Heat-shock proteins of 70 kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70_R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70_S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure.

70 kDa (Hsp70s) 的热休克蛋白对所有生命都至关重要，并且在蛋白质折叠中尤为重要。Hsp70s 在核苷酸结合域 (NBD) 使用 ATP 结合和水解来控制客户多肽在底物结合域 (SBD) 的结合和释放；然而，这种变构的机械基础一直难以捉摸。在这里，我们首先表征细菌 Hsp70 DnaK 中选定的域界面突变体的生化特性。然后，我们开发了 Hsp70 构象状态之间变构平衡的理论模型来解释观察结果：抑制状态 Hsp70 _R -ATP 限制 ATP 水解并且与肽的结合很差，而刺激状态 Hsp70 _S-ATP，快速水解 ATP，具有高内在底物亲和力，但结合动力学迅速。我们支持这种变构调节模型，即在假定的刺激状态 S 中获得的 DnaK 结构，通过 S 状态界面的生化测试，以及在 R 状态结构中改进肽结合位点定义。