Subunit interactions influence the biochemical and biological properties of Hsp104.
Subunit interactions influence the biochemical and biological properties of Hsp104.
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亚基相互作用影响 Hsp104 的生化和生物学特性。
DOI：
10.1073/pnas.98.3.914
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发表时间：
2001
影响因子：
11.1
通讯作者：
Lindquist,SL
中科院分区：
文献类型：
--
作者：
Schirmer,EC;Ware,DM;Queitsch,C;Kowal,AS;Lindquist,SL
Hsp 104的两个核苷酸结合结构域（NBD）（NBD 1和NBD 2）的点突变在体内消除了其耐热性功能，在体外，NBD 1突变几乎消除了ATP水解，对六聚化作用影响不大;类似的NBD 2突变降低了ATP酶活性，严重损害六聚化作用。我们报道了高蛋白浓度克服了NBD 2突变体的组装缺陷，并使ATP水解增加了几倍，改变了Vmax，但对Km的影响很小。以互补的方式，去污剂3-[（3-胆酰胺丙基）二甲基铵基]-1-丙磺酸盐抑制野生型（WT）Hsp 104的六聚化，降低Vmax而对Km几乎没有影响。ATP水解表现出希尔系数在1.5和2之间，表明它是由合作的亚基相互作用的影响。为了进一步分析亚基相互作用对Hsp 104的影响，我们评估了突变型Hsp 104蛋白对WT Hsp 104活性的影响。NBD 1突变体在体外可使WT Hsp 104的ATP酶活性降低，在体内对WT Hsp 104无毒性，但特异性抑制WT Hsp 104的耐热功能。因此，亚基间的相互作用影响Hsp 104的ATP酶活性，在其生物学功能中起着至关重要的作用，并为Hsp 104在体内的功能提供了条件失活机制。
Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance functionin 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, changingVmaxwith little effect onKm. In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, loweringVmaxwith little effect onKm. 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 Hsp104in 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 functionin vivo.