Fig. 2: Model for the JD–CTD contacts in a DnaJB8 monomer.

a XL–MS-based refinement of full-length expanded DnaJB8 monomer. Cartoon representation of DnaJB8 in fully expanded conformation (left) and collapsed conformation (right), colored by domain as in Fig. 1. Aromatic amino acids in the G/F and S/T domains are shown as spheres and colored according to the domain. Residues in helix 5 (H5) are shown as magenta spheres. Collapsed conformation model was selected from 1000 Rosetta ab initio generated models using a relax protocol. R_g and R_h values were calculated from the structural model in Rosetta and HYDROPRO, respectively. b Charge complementary surfaces on the JD and CTD mediate the interaction. Highly acidic potential is shown in red (− sign) and highly basic in blue (+ sign). c Net charge per residue (NCPR) distribution, defined as the average charge over a 10-residue window, highlights charge complementarity between basic and acidic residues on the JD and CTD, respectively (coloring as in Fig. 1). Helices in the JD with basic character are denoted as H2, H3, and H4. d GREMLIN sequence-based covariance analysis identified high confidence covarying amino acids on DnaJB8 that localize within the JD (red), within CTD (green), with H5 (brown), and across JD–CTD (gray). XL–MS links for full-length DnaJB8 (black dots) overlap with the covarying regions. Covarying positions localizing to amino acids in G/F domain are shown in brown and co-localize with XL–MS cross-links. e Domain map of the DnaJB8_F→S mutant, with mutated phenylalanine positions marked by cyan ticks. f DLS time course of the DnaJB8_F→S mutant. The average R_h was calculated to be 3.53 ± 0.05 nm. g SEC-MALS of CTD_170–232 shows a single peak that was calculated to have a molar mass of 24,530 ± 30 g/mol consistent with a monomer. h XL–MS contact map showing ADH/DMTMM cross-links for WT DnaJB8 and DnaJB8_F→S mutant. The axes are colored in red and green for JD and CTD, respectively. Cross-link pairs between JD–CTD, JD–JD, and CTD–CTD are shown in a dashed box colored gray, red, and green, respectively. Contacts to helix 5 in WT DnaJB8 are denoted with H5. i Schematic of DnaJB8 species observed in solution based on DLS dimensions. Domains are shown as JD (red spheres), CTD (green spheres), and G/F + S/T (light blue spheres). The average R_h of DnaJB8_F→S (3.53 ± 0.05 nm) and DnaJB8 ab initio Rosetta model (4.02 nm) are assigned to the monomer. The R_h of WT DnaJB8 begins as a 4.28-nm species and grows to 5.77 nm over 20 h. Size and volume estimates from the structural models suggest DnaJB8 exists as small species ranging from a monomer to octamer likely dominated by a dimer and over time maturing into large oligomers.