Fig. 2.

Direct binding of AdoCbl to LRRK2 protein. a Binding of strep-tagged LRRK2 to AdoCbl-agarose in the presence of AdoCbl. Input represents the amount of protein that was added to beads, while pull-down denotes the amount of protein left on the beads after washes. Significance was calculated by one-way ANOVA using the mean (±s.d.) of three biological replicates. *p ≤ 0.05, **p ≤ 0.005. b Thermal shift assays showing melting temperatures of strep-LRRK2 in the presence of AdoCbl or PF-06447475. c Microscale thermophoretic analysis of the interaction between AdoCbl or d PF-06447475 with strep-tagged LRRK2. e Coomassie stained SDS-PAGE of the Roco4 kinase domain purified from E. coli. f Dose-response curve of Roco4 kinase activity as a function of AdoCbl. g ATP STD-NMR shows direct binding of AdoCbl to the Roco4 kinase domain and competition with ATP. From top to bottom, the spectra are as follows: 1D 1 H NMR of ATP (blue), AdoCbl (red), STD negative control with ATP + AdoCbl only (green), STD positive control with ATP and Roco4 kinase domain (orange), STD of AdoCbl and Roco4 kinase domain with 1:1 ratio of AdoCbl to ATP (purple), and STD of AdoCbl and Roco4 kinase domain with 10:1 ratio of AdoCbl to ATP (yellow). AdoCbl protons showing strong STD signals are labeled with assignment. All experiments were collected at 4 °C on a Bruker 800 MHz spectrometer equipped with a cryoprobe. h Protons with strong STD signals (highlighted in red) mapped onto the structure of AdoCbl. The NMR assignment and nomenclature of vitamin B12 is from Summers et al.⁹⁴ Data points in a, c, d, f represent the mean (±s.d.) of three biological replicates