Kamath-Loeb A S, Lu C Z, Suh W C, Lonetto M A, Gross C A
Department of Microbiology, University of California, San Francisco 94143, USA.
J Biol Chem. 1995 Dec 15;270(50):30051-9. doi: 10.1074/jbc.270.50.30051.
DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is an ATP-dependent chaperone whose basal ATPase is stimulated by synthetic peptides and its cohort heat shock proteins, DnaJ and GrpE. We have used three mutant DnaK proteins, E171K, D201N, and A174T (corresponding to Glu175, Asp206, and Ala179, respectively, in bovine heat stable cognate 70) to probe the ATPase cycle. All of the mutant proteins exhibit some alteration in basal ATP hydrolysis. However, they all exhibit more severe defects in the regulated activities. D201N and E171K are completely defective in all regulated activities of the protein and also in making the conformational change exhibited by the wt protein upon binding ATP. We suggest that the inability of D201N and E171K to achieve the ATP activated conformation prevents both stimulation by all effectors and the ATP-mediated release of GrpE. In contrast, the defect of A174T is much more specific. It exhibits normal binding and release of GrpE and normal stimulation of ATPase activity by DnaJ. However, it is defective in the synergistic activation of its ATPase by DnaJ and GrpE. We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK necessary for the synergistic action of DnaJ+GrpE.
DnaK是真核生物hsp70蛋白的细菌同源物，是一种依赖ATP的伴侣蛋白，其基础ATP酶受到合成肽及其伴侣热休克蛋白DnaJ和GrpE的刺激。我们使用了三种突变的DnaK蛋白，即E171K、D201N和A174T（分别对应于牛热稳定同源70中的Glu175、Asp206和Ala179）来探究ATP酶循环。所有突变蛋白在基础ATP水解方面都表现出一些改变。然而，它们在调节活性方面都表现出更严重的缺陷。D201N和E171K在该蛋白的所有调节活性方面以及在结合ATP时野生型蛋白所表现出的构象变化方面均完全缺陷。我们认为，D201N和E171K无法实现ATP激活的构象，这既阻止了所有效应物的刺激，也阻止了GrpE的ATP介导释放。相比之下，A174T的缺陷则更为特异。它表现出GrpE的正常结合和释放以及DnaJ对ATP酶活性的正常刺激。然而，它在DnaJ和GrpE对其ATP酶的协同激活方面存在缺陷。我们认为，这种突变蛋白在DnaJ + GrpE协同作用所必需的DnaJ/GrpE介导的DnaK构象变化方面存在特异性缺陷。
