Extended Data Fig. 10: The GR_pre-Helix1 strand is engaged with the client-loading Hsp70 (Hsp70C) in the GR-loading complex.

a, The photoreactive cross-linker (pBpa) was placed at two positions in the GR_{pre-Helix 1} strand, residues before (GR^P517, sphere presentation) and after (GR^Q527, sphere representation) the predicted Hsp70-binding site (GR^519–526, stick representation). b, c, Whole fractions of GR-loading complex eluted from the size-exclusion column were exposed to UV using a gel imager (see also Methods). As expected, at both positions, cross-links between GR and Hsp70 were formed in the GR-loading complex, indicated by high molecular weight species. Left, SDS–PAGE stained with Coomassie blue. Middle, anti-Hsp70 Western blot. Right, anti-Hop Western blot. Data from (b, c) are from one experiment. Hsp70 cross-linking efficiency was higher for the GR^pBpa517position (b, middle) than for the GR^pBpa527 position (c, middle). It is likely because 1) the C-terminal end of the Hsp70-bound substrate tends to be flexible in the reverse binding mode, as indicated by high atomic B-factors and missing density from previously determined Hsp70 crystal structures with a reverse peptide bound (PDB ID: 4EZZ, 4EZQ, 4EZT, and 4EZY)⁶⁰, and 2) GR⁵²⁷ is closer to Hop⁵⁴³ than Hsp70. In addition, the two positions were able to cross-link with Hop (b, c, right), indicating it is the client-loading Hsp70 that the GR_{pre-Helix 1} strand cross-linked with, rather than the scaffolding Hsp70. Note that although the GR⁵¹⁷position is not adjacent to Hop in the GR-loading complex model, we reasoned that the cross-link may be formed in the one-Hsp70 loading complex (b, right). These results demonstrate that it is the GR_{pre-Helix 1} strand bound to Hsp70C, supporting our structural model. d, e, Raw western blots from middle and right panels in b, c. Red pixels in the western blots shown indicate overexposure.