Molecular characterization of direct interactions between MPP1 and flotillins

Figure 1. Structure of MPP1. Schematic representation (a) of the overall domain composition and ribbon model (b) of the structure of MPP1. PDZ blue, SH3 green, D5 light blue, GUK red. Domain boundaries were marked based on Quinn et al.11. Structural model redrawn according to Listowski et al.44.

Figure 2. Recombinant MPP1 binds recombinant flotillin 1 or flotillin 2 with equilibrium dissociation constant, KD, values in nanomolar range. HIS-tagged flotillins were immobilized on the Ni–NTA chip and its interaction with full-length recombinant, untagged MPP1 (series of concentration 0–2000 nM) was analyzed by SPR using a BIAcore T200. Black dotted lines represent fitted 1:1 Langmuir binding model. Residual plots beneath every sensorgram series were evaluated for systematic divergences from the fitting curves. Other details in “Materials and methods” section.

Figure 3. Visualization of MPP1-flotillin 1 binding sites mapped by molecular dynamics simulations. (a–d) Binding sites of MPP1 and FL1. MPP1 amino acid residues are marked in red, FL1 in black. Water molecules were removed for clarity.

Figure 4. Visualization of MPP1-flotillin 2 binding sites mapped by molecular dynamics simulations. (a–c) Binding sites of MPP1 and FL2. MPP1 amino acid residues are marked in red, FL2 in black. Water molecules were removed for clarity.

Figure 5. The D5 domain of MPP1 is of key importance for binding of flotillins. (a–e) HIS-tagged flotillins were immobilized on the Ni–NTA chip and their interactions with untagged MPP1 truncated mutants (MPP1-Mut1-5) in a series of concentrations 0–1600 nM were analyzed using a BIAcore T200. Other details as in Fig. 2 legend.

Figure 6. MPP1-Mut4 affects membrane order in HEL cells. Representative FLIM images (a) and quantitative analysis (b) of di-4 lifetime distribution collected from plasma membrane (ROI) of control (FLAG-transfected) and MPP1-Mut4-FLAG transfected HEL cells indicating significant (p < 0.0001) reduction in membrane order parameters after inhibition of endogenous MPP1-flotillin interactions. Each dot on a graph represents a single image of control (n = 38) and MPP1-Mut4-FLAG transfected cells (n = 42). Uncropped images, lifetime histograms as well as decay curves for representative images are shown in Fig. S8. Statistical analysis was performed using two-tailed unpaired t-test. Scale bar, 5 µm.

Figure 7. MPP1-Mut4 inhibits signal transduction from raft-dependent IR receptor in HEL cells. Control (FLAG-transfected) and MPP1-Mut4 transfected cells were treated with insulin and the whole-cell extracts were subjected to Western blot analysis and probed with antibodies against indicated proteins (a). Quantification of the relative phosphorylation levels of ERK1/2 in control and MPP1-Mut4 transfected cells (average ± S.D. from four independent experiments; statistical analysis was performed using non-parametric t-test p < 0.05) were performed (b). Uncropped blots are shown in Fig. S9.