Stepwise recruitment of Hsc70 by DNAJB1 produces ordered arrays primed for bursts of amyloid fibre disassembly

To understand the action of co-chaperones of the J-domain protein family in the assembly of disaggregation-active Hsc70 complexes on the surface of amyloid fibres, we used cryo-EM and tomography to compare the assemblies with wild-type co-chaperones or an activity deficient mutant. We show that human DNAJB1 binds uniformly and densely along α-synuclein amyloid fibres in an asymmetric orientation, with one subunit of the DNAJB1 dimer lying along the fibre surface and the other subunit further away. It acts in a 2-step recruitment of Hsc70 to the fibres, first releasing DNAJB1 auto-inhibition and then activating the Hsc70 ATPase by the J domain, with ATPase recycling stimulated by the nucleotide exchange factor Apg2. When the auto-inhibition is removed by mutating the H5 inhibitory binding site on DNAJB1 (Δ1H5 DNAJB1 mutant), Hsc70 is recruited to the fibres at a normal level, but the resulting complex is inactive in disaggregation. Cryo tomography of the wild-type DNAJB1:Hsc70:Apg2:αSyn fibre complex shows dense arrays of the chaperones extending out from the fibre surface along spiral tracks, with the Hsc70 density on the outer surface of the DNAJB1 layer. The Δ1H5 DNAJB1:Hsc70:Apg2:αSyn fibre complex results in equally dense but less organised binding all over the fibre surface and lacks the ordered clusters. On the basis of these findings, we propose that 2-step activation of DNAJB1 regulates Hsc70 access to the fibre substrate. Since the DNAJB1 dimers are bound every 40 Å, the J domain released by Hsc70 binding to one dimer could activate the ATPase of an Hsc70 bound to the adjacent dimer. This could trigger a cascade of recruitment and activation in a localised, dense cloud of Hsc70 molecules to give coordinated, sequential binding and disaggregation from an exposed fibre end, as observed by cryo-EM and earlier fluorescence and AFM studies. ### Competing Interest Statement The authors have declared no competing interest.