex; b, binary UL9ct-DNA complex; and c, free DNA. A native page of fractions a–c is given in the supplemental material. B, gel filtration of UL9ct:DNA mixtures at increasing protein concentration. Profiles shown are at protein:DNA ratios of 1:1 (blue) and 2:1 (pink) together with free DNA (green).

FIGURE 6.

Binding of UL9ct and UL9ct-ICP8ΔC complex to dsDNA_15-mer was analyzed using agarose gel electrophoresis (1% TBE). We measured the ratio of shifted duplex DNA by either a preincubation of UL9ct with the duplex DNA (A and B) and then addition of ICP8ΔC, or with a preincubation of UL9ct with ICP8ΔC (C and D) and then addition of duplex DNA. A and C show ethidium bromide-stained gels, and B and D show the Coomassie staining of the same gels. S1 corresponds to the binary UL9ct-DNA complex, and S2 corresponds to the ternary ICP8ΔC-UL9ct-DNA complex. + denotes the presence of one of the components, and − denotes its absence. 1.5 and 2 indicate the molar ratios of these components in the mixture. Where a number is absent the molar ratio is unity. Asterisk shows “free” pre-formed 2:1 UL9ct-DNA complex that is not bound to ICP8ΔC.

FIGURE 7.

ICP8ΔC/UL9ct binding to dsDNA containing the full HSV-1 replication origin (oriS). ICP8ΔC-UL9ct complex was incubated with either a super-twisted plasmid pGEM822 (A and B) or BamHI fragment (822 bp) purified from pGEM822 plasmid containing HSV-1 oriS and prepared for EM analysis as described under “Experimental Procedures” (C and D). The scale bar represents 100 nm.