Fig 2. Biochemical and structural characterization of the kinase domain of VpsO and its cognate phosphatase VpsU.

(A) Diagram of VpsO-503 and the position of the catalytic lysine residue (K551) and the potential C-terminal tail phosphorylation sites. VpsO kinase activity was evaluated by the Phos-Tag SDS-PAGE migration pattern of VpsO-503 and point mutants; n≥2. VpsO was incubated with 10 mM MgCl₂ in the presence and absence of ATP for 30 minutes at 37°C, prior to the Phos-Tag electrophoresis. P refers to the tyrosine phosphorylated state and U refers to the unphosphorylated state. Coomassie gel staining was used for protein visualization. In this experiment, the purified proteins were prepared such that they were initially unphosphorylated. CytoPlasmic Kinase Domain (CP-KD) (B) The 2.9-Å crystal structure of the VpsO^{E519A, R522A, R525A} kinase domain (cyan) compared to the structure of the Wzc kinase domain (magenta). Residues at the N-terminus of α1 in VpsO likely replace the N-terminal extension in Wzc (pink) in binding adenosine. The ADP is modeled in from the Wzc structure (PDB: 3LA6). (C) Electron density in the active site of VpsO modeled with a phosphotyrosine. The electron density is shown only near the phosphotyrosine from a 2mf_o−Df_c simulated-annealing composite omit map contoured at 1σ. (D) Determination of the catalytic rates of VpsU and VpsU^C12S in the presence of the generic substrate pNPP; n≥4. (E) VpsU crystal structure aligned with the E. coli homolog Wzb (PDB: 2WJA). (F) Phos-Tag SDS-PAGE migration pattern of WT and mutated versions of VpsO-503 proteins with or without prior incubation with VpsU for 30 minutes at 37°C; n≥2. P refers to the phosphorylated state and U refers to the unphosphorylated state. Coomassie gel staining was used for protein visualization. The WT VpsO-503 in this experiment is initially heterogeneously phosphorylated following recombinant expression and purification.