Structure-based analysis of the interaction between the simian virus 40 T-antigen origin binding domain and single-stranded DNA

Abstract

The origin-binding domain (OBD) of simian virus 40 (SV40) large T-antigen (T-Ag) is essential for many of T-Ag's interactions with DNA. Nevertheless, many important issues related to DNA binding, for example, how single-stranded DNA (ssDNA) transits along the T-Ag OBD, have yet to be established. Therefore, X-ray crystallography was used to determine the costructure of the T-Ag OBD bound to DNA substrates such as the single-stranded region of a forked oligonucleotide. A second structure of the T-Ag OBD crystallized in the presence of poly(dT)(12) is also reported. To test the conclusions derived from these structures, residues identified as being involved in binding to ssDNA by crystallography or by an earlier nuclear magnetic resonance study were mutated, and their binding to DNA was characterized via fluorescence anisotropy. In addition, these mutations were introduced into full-length T-Ag, and these mutants were tested for their ability to support replication. When considered in terms of additional homology-based sequence alignments, our studies refine our understanding of how the T-Ag OBDs encoded by the polyomavirus family interact with ssDNA, a critical step during the initiation of DNA replication.

FIG. 1.

The costructure of the SV40 T-Ag OBD bound to a forked DNA oligonucleotide. (A) The sequence of the forked DNA target used in crystallization indicating duplex DNA and ssDNA regions. The top strand is magenta, and the bottom strand is cyan. The first and last bases are numbered. Shown below, the labeled crystal structure of the DNA clearly depicts the frayed ends of the DNA. (B) Ribbon representation of T-Ag OBD interactions with the duplex portion of the DNA, including the 3′ terminal dA of the top strand. The T-Ag OBD molecules are shown in yellow and orange. The interacting protein residues are shown as red sticks. A Van der Waals dot surface is shown for the 3′ terminal adenine base to highlight the stacking interactions provided by the T-Ag OBDs. (C) Ribbon representation of T-Ag OBD interactions with single-stranded DNA. The T-Ag OBDs are colored as in panel B. A neighbor (symmetry-related) DNA molecule that interacts with the orange T-Ag OBD is shown at left. A close-up of the boxed region is shown (and rotated ∼90°) at right, where only the interacting ssDNA is shown for clarity. Van der Waals spheres are shown in tan for the side chains of the residues that interact with the ssDNA phosphate backbone.

FIG. 2.

The crystal structure of the SV40 T-Ag OBD grown in the presence of poly(dT)₁₂ substrate. (A) View of the crystallographic lattice. The arrangement of T-Ag OBD molecules in the lattice is shown as ribbon diagrams, and the poorly modeled A1 motif residues are shown as spheres. The disordered A1 residues occur within a solvent channel that is made up of four T-Ag OBD molecules, highlighted by the square, and the four T-Ag OBDs are shown in yellow, cyan, brown, and blue. The circles indicate the location of the solvent channels in the crystal structure. This arrangement positions two A1 motifs inside the channel; the other two participate in neighboring channels. (B) Electron density map of the T-Ag OBD crystallographic tetramer. This view shows a 2F_o-F_c (observed and calculated structure factor amplitudes, respectively) electron density map (blue) covering four T-Ag OBD molecules, shown in cyan, green, yellow, and red. The position of the disordered A1 residues are shown as gray dot surfaces and occur within a positively charged solvent channel.

FIG. 3.

Residues implicated in binding to ssDNA by either X-ray crystallography or NMR methods and therefore mutated to alanine. (A) T-Ag OBD is shown as a surface representation in yellow. The residues mutated to alanine are shown in blue and labeled. These include A1 residues 150 to 152, 154, and 156, B2 residues 203 and 204, and additional residues 199, 201, 210, and 214. (B) SDS-PAGE analysis of purified T-Ag OBD samples containing the indicated point mutations. As a control, an aliquot of wt T-Ag OBD_131-260 (5.0 μg) was loaded in lane 1. Lane 2 contains See Blue Plus 2 prestained protein size markers (10 μl; Invitrogen); the molecular masses of the individual markers are indicated in kDa. Aliquots (5.0 μg) of the individual T-Ag OBD_131-260 mutants are displayed in lanes 3 to 13; the particular mutation present in a given T-Ag OBD molecule is indicated. The gel is stained with Coomassie blue. (C) Evidence that, relative to wt T-Ag OBD_131-260, the individual T-Ag OBD_131-260 mutants are not structurally altered. Chromatograms are shown of the wt T-Ag OBD_131-260 and the T-Ag OBD_131-260 mutants eluted from a Superdex 75 10/300 GL column in T-Ag storage buffer (see Materials and Methods). The baselines of the chromatograms are offset on the y axis (absorbance at 280 nm [Abs280]) in order to separate them. The wt T-Ag OBD_131-260 chromatogram (green) is at the bottom of the Abs280 axis, followed by (in ascending order, to the top of the series) the mutants V150A, F151A, S152A, R154A, L156A, T199A, H201A, H203A, R204A, N210A, and K214A. Elution position and molecular mass (in kDa) of gel filtration standards are indicated at the top of the graph. (D) CD analyses of wt T-Ag OBD_131-260 and the T-Ag OBD_131-260 mutants. The spectra associated with the individual mutants are identified on the figure.

FIG. 4.

Binding of T-Ag OBD proteins to poly(dT) DNA measured by fluorescence anisotropy. (A) Sequence of the 24-nucleotide poly(dT) probe used in this study. (B) Measurement of the T-Ag OBD-poly(dT) interaction via fluorescent anisotropy. Binding isotherms were measured in triplicate with 15 nM single-stranded DNA probe and increasing concentrations of the wild-type T-Ag-OBD (○) or of the V150A (□), F151A (Δ), S152A (▿), R154A (), L156A (+), T199A (⋄), H201A (*), H203A (×), R204A (▴), N210A (▪), or K214A (•) mutant protein. Curves were obtained by a nonlinear regression and fitting to a one-binding-site equilibrium. Error bars are not visible on the graph as they are smaller than the size of the symbols. K_ds (dissociation constants) were not calculated for these binding curves because they are not saturated although the relative affinities may be compared.

FIG. 5.

Binding of T-Ag OBD proteins to forked DNA measured by fluorescence anisotropy. (A) Nucleotide sequence of the forked DNA probe used in this study. The double-stranded region is underlined. (B) Measurement of the T-Ag OBD-forked DNA interaction via fluorescent anisotropy. Binding isotherms were measured in triplicate with 15 nM probe and increasing concentrations of the wild-type T-Ag OBD (○) or of the V150A (□), F151A (▵), S152A (▿), R154A (gray square), L156A (+), T199A (⋄), H201A (*), H203A (×), R204A (▴), N210A (▪), or K214A (•) mutant protein. Curves were obtained by a nonlinear regression and fitting to a one-binding-site equilibrium. Error bars are not visible on the graph as they are smaller than the size of the symbols.

FIG. 6.

DNA replication activity of large T-Ag mutant proteins. (A) Transient DNA replication activities of the indicated wild-type or mutant T-Ag proteins in C33A cells. DNA replication activities were measured by determining the ratio of firefly (Fluc-ori plasmid) to Renilla (Rluc control plasmid) luciferase activity as described in Materials and Methods. Replication activities are reported as a percentage of the Fluc/Rluc ratio obtained with the largest amount of wild-type T-Ag expression vector. Cells transfected with vector only (No T-Ag) were used as a negative control. (B) Expression of T-Ag proteins. Western blot analysis of total protein extracts prepared from transfected C33A cells expressing the wild-type or the indicated T-Ag mutant proteins. α, anti.

FIG. 7.

Sequence alignment of several T-Ag OBDs. The programs Clustal W (27) and Jalview (50) were used to calculate and display the sequence alignment of several representative T-Ag OBDs. The amino acids are colored by sequence identity (a color gradient from dark blue, indicating 100% identity, to white, indicating 0% identity). The red boxes indicate the location of the four residues identified as being important in ssDNA binding in this study; the SV40 T-Ag amino acid numbers are also presented. The sequences used in this alignment are from the polyomavirus members SV40, BK, JC, mouse (Mus), and Merkel cell carcinoma.

FIG. 8.

ssDNA binding mutations mapped onto the surface of the T-Ag OBD spiral hexamer. (A) The spiral hexamer of T-Ag OBD generated from the crystal structure (RCSB PDB code 2FUF) is shown as a solvent-accessible surface representation. The individual subunits are shown in alternating tan and gray. Residues that were mutated in this study are shown as blue or magenta. The blue residues are critical for binding ssDNA and are labeled in the terminal subunit. The magenta residues are not critical for ssDNA binding and are not labeled. (B) Depiction of ssDNA transiting over the lock-washer structure of T-Ag OBD via the path described herein. This figure was made by superposition of coordinates of the T-Ag OBD of the forked DNA complex onto the T-Ag OBD of the lock-washer structure. The resulting position of the short sequence of ssDNA (5 bases) is shown in red and displayed as a surface representation. This figure illustrates the relative size of ssDNA to the channel.