Fig 1.
Three-dimensional model of the complex (KCTD11BTB-Cul349-68)4.
(A) The model of the three-dimensional structure of the complex (KCTD11BTB-Cul349-68)4 that was used as starting model in the molecular dynamics simulation. The peptide Cul349-68 is coloured in red. The C-terminal helices (α5) which protrude toward the solvent and the interacting loop α2-β3 of the four KCTD11BTB chains are shown in cyan and yellow, respectively. (B) A snapshot of a monomer KCTD11BTB-Cul349-68 which highlights the location of key elements of the model is reported.
Fig 2.
Buried area of Cul349-68 residues upon complex formation with KCTD11BTB.
Fig 3.
Aminoacid sequences of all peptides.
The replaced residues are highlighted in red. S5 corresponds to the non-standard amino acid (S)-2-(4’-pentenyl) alanine.
Fig 4.
Far-UV CD spectra of Cul349-68, Cul349-68EN, Cul349-68LA, and Cul349-68SL. Spectra were acquired in 10 mM phosphate buffer pH 7.0.
Fig 5.
Binding curves obtained from ELISA on KCTD11BTB using the biotinylated peptides Cul349-68 (▲),Cul349-68LA (●),Cul349-68EN (▀),Cul349-68SL (▼) and Cul349-68AA(♦).
Fig 6.
Normalized Fluorescence Polarization data for peptides as a function of [KCTD11BTB].
Peptides were plated at a final concentration of 2 μM, and the interaction with KCTD11BTB was tested over a concentration range of 0.1 nM to 20μM. Dissociation constants were calculated using nonlinear regression and are presented as mean ± standard error of triplicates.
Fig 7.
Normalized Fluorescence Polarization data for Cul349-68LA as a function of [KCTD5BTB].
Peptide was plated at a final concentration of 1 μM, and the interaction with KCTD5BTB was tested over a concentration range of 0.1 nM to 5 μM. Dissociation constants were calculated using nonlinear regression and are presented as mean ± standard error of triplicates.
Fig 8.
Hα secondary chemical shift for Cul349-68 (a), Cul349-68LA (b) and Cul349-68EN (c) peptides.
The red line indicates the average upfield shifts observed in peptides database for α-helix.
Fig 9.
The NMR structure of Cul349-68EN.
(A) NMR ensemble of the best 20 structures of Cul349-68EN peptide. In the figure the i, i + 4 staple bridge produced by the combination of cross-linking (S)-2-(2’-pentenyl) alanine is shown in green. (B): Structural statistics for the Cul349-68EN peptide.
Fig 10.
The NMR structure of Cul349-68LA.
(A) NMR ensemble of the best 20 structures of Cul349-68LA peptide. In the figure the i, i + 4 staple bridge produced by the combination of cross-linking (S)-2-(2’-pentenyl) alanine is shown in green. (B) Structural statistics for the Cul349-68LA peptide.
Table 1.
Serum stability of the peptides and assignment of the fragments derived from proteolysis by LC/MS analysis.
Fig 11.
Superposition of the stapled peptides with Cul3.
Superposition of the most representative conformer of Cul349-68EN (left panel; blue) and Cul349-68LA (right panel; yellow) respectively with the region (in red) encompassing residues Phe54-Leu66 of the crystal structure of Cul3 (PDB code 4EOZ).