Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA.
Immunology Program, Sloan Kettering Institute, New York, NY, USA.
Nat Commun. 2024 Jan 22;15(1):660. doi: 10.1038/s41467-024-44933-9.
The molecular chaperone DnaK is essential for viability of Mycobacterium tuberculosis (Mtb). DnaK hydrolyzes ATP to fold substrates, and the resulting ADP is exchanged for ATP by the nucleotide exchange factor GrpE. It has been unclear how GrpE couples DnaK's nucleotide exchange with substrate release. Here we report a cryo-EM analysis of GrpE bound to an intact Mtb DnaK, revealing an asymmetric 1:2 DnaK-GrpE complex. The GrpE dimer ratchets to modulate both DnaK nucleotide-binding domain and the substrate-binding domain. We further show that the disordered GrpE N-terminus is critical for substrate release, and that the DnaK-GrpE interface is essential for protein folding activity both in vitro and in vivo. Therefore, the Mtb GrpE dimer allosterically regulates DnaK to concomitantly release ADP in the nucleotide-binding domain and substrate peptide in the substrate-binding domain.
分子伴侣 DnaK 对结核分枝杆菌（Mtb）的生存至关重要。DnaK 通过水解 ATP 来折叠底物，然后由核苷酸交换因子 GrpE 将生成的 ADP 交换为 ATP。目前尚不清楚 GrpE 如何将 DnaK 的核苷酸交换与底物释放偶联。在这里，我们报告了与完整的 Mtb DnaK 结合的 GrpE 的冷冻电镜分析，揭示了不对称的 1:2 DnaK-GrpE 复合物。GrpE 二聚体的棘轮运动调节 DnaK 的核苷酸结合结构域和底物结合结构域。我们进一步表明，无规 GrpE N 端对于底物释放至关重要，并且 DnaK-GrpE 界面对于体外和体内的蛋白质折叠活性都是必不可少的。因此，Mtb GrpE 二聚体通过变构调节 DnaK，同时在核苷酸结合结构域中释放 ADP，在底物结合结构域中释放底物肽。
