Hattendorf Douglas A, Lindquist Susan L
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2732-7. doi: 10.1073/pnas.261693199. Epub 2002 Feb 26.
Hsp104 from Saccharomyces cerevisiae is a hexameric protein with two AAA ATPase domains (N- and C-terminal nucleotide-binding domains NBD1 and NBD2, respectively) per monomer. Our previous analysis of the Hsp104 ATP hydrolysis cycle revealed that NBD1 and NBD2 have very different catalytic properties, but each shows positive cooperativity in hydrolysis. There is also communication between the two domains, in that ATP hydrolysis at NBD1 depends on the nucleotide that is bound to NBD2. Here, we extend our understanding of the Hsp104 ATP hydrolysis cycle through mutagenesis of the AAA sensor-2 motif in NBD2. To do so, we took advantage of the lack of tryptophan residues in Hsp104 to place a single tryptophan in the C-terminal domain (Y819W). The Y819W substitution has no significant effects on folding stability of the C-terminal domain or on ATP hydrolysis by NBD1 or NBD2. The fluorescence of this tryptophan changes in response to ATP and ADP binding, allowing the K(d) and Hill coefficient to be determined for each nucleotide. By using this site-specific probe of binding, we analyze the effect of mutating the conserved arginine residue in the sensor-2 motif in Hsp104 NBD2. An R826M mutation causes nearly equal decreases in affinity of NBD2 for both ATP and ADP, indicating that at this site, the sensor-2 provides binding energy, but does not act to sense the difference between these nucleotides. In addition, the rate of ATP hydrolysis at NBD1 is decreased by the R826M mutation, providing further evidence for interdomain communication in the Hsp104 ATP hydrolysis cycle.
来自酿酒酵母的Hsp104是一种六聚体蛋白，每个单体含有两个AAA型ATP酶结构域（分别为N端和C端核苷酸结合结构域NBD1和NBD2）。我们之前对Hsp104 ATP水解循环的分析表明，NBD1和NBD2具有非常不同的催化特性，但每个结构域在水解过程中都表现出正协同性。两个结构域之间也存在通讯，因为NBD1处的ATP水解取决于与NBD2结合的核苷酸。在这里，我们通过对NBD2中AAA传感器-2基序进行诱变，扩展了对Hsp104 ATP水解循环的理解。为此，我们利用Hsp104中缺乏色氨酸残基的特点，在C端结构域中引入了一个单一的色氨酸（Y819W）。Y819W取代对C端结构域的折叠稳定性或NBD1或NBD2的ATP水解没有显著影响。该色氨酸的荧光会随着ATP和ADP的结合而变化，从而可以确定每种核苷酸的解离常数（K(d)）和希尔系数。通过使用这种位点特异性的结合探针，我们分析了Hsp104 NBD2中传感器-2基序中保守精氨酸残基突变的影响。R826M突变导致NBD2对ATP和ADP的亲和力几乎同等程度降低，这表明在这个位点，传感器-2提供结合能量，但并不负责感知这些核苷酸之间的差异。此外，R826M突变降低了NBD1处的ATP水解速率，为Hsp104 ATP水解循环中的结构域间通讯提供了进一步的证据。
