Schirmer E C, Ware D M, Queitsch C, Kowal A S, Lindquist S L
Department of Molecular Genetics and Cell Biology and Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2001 Jan 30;98(3):914-9. doi: 10.1073/pnas.98.3.914. Epub 2001 Jan 23.
Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization. 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 K(m). In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, lowering V(max) with little effect on K(m). 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 reduces 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 functions, and provide a mechanism for conditionally inactivating Hsp104 function in vivo.
热休克蛋白104（Hsp104）的两个核苷酸结合结构域（NBD1和NBD2）中任何一个发生点突变，都会在体内消除其耐热功能。在体外，NBD1突变几乎完全消除ATP水解，对六聚体形成影响很小；类似的NBD2突变会降低ATP酶活性并严重损害六聚体形成。我们报道，高蛋白浓度可克服NBD2突变体的组装缺陷，并使ATP水解增加几倍，改变V(max)而对K(m)影响很小。以互补的方式，去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐抑制野生型（WT）Hsp104的六聚体形成，降低V(max)而对K(m)影响很小。ATP水解的希尔系数在1.5至2之间，表明它受亚基协同相互作用的影响。为了进一步分析亚基相互作用对Hsp104的影响，我们评估了突变型Hsp104蛋白对WT Hsp104活性的影响。一种能形成六聚体但不水解ATP的NBD1突变体，在体外可降低WT Hsp104的ATP酶活性。在体内，这种突变体无毒，但能特异性抑制WT Hsp104的耐热功能。因此，亚基之间的相互作用影响Hsp104的ATP酶活性，在其生物学功能中起重要作用，并为在体内有条件地使Hsp104功能失活提供了一种机制。
