The crystal structure of the SV40 T-antigen origin binding domain in complex with DNA

Abstract

DNA replication is initiated upon binding of "initiators" to origins of replication. In simian virus 40 (SV40), the core origin contains four pentanucleotide binding sites organized as pairs of inverted repeats. Here we describe the crystal structures of the origin binding domain (obd) of the SV40 large T-antigen (T-ag) both with and without a subfragment of origin-containing DNA. In the co-structure, two T-ag obds are oriented in a head-to-head fashion on the same face of the DNA, and each T-ag obd engages the major groove. Although the obds are very close to each other when bound to this DNA target, they do not contact one another. These data provide a high-resolution structural model that explains site-specific binding to the origin and suggests how these interactions help direct the oligomerization events that culminate in assembly of the helicase-active dodecameric complex of T-ag.

Figure 1. DNA and T-ag Sequences

(A) The SV40 64-bp core-origin sequence. The pentanucleotides P1 through P4 are indicated above the sequence. Each GAGGC sequence is colored magenta, and its complement is cyan. The arrows indicate the 5′ → 3′ direction of the pentanucleotide sequence GAGGC. The AT-rich and early palindrome regions of the SV40 core origin are labeled. (B) The DNA duplex used in crystallization of the T-ag obd–DNA complex. This 21-mer contains the palindromic binding sites P1 and P3 and a mutated pentamer P2 site. The GAGGC sequences and their complements are indicated by magenta and cyan boxes, respectively. The altered P2 pentamer is indicated by hash-marks in magenta and cyan. As above, the arrows indicate the 5′ → 3′ direction of the pentanucleotide sequences, and the red X indicates that the P2 sequence is altered. (C) The BPV origin shows the E1 binding sites termed E1–1 through E1–4. The E1 binding sites are imperfect 5′-ATTGTT-3′ hexameric sequences. Boxes outline each binding site, and the binding sites are labeled. The arrows indicate the 5′ → 3′ direction of the binding site. The direct repeats (sites E1–1 and E1–2 or sites E1–3 and E1–4) overlap by 3 bp. The ATTGTT sequence (magenta) and its complement (cyan) are indicated. Lowercase letters are used for the portion of binding sites that do not overlap. (D) Structure-based sequence alignment of SV40 T-ag obd with BPV E1 obd. The secondary structure elements of the T-ag obd are shown above its amino acid sequence. Every tenth residue is indicated with a dot. T-ag obd residues that make base-specific contacts in the DNA co-structure are indicated by cyan boxes. T-ag obd residues that make phosphate interactions are indicated by red triangles above the amino acid sequence. There are two types of T-ag obd–obd interactions: the “head-to-head” type seen in the disulfide-linked dimer (possibly important in double-hexamer formation), and the “side-to-side” type (important in single-hexamer formation) seen in the spiral hexamer. T-ag obd residues that form the protein–protein interface in the disulfide-linked dimer structure are indicated by yellow boxes. T-ag obd residues that comprise the protein–protein interface in the spiral hexamer are indicated by an asterisk (*). Residues for BPV E1 obd that make base-specific contacts or phosphate contacts or participate in its dimer interface are indicated by pink boxes, magenta triangles below the E1 sequence, and green boxes, respectively. The information for E1 was obtained from the crystal structures of E1-obd with and without DNA.

Figure 2. Overall Architecture of T-ag obds Bound to P1 and P3 of the Origin of Replication

A ribbon diagram shows two T-ag obd monomers bound to the 21-mer DNA. The coloring of the DNA is the same as in Figure 1A. A ribbon diagram of an idealized B-form DNA is shown in orange superimposed on one binding site. The T-ag obd is colored as follows: red (A1 loop, amino acids 147–155), purple (B2 loop, amino acids 202–204), green (helix C), blue (helix B), orange (B3 loop amino acids 213–218), and yellow (Cys216 and the rest of the molecule). This orientation shows two T-ag obds arranged in a head-to-head fashion while engaged with DNA binding sites P3 and P1. This view shows A1 and B2 of each T-ag obd in the major groove of the DNA. All figures of molecules were generated with the molecular graphics program PyMOL [60].

Figure 3. T-ag obd–DNA Interactions

(A) Three close-up views of the protein–DNA interaction observed in the T-ag obd–DNA co-structure. In this figure, the GAGGC duplex is numbered and colored as shown in the boxed sequence. The top view shows both the A1 and B2 loops in the major groove of the DNA. The A1 and B2 loops are shown as sticks colored by atom type (carbon green, nitrogen blue, oxygen red). The DNA is shown as sticks with a translucent molecular surface and is colored as in Figure 2A. Red dashed lines indicate hydrogen bonds or electrostatic interactions. Residues are labeled. The middle and bottom views show the protein–DNA contacts from the A1 loop and the B2 loop, respectively. Atoms from the DNA involved in hydrogen bonds (yellow dashed lines) are shown as spheres: phosphate (red), oxygen (orange), and nitrogen (blue). (B) Schematic representation of protein–DNA interactions in the crystal structure. The DNA is numbered as shown. The GAGGC sequences of P1 and P3 are colored pink and their complements are colored cyan. The mutated P2 sequence is shown with the same coloring but with hash-marks. The P1 and P3 pentamers are placed in a yellow box. Arrows indicate the 5′ → 3′ direction of the GAGGC sequence. The red dotted lines indicate contacts with the phosphate backbone, and the blue solid arrows indicate sequence-specific H-bonds or salt-bridges. Blue dashed lines indicate observed water (W) mediated hydrogen bonds. (The dash-dot-dash line from Arg 202 indicates a phosphate interaction that could easily occur between the guanidinium group and a phosphate but is not observed in the structure.) (C) T-ag obd–DNA interface. The T-ag obd and DNA molecules are separated to show the interaction surface. Residues of T-ag obd which interact with DNA (and vice versa) are shown in green, and the rest of the T-ag obd molecule is magenta. The DNA is colored as in Figure 2A, but the surfaces which interact with T-ag obd are colored green. This exploded view clearly shows the two buried interaction surfaces (one for each T-ag obd) fill the major groove of the pentameric binding sites and that they occur on the same face of the DNA.

Figure 4. Comparison of T-ag obd and BPV E1 obd Co-Structures

(A) The BPV E1 obd (cyan) was superimposed onto the T-ag obd (yellow). The molecules are displayed as ribbon diagrams looking down the helical axis of the DNA. The A1 and B2 loops (and their E1 equivalents) are shown in red and magenta, respectively. The residues that make most of the sequence-specific contacts in T-ag obd (Asn153 and Arg154) and the analogous E1 residues (Lys186 and Thr187) are shown as sticks. Part of the DNA in front of the A1 loop has been omitted from the figure. This view illustrates that even though the protein loops interacting with the DNA superimpose reasonably well, the DNA does not. (B) Comparison of the relative orientation of the SV40 and BPV obds bound to DNA. A model of four SV40 T-ag obds (yellow and cyan) engaged with the four GAGGC sites is shown. (Details of the construction of the model are in the legend for Figure 7.) The four BPV E1 obds (magenta and green) bound to the four E1 binding sites are shown below. The DNA is depicted as a ribbon diagram. The respective binding sites are labeled, and the number of nucleotides between the inverted repeats is shown. The T-ag obds do not interact, whereas the E1 obds form a dimer while bound to the inverted repeat E1–3 and E1–1 or E1–4 and E1–2. The T-ag obds bound to P1 and P2 (or P3 and P4) differ by approximately 180°, whereas the E1 obds bound to E1–1 and E1–2 (or E1–3 and E1–4) differ by approximately 120°. The two views, one looking down the helical axis of the DNA, illustrate the different spatial arrangement of the T-ag and E1 obds when bound to their respective ori sequences.

Figure 5. T-ag obd Disulfide-Linked Dimer Structure

(A) T-ag obd dimer. Ribbon diagram of the T-ag obd dimer an orientation similar to Figure 2A. The A1 (red), B2 (purple), B3 (orange) loops, helix αB (blue), and helix αC (green) are colored as in Figure 2A. Cys216–Cys216 disulfide linkage is shown as yellow van der Waal spheres. (B) A ribbon diagram of a close-up of the dimer interface using the same coloring as in (A). Side chains of residues that participate in the interface are shown. (C) Schematic of dimer interface. The residues (less than 4 Å apart) involved in the dimer interface are shown. The disulfide bond is shown as a yellow line connecting the two Cys residues. The hydrogen bonds are indicated as red dashed lines, and van der Waals (carbon–carbon) interactions are depicted by solid green lines.

Figure 6. Superposition of T-ag obd Monomers

(A) The five nonequivalent crystallographically observed T-ag obd monomers (two from the co-structure, two from the dimer structure, and one from the spiral hexamer structure) are superimposed and displayed in a tube representation. The coloring is as follows: A1 (red), B2 (purple), B3 loop (amino acids 215–220, orange), Cys216 (yellow), and everything else (green). The side chain of Phe151 is shown to indicate the range of motion of A1 loop. The DNA-free (“up”) and DNA-bound (“down”) positions of the A1 loop are indicated. (B) Comparison of T-ag obd co-structure and disulfide-linked dimer structure. A superposition of the T-ag obd dimer onto the T-ag obd co-structure is displayed as a ribbon diagram. Loops A1, B2, and B3, helix αB, and helix αC are colored as above. The rest of the T-ag obd in the co-structure is colored yellow; the rest of the disulfide-linked dimer is gray. One T-ag obd monomer of the disulfide-linked dimer (gray) was superimposed on one T-ag obd monomer (yellow) from the co-structure. The superimposed monomer is shown on the left. The relative orientation of the second monomers differs by 104°.

Figure 7. Modeling Studies of the T-ag obd

A model of four T-ag obds engaged with DNA containing P1 through P4. Starting with the x-ray coordinates of the co-structure of two T-ag obds on P1 and P3, a model was generated of four T-ag obds engaged with the four pentameric binding sites P1–P4. As stated in the text, assembly of the double hexamer of T-ag does not require all four pentanucleotides, although all four are required for unwinding. However, given that the origin contains four pentanucleotides and the structure does not predict any steric clashes when all four are sites are occupied, it is likely that all four are occupied by the obd at some point during assembly. The DNA is colored as Figure 2A. The T-ag obds are shown as van der Waals spheres. The obds that engage with P1 and P2 will presumably comprise one hexamer (yellow). The obds that engage with P3 and P4 will presumably comprise the second hexamer (green). The A1 and B2 loops that engage with the DNA are colored red and purple, as in Figure 2A. The 5′ → 3′ direction of the GAGGC sequences is indicated by arrows and labeled. (Left) In this view, the obds bound to P1 and P3 (or P2 and P4) are oriented head-to head. As stated in the text, obds bound to P1 and P3 (or by extension, P2 and P4) are close but do not contact. This model also shows that the obds on adjacent pentamers (P1 and P2 or P3 and P4) do not interact with each other. (Center) This view is looking down the axis of the DNA and shows clearly that the obds bound to adjacent pentamers occur on opposite faces of the DNA. (Right) This is a view of spiral hexamer of T-ag obd, with the DNA interacting loops colored the same as in the other panels. Duplex DNA is modeled along the central channel of the spiral. The obds that are 180° apart and could have previously interacted with binding sites (eg, P1 and P2) are indicated. This figure illustrates two important points. First, the approximately 30-Å-diameter channel of the spiral hexamer positions the obds farther from the DNA than when engaged site-specifically. Second, the position of the DNA binding loops in red and purple indicates that a significant rotation of the obds must occur to transit from the sequence-specific DNA binding structure to the spiral structure.

Figure 8. Schematic of SV40 T-ag Assembly on Origin DNA

The SV40 origin dsDNA is depicted as two ribbons. The SV40 T-ag N-terminal J domain is omitted for clarity. The SV40 T-ag domains are depicted as follows: the obd (yellow spheres), the helicase domain (yellow ellipsoids), and the flexible linker that connects them (green). (A) The T-ag obd binds its high-affinity GAGGC sites. The T-ag obd anchors the protein on the four GAGGC pentamers and thus orients the helicase domain for appropriate DNA strand selection in the subsequent steps. The obds on P2 and P4 are shown as transparent spheres to indicate that they are not crucial for single-hexamer formation but are required for unwinding. The helicase domains may interact as monomers with the DNA in a non–sequence-specific manner at this point. (B) Once the origin has been recognized by the obds, the two helicase domains each hexamerize around one strand of DNA. As a result, one strand goes through the central channel of the helicase domain, and the other traverses the surface of the helicase domain. This is consistent with the crystal structure of the E1 helicase domain with ssDNA [20], and this model is similar to that proposed for other hexameric helicases [50]. It is not known whether the DNA at site II is melted at this point or not. Twelve obds are now in close proximity and may now interact with one another despite relatively weak affinities. (C) Interaction between the two hexamers could occur through a series of obd–obd structural rearrangements wherein these domains transition from the site-specific complex with the A1 and B2 loops fully engaged with the DNA, to a structure where loop B3 makes contacts across a pair of obds (possibly as spirals). The A1 and B2 loops are now oriented away from the central channel (and are proximal to the helicase domains). The open ring spiral hexamer of the T-ag obd would allow access of ssDNA from the outside surface of the helicase domain to the center of the channel. This channel is positively charged and sufficiently wide (approximately 30 Å) to accommodate two ssDNA strands moving in opposite directions. It is likely that the obds are dynamic and fluctuate between differing states of interaction, including aclosed hexameric ring, depending on the requirement to interact with ssDNA, dsDNA, or other factors, such as the SSB hRPA. The double hexamer is now assembled and ready to recruit other host factors necessary for replication (for a review, see [3]).