Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone
Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone
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DOI：
10.1074/jbc.272.44.27796
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发表时间：
1997-10-31
影响因子：
4.8
通讯作者：
Wilbanks, SM
中科院分区：
文献类型：
--
作者：
Ha, JH;Hellman, U;Wilbanks, SM
我们比较了从牛脑中分离的70 kda热休克同源蛋白（Hsc70）与重组野生型蛋白和突变型E543K蛋白（之前在我们的实验室中作为野生型研究），野生型牛和重组蛋白的不同之处是赖氨酸561的翻译后修饰，但与短肽（荧光素标记的FYQLALT）和变性葡萄球菌核酸酶-(Delta 135-149)的相互作用相似。突变E543K导致肽释放速度加快4.5倍，与葡萄球菌核酸酶-复合物的稳定性降低（Delta 135-149）。野生型蛋白的ATP水解率通过添加肽提高了6-10倍。E543K突变体的肽刺激水解率与野生型相似，但未刺激的水解率更高，仅提高了2倍。三种版本的Hsc70都具有相似的atp依赖性构象移位，并且都表现出钾离子依赖性。这些数据支持以下模型：(i)在K+、Mg2+和ATP存在的情况下，肽结合域抑制ATP酶；（ii）肽的结合减轻了这种抑制；（iii） E543K突变显著减弱了肽结合域的抑制作用，破坏了hsc70肽复合物的稳定性。
We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory), Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta 135-149). Mutation E543K results in 4.5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta 135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.