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.

Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

“…Apart from the HPD motif, the other amino acids on the J-domain of Hsp40 proteins that are involved in the binding to a partner Hsp70 are less precisely defined. However, as a result of our work (Hennessy, Cheetham, Dirr, & Blatch, 2000; Hennessy, Boshoff, & Blatch, 2005a) and that of other researchers (Garimella et al, 2006; Genevaux, Schwager, Georgopoulos, & Kelley, 2002; Genevaux et al, 2003; Lu & Cyr, 1998; Suh et al, 1999), other residues and regions outside the HPD motif, especially helices II, III and IV, are gradually being implicated in the general binding and specificity of interaction of Hsp40 proteins with Hsp70 proteins.…”

The Four Hydrophobic Residues on the Hsp70 Inter-Domain Linker Have Two Distinct Roles

“…18 It is possible that this J domain is not from a canonical Hsp40, which has been shown to interact differently with Hsp70s. 17,54 More importantly, the inter-domain linker is missing in their structures. Further biochemical and structural studies are needed to clarify this issue.…”

MMP-12 Catalytic Domain Recognizes Triple Helical Peptide Models of Collagen V with Exosites and High Activity

“…To recognize the affinity for ␣1(V)436 -450 THP and sites it affects in 15 N-labeled MMP-12(E219A), the perturbations of amide NMR chemical shifts by THP were monitored. For more accurate mapping of the residues in the interface, ␣1(V) THP protection of MMP-12(E219A) residues from paramagnetic line broadening by inert EDTA-chelated Gd(III) was monitored (47,48,50). …”

“…We applied an NMR footprinting method to map sites of ␣1(V)436 -450 THP protection of MMP-12 from the paramagnetic probe of Gd⅐EDTA. This NMR method has accurately mapped interfaces of protein-protein complexes with affinities ranging from 3 nM (47,48) to about 10 M (49, 50) and has successfully guided mutagenesis to an unexpected "hot spot" in a protein-protein interface (50). NMR mapping identified THP contacts not only around the MMP-12 active site but also at THP-protected exosites on the ␤-sheet, the S-shaped cation-binding III-IV loop, and V-B loop.…”