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Structural characterization of the human DjC20/HscB cochaperone in solution

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Coto, Amanda Lais de Souza ; Pereira, Arthur Alexandre ; Oliveira, Sabrina Dorta ; Moritz, Milene Nobrega de Oliveira ; da Rocha, Arthur Moraes Franco ; Dores-Silva, Paulo Roberto ; da Silva, Noeli Soares Melo ; Nogueira, Ana Rita de Araujo ; Gava, Lisandra Marques ; Seraphim, Thiago Vagas ; Borges, Julio Cesar
Total Authors: 11
Document type: Journal article
Source: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS; v. 1872, n. 1, p. 12-pg., 2023-10-26.
Abstract

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 cochaperones 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. (AU)

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