Hsc70 Contacts Helix III of DnaJ‐like Domain from Polyomavirus T Antigens

Abstract

The J domain of large T antigen, Hsc70, and its hydrolysis of ATP were found necessary for polyomavirus infection to release E2Fs from pRb family members. Hsc70's expected binding site on helix II of the DnaJ-like domain of T antigens is blocked in the crystal structure of a T antigen fragment bound to tumor suppressor pRb. We used NMR to find recognition sites for mammalian Hsc70 upon the J domain of murine polyomavirus T antigens (PyJ). The Hsc70 ATPase domain unexpectedly shifts most of the amide NMR peaks of the C-terminal end of helix III of PyJ. The Hsc70 ATPase domain buries the C-terminal end of helix III of PyJ, protecting it from an uncharged paramagnetic probe. This region corresponds to the QKRAA motif of orthodox J domains. Evolutionary trace analysis suggests helix III of J domains to be of wide importance to specificity for hsp70s. The NMR-detected Hsc70 contacts map to a PyJ surface homologous to a solvent-accessible surface of the SV40 T antigen fragment bound to pRb. This surface of helix III and the HPD loop may allow Hsc70 to form a ternary complex with T antigen and pRb, avoiding obstructions to helix II. The helix III docking site is consistent with large T antigen being able to free an E2F from pRb by positioning the substrate-binding domain of Hsc70 for direct contact with the E2F. NMR spectra show the pRb-binding region of E2F1 (aa243–437) to be unfolded in the absence of DP1. Hsc70 is known to bind unfolded proteins. In the absence of DP1, E2F1(aa243–437) acts as a substrate for Hsc70 in vitro, stimulating its ATPase activity in a manner dependent on PyJ from T antigen. If segments of an E2F are unstructured in vivo, it is conceivable that Hsc70, recruited by large T antigen to a complex with pRb, might act on the unstructured portion of the E2F when liberating it from the pRb family member.