Fig. 4: Detection of distinct HSPA8 conformations by residue labeling approaches.

a Ribbon structure of HSPA8 (models built with PDB 4H5R⁶¹, 3AGY²⁹ and 4KBQ⁶²). Nucleotide binding domain (NBD; ruby), substrate binding domains (SBDβ green, SBDα dark-gray) and cofactor binding motif (EEVD motif; teal) are shown on protein backbones. b Ribbon structure of DNAJB1 (model built with PDB 3AGZ²⁹ and 1HDJ⁶³), with dimer composed of two monomers colored dark and light gray respectively. J-domain (salmon) and HSPA8 binding region (teal) are shown on protein backbones, and C-terminal domains (CTDI and CTDII) are indicated by brackets. c Ribbon structure of MDH2 (PDB 1MLD⁶⁴). Dotted lines represent sequence regions with missing structural information d Changes in thiol reactivity of HSPA8 and DNAJB1 peptides in Neuro2a lysate titrated with urea. e Schematic for recombinant client-binding assay. Human HSPA8 and DNAJB1 were incubated with native (above line) or heat-denatured (below line) client, MDH2. Nucleotide binding domain (NBD; ruby), substrate binding domains (SBDβ; green and SBDα; gray) and cofactor interaction region (IR; teal) are shown on protein backbones. In the case of DNAJB1, dimer is shown with second monomer desaturated. After incubation, samples were labeled with TPE-MI then prepared for mass spectrometry. Finally, the corrected cys ratio for each of the HSPA8 and DNAJB1 was compared between samples containing native and denatured client, where the corrected cys ratio will increase for those residues that become less reactive. f Change in cysteine reactivity of peptides derived from native human HSPA8 and DNAJB1 when incubated with heat-denatured MDH2, in the absence or presence of exogenous ATP prior to TPE-MI labeling. Means ± S.D. (n = 4 biological replicates) are shown, and deviations from the expected mean of 1 were tested using a one-sample t-test (* denotes p < 0.05, ns denotes p > 0.05). Exact p values are provided in Supplementary Data 1. In all panels, cysteine residues are labeled and colored according to the cluster their respective peptides were assigned (orange, red, purple and blue correspond to clusters 1–4 respectively, black was not observed). Source data are provided as a Source Data file.