The samples were crystallized after removing the tag with thrombin, which generated a four-residue (Gly–Ser–His–Met) peptide N-terminal to the native Asp5 residue.
Based on the APPL1 BAR-PH crystal structure, we find that, in general, the PH domain is more conserved than the BAR domain, and most of the highly conserved positions are located closer to the BAR−PH interface rather than the central region of the symmetric dimer.We have determined the crystal structure of full-length SV40 ST in complex with the full-length Aα subunit of PP2A. This structure reveals two novel zinc-binding motifs formed by the unique C-terminal domain, the structural linkage of the J and unique domain of ST, and the interaction site of ST with the structural A subunit. Together with our biochemical data, we provide a structural basis for understanding the tumorigenic activity of ST protein.
Crystal structure of the complex was determined by a combination of molecular replacement, using the PP2A A subunit structure as the searching model, and single-wavelength anomalous dispersion of intrinsic zinc atoms in ST, and was refined at 3.1 Å resolution (Table 1).
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.
Structural comparison of the A-ST crystal structure with previously reported crystal structures of A, AC complex, and A-B56-C complexes [25,26,45,46] also support the conclusion that HEAT repeats 2–10 and HEAT repeats 13–15 form two relatively rigid blocks. However, there is substantial structural flexibility between these two structural blocks, due to the result of accumulative conformational changes in HEAT repeats 10–13 (Figures S1 and S2).
Here we report the crystal structure of SV40 ST in complex with the murine PP2A A subunit. It is striking that all four A-ST complexes in the asymmetric unit of our crystal lattice have essentially the same structure, except the flexible HEAT repeats 11–15 of the A subunit that are not involved in the A-ST interaction. This observation argues strongly that the ST structure as well as the A-ST interface observed in our crystal structure are independent of crystal packing and should be physiologically relevant.
In our ST crystal structure, instead of forming a binuclear cluster, these two zinc ions are located in two separate positions and form two novel zinc-binding motifs. The first and second cysteine clusters (residues 111–116, 138–143, respectively) form two zinc-binding motifs together with the conserved Cys103 and His122, respectively. Both zinc ions interact with helix α5 and stabilize the structure of the C-terminal unique domain.
However, the first zinc-binding motif does not interact with the A subunit of PP2A in the crystal structure. Instead, the crystal structure suggests that the first zinc-binding motif may directly interact with the C subunit near its active site, since the first zinc-binding motif is spatially close to the active site of the PP2A C subunit in the structural superposition of PP2A and A-ST complexes (Figure 5).
In addition to the inhibition of the phosphatase activity of the PP2A AC dimer, the J domain may also play a role in the oncogenic activity of ST by providing an additional binding site for Hsp70, even when in complex with the PP2A AC complex, as suggested by our crystal structure.