Figure 2
Characterization of PREP1 leucine and isoleucine mutants. Panel A: Mapping of the heptad repeats within HR1, HR2 and HR3 regions of PREP1. The areas in magenta represent predicted alpha-helical regions within PREP1. The heptad repeats of HR1 and HR2 domains are represented as alpha-helical turns above the linear sequence of the protein, highlighting the hydrophobic residues pointing toward the same face of the helix. The position and sequence of the hydrophobic “HR3” stretch are indicated. Panel B: Mapping of the heptad repeats within HR1 and HR2 of MEIS1. Predicted alpha-helical regions are represented as light-blue areas above the linear sequence. Panel C: Binding of recombinant wild-type and mutants PREP1 to recombinant wild-type PBX1 measured by Time-resolved fluorescence immunoassay (TR-FIA). Single mutations analysed in the assay are indicated; HR1m is the quadruple L63A/L66A/L67A/L70A mutant; HR2m is the quadruple I122A/L125A/L129A/L132A mutant. HR1, HR2 and “HR3” domains are mapped below the bars for clarity. The Y axis reports the binding to PBX1 of the PREP1 mutants, normalized to wild-type PREP1 taken as 1.0. The results represent the average of four independent assays, + /− the standard deviation. T-TEST was performed as described in the Methods section. Panel D ELISA determination of the affinity between N-terminal GST-fused wild-type PREP1 (black line), GST-fused wild-type MEIS1 (blue line), GST-fused HR1m (L63A/L66A/L67A/L70A, red squares) and HR2m (I122A/L125A/L129A/L132A, green triangles) PREP1 mutants. Excess proteins were added to plates coated with increasing concentrations of wild-type PBX1. The values on the Y axis are the signals obtained with the individual proteins subtracted of the GST used as control. Results represent the average of three independent assays + /− the standard deviation.
