Glutamic acid 446 and Arginine 447 in heat shock protein 70 (hsp70) are critical for regulating superoxide dismutase‐2 (SOD2) function

Background Stress‐inducible hsp70 interacts with SOD2 after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the amino acid residues in hsp70 that are critical for SOD2 interactions remain unknown. Methods and results We map the SOD2‐binding site in hsp70 by making a series of hsp70 mutants (GST‐tagged 1–400, 393–537 and 537–641) and tested for their ability to bind SOD2. The GST‐tagged hsp70 mutants and EGFP‐tagged wild type SOD2 were co‐expressed in HEK293T cells. Using pull down assays, we showed that SOD2 preferentially bound to the hsp70 mutant corresponding to amino acid 393–537 region in hsp70 ( Fig. 1). SOD2 did not bind to the other regions tested. This exclusivity suggests that SOD2 associates with the 393 hsp70 537 domain. To better define the binding site, nine overlapping decoy peptides (p1–p9) were made spanning the entire length of the 393 hsp70 537 region, where SOD2 is suspected to bind to hsp70. The peptides were screened to determine which one could inhibit SOD2/hsp70 interactions. Out of the nine decoy peptides tested, p4 (YSDNOPGVLIQVYEGERAMT: amino acid residues 423–443) and p5 (GERAMTRDNNLLGRFELSGIP; amino acid residues 439–459) significantly disrupted SOD2 association with hsp70 in endothelial cell lysates ( Fig 2A). Since GERAMT is common to both p4 and p5 peptides, we speculated that this sequence may represent the minimal portion responsible for disrupting SOD2 interactions with hsp70. To test this hypothesis, we added the GERAMT peptide alone or GERAMT‐less P4 and P5 peptides (Δp4 and Δp5) individually to aliquots of cell lysates. Colorimetry analysis revealed that GERAMT‐treated lysates had ~3‐fold decrease in SOD2 activity compared to the GERAMT‐less peptides ( Fig 2B). Small peptides containing the GERAMT sequence decreased the activity of SOD2 in vitro and in vivo . To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine (A) in the GERAMT‐binding site in a step‐wise fashion and tested the effects of mutations on SOD2‐hsp70 interactions. Substitutions of alanine for glutamic acid (E446A) and arginine (R447A) inhibited the ability of hsp70 to bind SOD2 and decreased SOD2 activity, more than other substitutions ( Fig 3). Conclusions Together, these findings indicate that the GERAMT sequence is critical for the chaperone‐mediated regulation of SOD2 antioxidant function and that E446 and R447 cooperate with other residues in the GERAMT‐binding site for proper hsp70 chaperone functions. Support or Funding Information Funding supports from Department of Pediatrics and Children's Research institute (CRI) at the Medical College of Wisconsin and 1K08HL133379‐01 (Adeleye Afolayan)