Figure 1.
PPIL1 binds short epitopes of SKIP in peptide scanning experiments.
(A) Peptide scan analysis of the PPIL1·SKIP interaction. Cellulose-bound peptide arrays were successively incubated with GST-PPIL1 fusion protein (upper panel) or GST alone (lower panel), rabbit α-GST antibody and horseradish peroxidase-coupled goat α-rabbit antibody. Images were obtained after luminol reaction. The locations of the spotted peptides are indicated by red circles. (B) Binding peptides exhibiting luminol signal above background. The consensus sequence common to all binding peptides is highlighted.
Figure 2.
SKIP43–79 is sufficient to pull-down PPIL1.
Pull-down of purified PPIL1 with glutathione-sepharose beads pre-coated with recombinant GST-SKIP43–79 (lanes 2 and 3) shows that PPIL1 binds to the mapped SKIP epitope, while GST alone (lanes 4 and 5) does not bind PPIL1. NaCl concentration in the washing buffer was varied between 150 mM (lanes 1, 2 and 4) and 1 M (lanes 3 and 5). Input (lane 1) and bound fractions (lanes 2 to 5) were analyzed by SDS-PAGE and stained with Coomassie. The band below the GST-SKIP fusion protein in lanes 2 and 3 is likely an abortive translation product corresponding to GST alone.
Figure 3.
Crystal structure of the PPIL1·CsA complex.
(A) Cartoon representation of PPIL1 with bound CsA (carbon atoms: brown) and two Cd2+ ions (golden spheres). (B) Experimental electron density obtained by SAD phasing contoured at the 2σ level (grey mesh). The final model is displayed as sticks.
Table 1.
Crystallographic data and refinement.
Figure 4.
Cadmium ions are coordinated by two neighboring PPIL1 molecules and solvent molecules.
(A) Cd1 (gold sphere) is coordinated by Asp89 in a bidentate fashion and by His31 of one PPIL1 molecule (carbon atoms in light blue) as well as by Cys133 of the (1/2-x, y-1/2, -z)-symmetry-related molecule (carbon atoms in green). The fourth coordination is site taken by a solvent molecule (red sphere). Cd2 shown here is (1/2-x, y-1/2, -z)-symmetry-related and coordinated by His87 of the present molecule and Glu26 and Cys133 of the (1/2-x, y-1/2, -z)-symmetry-related molecule as well as a solvent molecule. (B) Carbon atoms of PPIL1 residues that have been previously mapped to interact with a SKIP peptide by HSQC-NMR [1] are shown in magenta. These residues include the Cd2+-coordinating residues His87, Asp89 and Cys133.
Figure 5.
A docked model for the PPIL1·SKIP interaction.
The SKIP peptide (carbon atoms gold) adopts a loop structure, in which the central Pro65 is buried in a hydrophobic groove formed by Ile97 and the aliphatic region of the side chain of Arg131 of PPIL1 (PPIL1 carbon atoms light blue). The Glu66 side chain of the SKIP peptide engages in hydrogen bonds to PPIL1 residues Tyr28, Lys30 and Arg131. Furthermore, His68 of SKIP is bound by Asp89 and Thr93 of PPIL1.