Greene M K, Maskos K, Landry S J
Department of Biochemistry, Tulane University School of Medicine, New Orleans, LA 70112-2699, USA.
Proc Natl Acad Sci U S A. 1998 May 26;95(11):6108-13. doi: 10.1073/pnas.95.11.6108.
The Escherichia coli Hsp40 DnaJ and Hsp70 DnaK cooperate in the binding of proteins at intermediate stages of folding, assembly, and translocation across membranes. Binding of protein substrates to the DnaK C-terminal domain is controlled by ATP binding and hydrolysis in the N-terminal ATPase domain. The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ that includes residues 2-75. Heteronuclear NMR experiments with uniformly 15N-enriched DnaJ2-75 indicate that the chemical environment of residues located in helix II and the flanking loops is perturbed on interaction with DnaK or a truncated DnaK molecule, DnaK2-388. NMR signals corresponding to these residues broaden and exhibit changes in chemical shifts in the presence of DnaK(MgADP). Addition of MgATP largely reversed the broadening, indicating that NMR signals of DnaJ2-75 respond to ATP-dependent changes in DnaK. The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electrostatic interactions. The results suggest that the J-domain tethers DnaK to DnaJ-bound substrates, which DnaK then binds with its C-terminal peptide-binding domain.
大肠杆菌热休克蛋白40（Hsp40）DnaJ和热休克蛋白70（Hsp70）DnaK在蛋白质折叠、组装和跨膜转运的中间阶段协同作用，负责蛋白质的结合。蛋白质底物与DnaK C末端结构域的结合受N末端ATP酶结构域中ATP结合和水解的控制。DnaJ与DnaK的相互作用至少部分是由DnaJ高度保守的N末端J结构域介导的，该结构域包含2至75位氨基酸残基。对均匀富集15N的DnaJ2 - 75进行的异核核磁共振实验表明，位于螺旋II及其侧翼环中的残基与DnaK或截短的DnaK分子DnaK2 - 388相互作用时，其化学环境受到干扰。在存在DnaK（MgADP）的情况下，与这些残基对应的核磁共振信号变宽并表现出化学位移的变化。添加MgATP在很大程度上逆转了信号变宽，表明DnaJ2 - 75的核磁共振信号对DnaK中依赖ATP的变化有响应。J结构域的相互作用定位于DnaK的ATP酶结构域，并且可能主要由静电相互作用主导。结果表明，J结构域将DnaK与结合了DnaJ的底物连接起来，然后DnaK再用其C末端肽结合结构域与之结合。
