title: Hsc70 contacts helix III of the J domain from polyomavirus T antigens: addressing a dilemma in the chaperone hypothesis of how they release E2F from pRb.
abstract: Hsc70's expected binding site on helix II of the J domain of T antigens appears to be blocked in its structure bound to tumor suppressor pRb. We used NMR to map where mammalian Hsc70 binds the J domain of murine polyomavirus T antigens (PyJ). The ATPase domain of Hsc70 unexpectedly has its biggest effects on the NMR peak positions of the C-terminal end of helix III of PyJ. The Hsc70 ATPase domain protects the C-terminal end of helix III of PyJ from an uncharged paramagnetic probe of chelated Gd(III), clearly suggesting the interface. Effects on the conserved HPD loop and helix II of PyJ are smaller. The NMR results are supported by a novel assay of Hsc70's ATP hydrolysis showing that mutations of surface residues in PyJ helix III impair PyJ-dependent stimulation of Hsc70 activity. Evolutionary trace analysis of J domains suggests that helix III usually may join helix II in contributing specificities for cognate hsp70s. Our novel evidence implicating helix III differs from evidence that Escherichia coli DnaK primarily affects helix II and the HPD loop of DnaJ. We find the pRb-binding fragment of E2F1 to be intrinsically unfolded and a good substrate for Hsc70 in vitro. This suggests that E2F1 could be a substrate for Hsc70 recruited by T antigen to an Rb family member. Importantly, our results strengthen the chaperone hypothesis for E2F release from an Rb family member by Hsc70 recruited by large T antigen. That is, it now appears that Hsc70 can freely access helix III and the HPD motif of large T antigen bound to an Rb family member.
authors.affiliation: Department of Biochemistry, 117 Schweitzer Hall, University of Missouri, Columbia, Missouri 65211, USA.
authors.forename: Ravindranath
authors.initials: R
authors.lastname: Garimella
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Qiao
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Liang
authors.forename: Erik R P
authors.initials: ER
authors.lastname: Zuiderweg
authors.forename: Michael I
authors.initials: MI
authors.lastname: Riley
authors.forename: Steven R
authors.initials: SR
authors.lastname: Van Doren
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000737:chemistry* / Q000235:genetics; D050687:E2F1 Transcription Factor / Q000737:chemistry* / Q000378:metabolism; D019143:Evolution, Molecular; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D011120:Polyomavirus / Q000276:immunology*; D011487:Protein Conformation; D017510:Protein Folding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016160:Retinoblastoma Protein / Q000737:chemistry* / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000235:genetics
chemical_list: D000957:Antigens, Viral, Tumor; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D050883:HSC70 Heat-Shock Proteins; D018832:Molecular Chaperones; D016160:Retinoblastoma Protein; D013912:Threonine; D000251:Adenosine Triphosphatases; D008239:Lysine