Subunit interactions influence the biochemical and biological properties of Hsp104

摘要

Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vivo, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization: analogous NBD2 mutations reduce ATPase activity and severely impair hexameriza- tion. We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold. changing V_max.. with little effect on Km. In a comple- mentary fashion, the detergent 3-[(3-cholamidopropyl)dimethyl- ammonio]-1-propanesulfonate inhibits hexamerization of wild- type (WT) Hsp104, lowering V_max.. with little effect on Km. ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but does not hydrolyze ATP re- duces the ATPase activity of WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104. Thus. interactions between subunits influence the ATPase activity of Hsp104, play a vital role in its biological func- tions, and provide a mechanism for conditionally inactivating Hsp104 function in vivo.