Structural characterization of the human DjC20/HscB cochaperone in solution

• DOI: 10.1016/j.bbapap.2023.140970
• Publication Date: 2023-10-21T15:28:47Z
• Authors: Amanda Lais de So...; Arthur Alexandre ...; Sabrina Dorta Oli...; Milene Nobrega de...; Arthur Moraes Fra...; Paulo Roberto Dor...; Noeli Soares Melo...; Ana Rita de Araúj...; Lisandra Marques ...; Thiago Vagas Sera...; Júlio César Borges
• Keywords: Adenosine Triphosphatases; Humans; HSP70 Heat-Shock Proteins; Heat-Shock Proteins; Edetic Acid; Molecular Chaperones

J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.