A unique chaperoning mechanism in Class A JDPs recognizes and stabilizes mutant p53

Abstract

SUMMARYJ-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here we uncover a novel mechanism by which chaperones recognize misfolded clients, present in class-A JDPs. Through a newly-identified β-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class-A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the β-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class-A JDP β-hairpin as a highly specific target for cancer therapeutics.

Mechanism of chaperone coordination during cotranslational protein folding in bacteria

“…NCs likely expose a combination of unfolded, hydrophobic segments and near-native structures, and our data suggest that DnaJ binds NCs via a surprisingly large surface is not exclusively hydrophobic (Fig 7H). This is consistent with the ability of DnaJ to recognise substrates in different folding states 71,76 , (Fig 6) and the fact that Hsp40s generally bind intact proteins with much higher affinity than isolated peptides 64,77 . The binding mode of DnaJ to NCs is strikingly similar to that of TF, also an ATP-independent chaperone, potentially providing a molecular explanation for the well-established functional redundancy of TF and DnaJ/K 11,15,16 .…”

“…We used HDX-MS as a complementary approach to map NC binding sites on DnaJ (Fig 6D and S6D, Table S5). Binding to RNC 1-646 induced strong protection (>1 Da) in the G/F-rich region, ZBD and CTD I, all previously implicated in substrate binding 71–76 . Weaker protection (0.5-1 Da) was observed in the JD, including the region containing the second most crosslinked residue K 62 , supporting its involvement in NC binding.…”

“…From N-to C-terminus, DnaJ consists of a J domain (JD) which stimulates ATP hydrolysis by DnaK, G/F-rich region (G/F), zinc binding domain (ZBD), two C-terminal β-sandwich domains (CTD I and II), and a dimerization domain (DD) (Fig 6A). Several Hsp40s have been shown to bind constitutively unfolded proteins via CTD I and II 71 , although other domains have also been implicated in client recognition 72–76 . It remains unclear how Hsp40s bind folding intermediates 77 .…”

“…Use of this alternative interaction site could be dictated by the overall topology of the binding surface, and might reflect differences in binding modes between constitutively unfolded proteins/peptides and compact folding intermediates. Recent work showed that peptides derived from p53 bind the equivalent site in yeast DnaJA2 (Ydj1), suggesting that site B is a conserved peptide-binding interface among class A J-proteins 76 .…”

The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations

“…DNAJA2 was previously shown to help target mutant CFTR for degradation at the plasma membrane, with the CHIP E3 ligase (Kim Chiaw et al, 2019). A recent study also showed that DNAJA2 can stabilize mutant p53, which normally resides in the nucleus, and slow its degradation via sequestration of the mutant (Zoltsman et al, 2023). Importantly, the role of DNAJA2 in the degradation of misfolded cytosolic proteins more broadly has not been fully investigated.…”