Supplementary Figure 6: Leu146 is critical to the activation of Mtb PtpA by ubiquitin.

(a) Mutation at A140E abolishes the ubiquitin-stimulated phosphatase activity of Mtb PtpA towards p-VPS33B as shown in immunoblot analysis. p-VPS33B was titrated with purified wild-type Mtb PtpA or PtpA A140E mutant protein in the presence of 1.5 μM ubiquitin. (b) Mutation at I44A abolishes the ubiquitin-stimulated phosphatase activity of Mtb PtpA against p-VPS33B as shown in immunoblot analysis. (c) Mutation at A140E abolishes PtpA-mediated inhibition of phagosome acidification. The indicated mycobacterial strains were labeled with pH-sensitive fluorescent dye (pHrodo) and used to infect U937 cells. Phagosomal pH of the infected macrophages was measured with FACS. Wild-type BCG and the complemented strain △PtpA + PtpA maintained a phagosomal pH of 6.2 ~ 6.7, whereas phagosomes of △PtpA, △PtpA + D126A and △PtpA + A140E strains were acidified to pH 4.5 ~ 5.5. *P < 0.05 and **P < 0.01 (significant difference compared with wild-type BCG by two-tailed unpaired t-test). (d) A model for activation of the catalytic activity of Mtb PtpA by ubiquitin. The active site of Mtb PtpA (red and orange) is located between the α helixes 1 and 2, both of which are closely adjacent to α helix 5. Binding of ubiquitin to the UIML region on α helix 5 may thus change the structure of the active site of PtpA, leading to activation of its phosphatase activity. Hydrophobic Leu146, which is sandwiched by two other hydrophobic residues (Phe25 on α helix 1 and Phe113 next to α helix 4), is critical to the activation, probably by bridging α helix 5 with α helix 1 through a hydrophobic interaction. (e) Kinetic analysis of phosphatase activity of Mtb PtpA and its mutants. The small molecule substrate pNPP and the protein substrates p-Jnk, p-p38 and p-VPS33B were used as the substrates, and the released inorganic phosphate was measured by the nonradioactive molybdate dye-based phosphatase assay. The Lineweaver-Burk plots were used to determine the Michaelis-Menten kinetic parameters (k_cat and K_m). Data are representative of one experiment with at least three independent biological replicates (c; mean and s.e.m., n = 3).