Figure 1.
SPR kinetic analysis of pSpry2(A), ppSpry2(B), pEGFR(C), ppEGFR(D), tpEGFR(E) binding to c-Cbl TKB domain.
Six peptide concentrations ranging from 40 nM to 125 µM are used for each peptide in this study. Each concentration is repeated two times and the raw data is shown. The y axis shows the response unit (RU) differences (minus the reference) and the x axis shows the time (s). The kinetic values are listed in Table 1.
Table 1.
Binding parameters for the c-Cbl:peptide interactions.
Figure 2.
Crystal structure of TKB:ppSpry2 and TKB:ppEGFR complexes.
(A) Ribbon diagram of the TKB:ppSpry2 and (B) TKB:ppEGFR, N- and C- termini are labeled. c-Cbl-TKB is in gold. ppSpry2 (green) and ppEGFR (magenta) peptides are shown in stick represenations. These figures and the following figures in this manuscript were prepared using the program PyMol [40].
Table 2.
Crystallographic data and refinement statistics.
Figure 3.
Stereo-view of the 2Fo-Fc simulated annealing omit map of peptides from (A) ppSpry2-Cbl and (B) ppEGFR-Cbl.
All atoms within 3.5 Å of the peptides were omitted prior to refinement. Maps were contoured at a level of 1.0σ.
Figure 4.
The electrostatic surface potential at the region where the phosphorylated peptide binds to the c-Cbl TKB domain.
ppSpry2 peptide is shown in stick representation. The yellow arrows show the negatively-charged region on c-Cbl that electrostatically repels the phosphate group of the phosphorylated serine (pSer) residue.
Figure 5.
Superposition of TKB:peptide complexes.
Peptides are shown in colored stick representation, and c-Cbl-TKB is shown in ribbon representation (gold). (A) ppEGFR (purple) versus pEGFR (yellow). The rmsd between the superimposed TKB SH2 domains from both complexes is 0.351 Å for 81 Cα atoms. The enlarged view shows the peptide shift. The shift of the backbone atoms of the phosphorylated residues at the Y+1, Y+2 and Y+3 positions is approximately 0.7 to 1 Å, while the shift of the phosphorylated tyrosine backbone atom is about 0.1 Å. (B) ppSpry2 (green) versus pSpry2 (orange). The SH2 domain (81 Cα atoms) of the TKB from both complexes was superimposed with an rmsd of 0.219 Å.
Table 3.
Comparison of interaction surface and dissociation energy barrier predicted with PISA [33].