Extended Data Fig. 5: Tsparkin and pre-crystallization biochemistry for human parkin.

a, HDX-MS experiment comparing phospho-Tsparkin reacted with phospho-ubiqutin-C3Br and phospho-Tsparkin reacted with phospho-ubiquitin-C3Br and Ub-VS with identical colouring (blue, more protected from solvent exchange; red, less protected from solvent exchange; grey, not covered in all of the compared states, see Fig. 1). The experiment was performed in technical triplicate. The Tsparkin profile is highly similar to the profile of human parkin in an analogous state (Fig. 1e). Higher peptide resolution in this sample reveals protection of the RING2 interface by reacted Ub-VS, but the C terminus of RING2 that binds to the UPD interface is surface exposed. Both phospho-Ubl and the Ubl–UPD linker are protected in activated parkin. b, Limited proteolysis of Tsparkin with elastase, in different stages of activation. In unphosphorylated, autoinhibited Tsparkin, the Ubl is cleaved off in the Ubl–UPD linker. In activated forms of Tsparkin (phospho-Tsparkin, phospho-Tsparkin reacted with phospho-ubiquitin-C3Br, phospho-Tsparkin reacted with phospho-ubiquitin-C3Br and Ub-VS), the RING2 is readily cleaved off, while the Ubl is not efficiently removed. This suggests that the Ubl–UPD linker is not accessible in activated forms of Tsparkin. A representative gel from three independent experiments is shown. For gel source data, see Supplementary Fig. 1. c, A TEV cleavage site was introduced after the IBR domain, so that after activation by phospho-ubiquitin and Ubl-phosphorylation, the released RING2 domain can be removed. Once removed, RING2 is no longer stably associated with the remaining parkin core. Shown is a gel filtration profile illustrating this point. A representative profile from three independent experiments is shown. d, SDS–PAGE analysis of sample preparation process (see Methods). Asterisk denotes ubiquitin probe (Ub-C3Br)-reacted material that modifies the RING2 catalytic Cys, which explains the cleaved, probe-reacted RING2 band (asterisk in step 3). A representative gel from three independent experiments is shown. For gel source data, see Supplementary Fig. 1. e, HDX-MS experiment on Tsparkin, comparing phospho-Tsparkin reacted with phospho-ubiquitin-C3Br with phospho-Tsparkin reacted with phospho-ubiquitin-C3Br and Ub-VS (bottom) or with RING2-TEV-cleaved phospho-Tsparkin reacted with phospho-ubiquitin-C3Br (top), coloured as in a. Identical profiles were obtained, showing that RING2 removal has no effect on the activated core of parkin. This further indicates that RING2 acts independently of the parkin core upon full activation. Notably, in both comparisons, we observed concomitant protection of phospho-Ubl and the Ubl–UPD linker. The experiment was performed in technical triplicate.