Figure 2

Catalytic site of TcPINK1. (A) Recognition of the adenine ring of the bound ATP analogue. The adenine ring-interacting residues and bound ATP analogue are shown as sticks. The colouring scheme is the same as that in Fig. 1A. (B) In vitro kinase assays of TcPINK1 mutants that were designed to compromise the interaction with the adenine ring of the ATP analogue. The Ub phosphorylation (pUb) by wild-type or mutant GST-TcPINK1 was analysed by Phos-tag SDS-PAGE with Coomassie brilliant blue staining. (C) Mg²⁺-mediated interactions with the triphosphate group of the bound ATP analogue. The Mg²⁺-coordinating residues and bound ATP analogue are shown as sticks. The coordinated Mg ions and water molecules are shown as green and red spheres, respectively. The interactions are indicated by dashed lines. The colouring scheme is the same as that in Fig. 1A. (D) In vitro kinase assays of TcPINK1 mutants that were designed to compromise the coordination of the catalytic Mg ions. The Ub phosphorylation (pUb) by wild-type or mutant GST-TcPINK1 was analysed by Phos-tag SDS-PAGE with Coomassie brilliant blue staining. KD; kinase dead. 1/10; one-tenth dose of GST-TcPINK1.