HSPA5 forms specific complexes with copper.

Our previous study indicated that Hspa5 directly interacts with copper (Cu) to maintain Cu homeostasis in astrocytes. In this study, we explored the possibility that Cu forms a specific complex with Hspa5 by assaying stoichiometric binding of Cu and other metals to recombinant human HSPA5 (rh-HSPA5) in silico. Spectrophotometric analysis showed that incubation of rh-HSPA5 with Cu but not with Fe, Mn, Zn, or Pb in the presence of ascorbic acid produced an absorbance peak at 470 nm. Furthermore, the absorbance peak was absent when bovine serum albumin was incubated with Cu and when another recombinant protein YWHAZ-14-3-3-Zeta carrying a 6× histidine tag identical to the tag in the rh-HSPA5 was incubated with Cu. The absorbance peak produced by Cu and rh-HSPA5 was abolished by EDTA treatment and was stabilized at pH levels above 6.5. Assay of the stoichiometry of metal binding to the purified rh-HSPA5 showed that one molecule of the rh-HSPA5 could chelate 1 or 2 Cu, 13 iron (Fe), 5 zinc (Zn) and 10 lead (Pb) ions but not manganese (Mn). These data further support our previous finding that HSPA5 specifically forms a complex with Cu to help maintain Cu homeostasis.