New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE.
New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE.
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DOI：
10.1016/j.jbc.2023.105574
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发表时间：
2024-01
影响因子：
4.8
通讯作者：
Gierasch, Lila M
中科院分区：
文献类型：
--
作者：
Rossi, Maria-Agustina;Pozhidaeva, Alexandra K;Clerico, Eugenia M;Petridis, Constantine;Gierasch, Lila M
70 kDa热休克蛋白(Hsp70)通过其核苷酸结合域(NBD)和底物结合域(NBD)之间的变构通讯来调节它们与蛋白质客户的亲和力，从而在许多细胞功能中发挥关键作用。核苷酸交换因子对Hsp70变构循环的调节至关重要，它通过一种保守的机制发挥作用，包括与ADP结合的NBD结合和打开核苷酸结合裂隙以加速ADP的释放和ATP的结合。大肠杆菌Hsp70的NBD-DNAK与其NEF-GRPE之间的复合体的晶体结构已有报道，但该复合体中的GRPE带有点突变(G122D)。该突变对NEF的功能影响及其对NEF的位置导致我们使用AlphaFold建模和报告蛋白质构象和突变的溶液方法验证DNAK NBD/GRPE复合体的结构。这项工作导致了DNAK NBD in与GRPE络合物的新模型，其中NBD的IIB亚域旋转比在晶体结构中更多，导致核苷酸结合裂隙的开放构象，现在更类似于其他HSP/NEF复合体中看到的。此外，新模型与伴随GRPE结合而增加的ADP脱落率是一致的。令人兴奋的是，我们的发现指出了DNAK中由GRPE结合触发的域间变构信号。
The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D). Both the functional impact of this mutation and its location on the NEF led us to revisit the DnaK NBD/GrpE complex structurally using AlphaFold modeling and validation by solution methods that report on protein conformation and mutagenesis. This work resulted in a new model for the DnaK NBD in complex with GrpE in which subdomain IIB of the NBD rotates more than in the crystal structure, resulting in an open conformation of the nucleotide-binding cleft, which now resembles more closely what is seen in other Hsp/NEF complexes. Moreover, the new model is consistent with the increased ADP off-rate accompanying GrpE binding. Excitingly, our findings point to an interdomain allosteric signal in DnaK triggered by GrpE binding.