Structural basis of PP2A inhibition by small t antigen

Abstract

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 A resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3-7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST.

Figure 1. Overall Structure of SV40 ST in Complex with the A Subunit of PP2A

A cartoon illustration of the “front” and “top” views of the PP2A A subunit–SV40 ST complex. The scaffold Aα subunit of PP2A, the J domain, and the unique domain of SV40 ST are colored blue, green, and magenta, respectively. In addition, two zinc ions in the ST unique domain are yellow. ST interacts with the intrarepeat loops of HEAT repeats 3–7 of PP2A A subunit.

Figure 2. Structure of SV40 Small t Antigen

(A) Structural organization of SV40 ST and the zinc coordination of ST unique domain. The peptide chain is color-coded from blue to red, going through the rainbow colors, from the N terminus (blue) to the C terminus (red). (B) Interface of the J and unique domains. The J and unique domains are in green and pink, respectively. Key residues in the interface between the J domain and the unique domain—Trp24, Gly25, Pro28, His70, Trp135, Tyr139, Trp147, and Ile163—are labeled. (C) Sequence alignment of ST proteins from SV40 (strain VA45-54-2), baboon polyomavirus (PyV), BK polyomavirus (BKV), and JC polyomavirus (JCV). Sequence conservation is indicated below the aligned sequences. Secondary structures in the determined crystal structure are indicated above the aligned sequences. Solid and empty stars indicate residues interacting with PP2A A subunit using side-chain and main-chain, respectively. Solid and empty squares represent residues involved in interactions between the J domain and the unique domain with side-chain and main-chain atoms, respectively.

Figure 3. The Interface between SV40 ST and PP2A A Subunit

Color assignment for different subunit or domains is the same as in Figure 1. Hydrogen bonds between ST and the PP2A A subunit are indicated by dashed lines.

Figure 4. Structural Comparison of the A-ST Complex and the A-B56-C PP2A Holoenzyme

(A) Structural superposition of these two complexes. These two complexes are superimposed using A subunit HEAT repeats 2–10. The J and unique domain of ST are colored green and pink, respectively. The Cα trace of B56γ1 are shown in light orange. It is clear that ST and B56γ1 bind to the same sites on PP2A A subunit. (B) The PP2A A subunit residues involved in both ST and B56γ1 interactions. ST and B56γ1 share a common footprint on the ridge of A subunit.

Figure 5. The First Zinc-Binding Motif May Directly Interact with and Inhibit the Catalytic C Subunit of PP2A

The structures of the A-ST and A-B56-C complexes are superimposed. The PP2A catalytic subunit is shown in the surface model. The active site of the PP2A C subunit is indicated. The potential binding site of Hsp70 is represented by a gray sphere.

Figure 6. Structural Comparison of the PP2A A Subunit and the Structural Flexibility

(A) The structural alignment of PP2A A subunits. PP2A A subunit structures were aligned based on their structures of HEAT repeats 2–10. PP2A A subunit structures that used for the alignment are from the four A-ST complexes in the asymmetric unit, the A subunit structure alone (PDB code: 1B3U), the AC dimer structure (PDB code: 2IE3), and the A-B56-C trimeric structure (PDB code: 2IAE). The HEAT repeats 2–10 may form a rigid structural block since no significant structural variations were observed for this region among all A subunit structures. (B) The amplitude of conformational variations of the PP2A A subunit HEAT repeats 11–15. (C) HEAT repeats 13–15 may form the other relatively rigid structural block in the PP2A A subunit. There is no major conformational variation in HEAT repeats 13–15 among all A subunit structures. Therefore, the structural variations observed in (A) and (B) are mostly due to the conformational flexibility of HEAT repeats 10–13. (D) Superposition of PP2A A subunit structures from Aα alone, A-ST complex, and A-B56-C trimeric complex. The structure of the PP2A A subunit in the A-ST complex can have a conformation very similar with that of the A subunit in the A-B56-C trimeric complex.

Figure 7. Mutational Analysis of the A-ST Interface

(A) Mutation of ST residues predicted to interact with PP2A Aα. Top: Expression of ST in whole cell lysates (WCL). Middle: Isolation of PP2A Aα complexes and immunoblotting with anti-PP2A Aα antibodies. Bottom: Isolation of PP2A Aα complexes and immunoblotting for ST. (B) Mutation of PP2A Aα residues predicted to interact with ST. Top: Isolation of PP2A Aα complexes and immunoblotting with anti-PP2A Aα. Bottom: Isolation of PP2A Aα complexes and immunoblotting for ST.