Burkholder W F, Zhao X, Zhu X, Hendrickson W A, Gragerov A, Gottesman M E
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10632-7. doi: 10.1073/pnas.93.20.10632.
Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate.
大肠杆菌DnaK通过其ATP调节的多肽底物结合和释放来发挥分子伴侣的作用。过表达包含多肽底物结合结构域的DnaK的C末端片段（CTF，从 Gly-384 到 Lys-638）在野生型大肠杆菌中是致死的。这种显性负性表型可能是由于CTF与DnaK的细胞多肽靶标发生无效结合所致。通过选择无细胞毒性的CTF突变体，然后进行体外筛选，鉴定出影响DnaK底物结合的突变。这些突变在CTF三维结构中的聚集表明了这样一种模型，即环L1,2和L4,5形成了与底物相互作用至关重要的刚性核心结构。
