Auto-regulatory J-domain interactions control Hsp70 recruitment to the DnaJB8 chaperone

Abstract

In humans, Hsp40s are classified into A, B, and C sub-families, in which members 90 typically function as monomers, dimers, or polydisperse oligomers. For example, the A 91 family Hsp40s (DnaJA1-A4) and a subset of B family Hsp40s (DnaJB1/B4/B5), all encode 92 a globular domain architecture similar to the bacterial Hsp40 DnaJ. These classical Hsp40 93 members assemble into homo-dimers through conserved C-terminal motifs and bind 94 unfolded substrates through conserved b-barrel C-terminal domains (CTD) 20 . Recent 95 evidence suggests that this subset of Hsp40s can form mixed hetero-dimers driven by 96 electrostatic contacts to expand the substrate recognition repertoire 21 . 97 98 A different set of non-classical Hsp40 B family members (DnaJB2, DnaJB6b, DnaJB7 and 99DnaJB8) encode a domain architecture that is distinct from the classical dimeric DnaJ 100 orthologs. Rather than encoding a classical b-barrel CTD, their C-terminus encodes a 101 different architecture [22][23] . Of these 4 members, the DnaJB8 and DnaJB6b proteins have 102 been shown to assemble into oligomers in vitro and in vivo 22,[24][25] . The role of the CTD in 103 these chaperones remains unclear, and the literature reveals conflicting results, 104 suggesting either that it drives oligomerization or that it mediates intramolecular 105 contacts [22][23][24] 26 . The functional role of the oligomeric state of DnaJB6b and DnaJB8 106 remains unclear, although it has been proposed to serve as a type of phase-separated 107 storage form capable of capturing substrates 25 . The characteristic structural and dynamic 108 heterogeneity of the oligomers has greatly hindered efforts to study them, with recent 109 structural studies resorting to deletion mutants to gain structural insight [22][23] . A 110 combination of variables, including large size and polydispersity, has made these 111 chaperones recalcitrant to both classical and modern structural biology methods, 112 including cryoEM 25 . 113 114 The oligomeric chaperones DnaJB6b and DnaJB8 have been shown to be potent 115 suppressors of aggregation of amyloid proteins in cells and in vitro 22, 26 . Despite several 116 studies demonstrating a role for DnaJB6b in suppressing assembly of amyloid-prone 117 substrates it is unclear which domains directly interact with substrates, and how 118 oligomerization plays a role in this process. For example, the serine/threonine domain of 119 DnaJB6b is important for suppression of mutant huntingtin protein aggregation in cells, 27 120 and its deletion limits oligomerization yielding a stable monomer at the expense of 121 activity 23 . In absence of a comprehensive understanding of the full-length structural 122 behavior for this class of DnaJ chaperones, it remains unclear how they fulfill their critical 123 function to protect the proteome from aggregation. 124 125Here we examine in unprecedented detail the DnaJB8 variant. DnaJB8 was shown to be 126 particularly effective at preventing formation of polyglutamine deposits, even more so than 127 DnaJB6b, despite...