Biochemical and structural studies on the high affinity of Hsp70 for ADP
Biochemical and structural studies on the high affinity of Hsp70 for ADP
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DOI：
10.1002/pro.663
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发表时间：
2011-08-01
期刊：
影响因子：
8
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
--
作者：
Arakawa, Akihiko;Handa, Noriko;Yokoyama, Shigeyuki
分子伴侣 70-kDa 热休克蛋白 (Hsp70) 由 ATP 水解和 ADP-ATP 交换驱动。据报道，在无机磷酸盐 (P-i) 存在的情况下，ADP 从 Hsp70 解离缓慢。在本研究中，我们通过等温滴定量热法测量详细研究了 Hsp70 及其核苷酸结合域 (NBD) 与 ADP 的相互作用，发现 Mg2+ 离子显着提高了 Hsp70 对 ADP 的亲和力。另一方面，P-i 在 Mg2+ 离子存在下增加了亲和力，但在不存在 Mg2+ 离子时则没有增加。因此，P-i 增强了 Mg2+ 离子对 ADP 结合的影响。接下来，我们确定了含有和不含 Mg2+ 离子的 ADP 结合 NBD 的晶体结构。与无Mg2+离子结构相比，ADP-和Mg2+离子结合的NBD含有一个Mg2+离子，它与ADP的β-磷酸基团配位，并通过四个水分子与Asp10、Glu175和Asp199结合。 Mg2+ 离子还与一个 P-i 分子配位，该分子与 Lys71、Glu175 和 Thr204 相互作用。事实上，无论 P-i 存在还是不存在，Asp10 和 Asp199 的突变都会降低 NBD 对 ADP 的亲和力。因此，Mg2+离子介导的网络，包括P-i和水分子，增加了Hsp70对ADP的亲和力，因此ADP的解离缓慢。在 ADP-ATP 交换中，缓慢的 ADP 解离可能会限制速率。然而，核苷酸交换因子实际上通过破坏 Mg2+ 离子介导的网络来增强 ADP 的释放。
The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P-i). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg2+ ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P-i increased the affinity in the presence of Mg2+ ion, but not in its absence. Thus, P-i enhances the effect of the Mg2+ ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg2+ ion. As compared with the Mg2+ ion-free structure, the ADP- and Mg2+ ion-bound NBD contains one Mg2+ ion, which is coordinated with the beta-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg2+ ion is also coordinated with one P-i molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P-i. Therefore, the Mg2+ ion-mediated network, including the P-i and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg2+ ion-mediated network.