Shida Meiri, Arakawa Akihiko, Ishii Ryohei, Kishishita Seiichiro, Takagi Tetsuo, Kukimoto-Niino Mutsuko, Sugano Sumio, Tanaka Akiko, Shirouzu Mikako, Yokoyama Shigeyuki
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Acta Crystallogr D Biol Crystallogr. 2010 Mar;66(Pt 3):223-32. doi: 10.1107/S0907444909053979. Epub 2010 Feb 12.
The 70 kDa heat-shock proteins (Hsp70s) are highly conserved chaperones that are involved in several cellular processes, such as protein folding, disaggregation and translocation. In this study, the crystal structures of the human Hsp70 nucleotide-binding domain (NBD) fragment were determined in the nucleotide-free state and in complex with adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP). The structure of the nucleotide-free NBD fragment is similar to that of the AMPPNP-bound NBD fragment and is designated as the ;closed form'. In the nucleotide-free NBD fragment the closed form is intrinsically supported through interactions between Tyr15, Lys56 and Glu268 which connect subdomains IA, IB and IIB at the centre of the protein. Interaction with the substrate-binding domain (SBD) of Hsp70 or the BAG domain of BAG1 impairs this subdomain connection and triggers the rotation of subdomain IIA around a hydrophobic helix from subdomain IA. The subdomain rotation is limited by Asp199 and Asp206 from subdomain IIA and clearly defines the open form of the NBD. The open form is further stabilized by a new interaction between Gly230 from subdomain IIB and Ser340 from subdomain IIA. The structure of the NBD in the nucleotide-free state is determined by switching of the inter-subdomain interactions.
70 kDa热休克蛋白（Hsp70s）是高度保守的分子伴侣，参与多种细胞过程，如蛋白质折叠、解聚和转运。在本研究中，测定了人Hsp70核苷酸结合结构域（NBD）片段在无核苷酸状态下以及与腺苷5'-（β，γ-亚氨基）三磷酸（AMPPNP）结合时的晶体结构。无核苷酸NBD片段的结构与结合AMPPNP的NBD片段相似，被称为“封闭形式”。在无核苷酸NBD片段中，封闭形式通过位于蛋白质中心的亚结构域IA、IB和IIB之间的Tyr15、Lys56和Glu268之间的相互作用内在地得以维持。与Hsp70的底物结合结构域（SBD）或BAG1的BAG结构域相互作用会破坏这种亚结构域连接，并触发亚结构域IIA围绕来自亚结构域IA的疏水螺旋旋转。亚结构域旋转受到来自亚结构域IIA的Asp199和Asp206的限制，并明确界定了NBD的开放形式。开放形式通过亚结构域IIB的Gly230与亚结构域IIA的Ser340之间的新相互作用进一步稳定。无核苷酸状态下NBD的结构由亚结构域间相互作用的转换决定。
