Subunit interactions influence the biochemical and biological properties of Hsp104

Point mutations in either of the two nucleotide-binding domains (NBD) of Hs p104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vit ro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severe ly impair hexamerization. We report that high protein concentrations overco me the assembly defects of NBDZ mutants and increase ATP hydrolysis several fold, changing V-max with little effect on K-m. In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in hibits hexamerization of wildtype (WT) Hsp104, lowering V-max with little e ffect on K-m. ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To f urther analyze the effects of subunit interactions on Hsp104, we assessed t he effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutan t that hexamerizes but does not hydrolyze ATP reduces the ATPase activity o f WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically in hibits the thermotolerance function of WT Hsp104. Thus, interactions betwee n subunits influence the ATPase activity of Hsp104 play a vital role in its biological functions, and provide a mechanism for conditionally inactivati ng Hsp104 function in vivo.