Supplementary Figure 5: YopO uses actin as bait for phosphorylation.
(a) Model of the ternary complex of YopO:actin:gelsolin (G4–G6), following the color scheme in Fig. 1a, with gelsolin in green (PDB code 1H1V). Note that gelsolin comprises six domains, two of which bind G-actin similarly, G1 and G4. CapG consists of three gelsolin domains. Models of YopO–actin–G1–3 (Fig. 4b) and YopO–actin–G4–6 represent potential models of the YopO–actin–CapG structure.
(b) Model of the ternary complex of YopO–actin–ADFH, with the ADFH of twinfilin in magenta (PDB code 3DAW). Note that this represents a model of the YopO–actin–cofilin structure. Full length twinfilin contains a second ADFH domain. CapG consists of three gelsolin domains and Twf1, of two ADF-H domains. Both CapG and Twf1 are not sufficiently elongated to span from the actin-binding site to the kinase catalytic cleft in order to be phosphorylated by YopO–actin.
(c) In vitro phosphorylation assay of YopO and mutants. YopO WT or mutants (4.7 μM) were incubated with or without Sf9 actin (4.7 μM), VASP as substrate (14.1 μM) or profilin (14.1 μM) as indicated, supplemented with 5μCi [γ-32P]ATP. Lane 1: YopO does not phosphorylate itself in the absence of actin. Lane 2: YopO autophosphorylates in the presence of actin. Lane 3: VASP is phosphorylated in the presence of actin and YopO; which is not impeded by the presence of profilin (lane 4). Lane 5: Kinase-dead (KD) YopO does not phosphorylate itself or VASP, and neither does nAct the reduced actin binding YopO mutant (lane 6). Lane 7: The non-Rac binding YopO mutant (nRac) phosphorylates itself and VASP.
(d) SDS-PAGE analysis of the proteins used in the in vitro phosphorylation assay in Fig. 5. YopO, VASP, EVL, gelsolin, CapG, twinfilin1 are without fusion tags. mDia1 (583–1262) and WASP (150–502) each contain both an N-terminal GST and a C-terminal His6 tag. Due to overlap in the molecular weights of YopO and GST-WASP, in Fig. 5, the phosphorylated GST-WASP was cleaved with thrombin prior to SDS-PAGE. In this figure, GST-WASP was not cleaved with thrombin. Red stars indicate the molecular weight of the substrates. Invitrogen Benchmark protein ladder is loaded in the first lane, with the molecular weights of bands in the ladder indicated. The gel was stained with Coomassie blue.
