Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, 113-0032, Japan.
Synchrotron Radiation Research Organization, The University of Tokyo, Tokyo, 113-0032, Japan.
Sci Rep. 2018 Jul 10;8(1):10382. doi: 10.1038/s41598-018-28656-8.
Mutations of PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin (Ub) ligase parkin can cause familial parkinsonism. These two proteins are essential for ubiquitylation of damaged mitochondria and subsequent degradation. PINK1 phosphorylates Ser65 of Ub and the Ub-like (UBL) domain of parkin to allosterically relieve the autoinhibition of parkin. To understand the structural mechanism of the Ub/UBL-specific phosphorylation by PINK1, we determined the crystal structure of Tribolium castaneum PINK1 kinase domain (TcPINK1) in complex with a nonhydrolyzable ATP analogue at 2.5 Å resolution. TcPINK1 consists of the N- and C-terminal lobes with the PINK1-specific extension. The ATP analogue is bound in the cleft between the N- and C-terminal lobes. The adenine ring of the ATP analogue is bound to a hydrophobic pocket, whereas the triphosphate group of the ATP analogue and two coordinated Mg ions interact with the catalytic hydrophilic residues. Comparison with protein kinases A and C (PKA and PKC, respectively) unveils a putative Ub/UBL-binding groove, which is wider than the peptide-binding groove of PKA or PKC to accommodate the globular head of Ub or UBL. Further crosslinking analyses suggested a PINK1-interacting surface of Ub. Structure-guided mutational analyses support the findings from the present structural analysis of PINK1.
PTEN 诱导的假定激酶 1（PINK1）和 E3 泛素（Ub）连接酶 parkin 的突变可导致家族性帕金森病。这两种蛋白对于损伤线粒体的泛素化和随后的降解至关重要。PINK1 磷酸化 Ub 的 Ser65 和 parkin 的 Ub 样（UBL）结构域，以变构方式解除 parkin 的自身抑制。为了理解 PINK1 对 Ub/UBL 的特异性磷酸化的结构机制，我们以 2.5Å 的分辨率确定了 Tribolium castaneum PINK1 激酶结构域（TcPINK1）与非水解型 ATP 类似物复合物的晶体结构。TcPINK1 由 N-和 C-末端结构域以及 PINK1 特异性延伸组成。ATP 类似物结合在 N-和 C-末端结构域之间的裂隙中。ATP 类似物的腺嘌呤环结合在一个疏水性口袋中，而 ATP 类似物的三磷酸基团和两个配位的 Mg 离子与催化亲水残基相互作用。与蛋白激酶 A（PKA）和蛋白激酶 C（PKC）的比较揭示了一个假定的 Ub/UBL 结合槽，它比 PKA 或 PKC 的肽结合槽更宽，以容纳 Ub 或 UBL 的球状头部。进一步的交联分析表明了 Ub 的 PINK1 相互作用表面。结构指导的突变分析支持了本研究中 PINK1 结构分析的结果。
