(A) Stereodiagram of the SOUL molecule. The eight-stranded β-sheet is shown in two colours, red and blue, to emphasize the presence of two repeated units, each with β-β-α-β-β topology, related by a pseudo-two-fold axis of symmetry. (B) A topological diagram of SOUL. The eight antiparallel β-strands form a distorted β-barrel with the two α-helices arranged on one face. Strands are labelled in the order of their appearance from the N-terminus to the C-terminus using the letters A–H. The eight strands span the following residues: A, 39–43; B, 46–55; C, 89–94; D, 103–110; E, 127–132; F, 135–142; G, 174–178; H, 190–195. The two helices span residues 58–73 and 148–164. (C) Ribbon representation of the SOUL monomer viewed in a direction rotated approximately 90 ° with respect to (A). The two helices are in yellow, and the strands of the β-sheet have been coloured as (A and B) to emphasize the presence of the pseudo-two-fold axis. (D) Ribbon diagram of SOUL with the peptide spanning the BH3 domain represented in light blue for the portion of the chain identified by sequence similarity with known BH3 domains and in yellow for the rest of the second helix of the molecule. (E) Ribbon diagram of the NMR model of BID (lowest energy structure, PDB code 2BID) with the BH3 peptide and domain oriented and colour-coded as in (D). The Figures of the models were prepared using the program PyMOL (http://www.pymol.org).

(A) ¹⁵N-¹H NMR correlation spectra of ¹⁵N-labelled human Bcl-xL titrated with increasing amounts of a 26-amino-acid-long peptide spanning residues 147–172 of human SOUL. The peptide sequence is SAQKNQEQLLTLASILREDGKVFDEK. Arrows indicate the direction of peak shifts. Only four spectra are represented in the Figure; the black one is before any addition, the green after the addition of 0.52 equivalents of the peptide, the blue after the addition of 1.38 equivalents and the red after adding 3.83 equivalents of the BH3 domain peptide. (B) Decoupled ¹H spectrum showing the shifts in two peaks in fast exchange in the methyl region used to calculate an approximate dissociation constant. The colour of the spectra is the same as in (A) and corresponds to the addition of the same amounts of peptide. (C) Magnitude of the change in the ¹H chemical shift of the two selected peaks plotted as a function of the total number of equivalents of BH3 domain peptide added. The best fit curve is shown along with the coefficient of determination (R²) and the calculated dissociation constants. The values determined using the two peaks are 47.8 and 41.3 μM. The equation used to fit the data is given in the Experimental section. (D) SPR studies of the same interaction. The sensorgram shows the binding of the BH3 peptide to truncated immobilized Bcl-xL. Relative units (RU) are plotted as a function of time (in s). (E) A plot of the response as a function of the peptide concentration used to estimate the dissociation constant. The diagram is the result of several experiments and higher peptide concentrations could not be used because the BH3 peptide had a tendency to aggregate.

(A) A dimer of human Bcl-xL (Δ27–82) showing swapping of the α1 domains. The two helical SOUL BH3 peptides are represented in red. (B) A protomer of Bcl-xL and the SOUL BH3 peptide in contact with it. The portion of the peptide predicted by sequence homology to be the BH3 domain is in blue, whereas the yellow part is the additional portion of the peptide, the beginning of the second helix of SOUL. (C) Electron density of the SOUL BH3 peptide bound to Bcl-xL oriented as in (B). The molecular surface of Bcl-xL shows the negatively charged residues in red and those positively charged in blue. The 2F_obs−F_c map was contoured at a 1.2 σ level. Selected BH3 peptide amino acids participating in important contacts with Bcl-xL have been labelled in single-letter amino acid notation. The Figure was prepared using the program PyMOL (http://www.pymol.org).

(A) Arg¹⁶³ and other side chains participating in one of the specific contacts of the SOUL BH3 peptide with Bcl-xL. Hydrogen bonds are indicated with green broken lines, whereas the amino acids that make hydrophobic contacts are only indicated but not represented as ball and stick models. (B) The same type of diagram as (A), but with another specific contact in which the key residue is Asp¹⁷⁰ of the SOUL BH3 peptide. (C) Sequence alignment of the SOUL BH3 peptide and eight BH3-only proteins. The BH3 domains are boxed and the amino acids that are identical in SOUL and at least two other sequences are in red, whereas those that are identical in at least three sequences are in blue. The leucine residue conserved in all of the sequences is indicated on a green background. (A and B) were prepared using the visualization program LIGPLOT [49].

(A) Comparison of the peptide bound to Bcl-xL (Δ27–82) (blue) and the same sequence in the intact SOUL molecule (red). Eight amino acids change their conformation to extend the BH3 helix towards its C-terminus. The co-ordinates were superimposed by using the CCP4 suit of programs. Three charged amino acids that give specificity to the interaction are represented as stick models in the two conformations. (B) Superposition of the SOUL BH3 peptide Bcl-xL (Δ27–82) complex (the peptide is in yellow and Bcl-xL is in orange) and the same complex of Bcl-xL (in light blue) and the Beclin 1 BH3 peptide (in green). The PDB code of the model of the Beclin 1 peptide complex is 2P1L [13]. The interactions of the SOUL amino acids Arg¹⁶³ and Asp¹⁷⁰ (shown in Figure 4) are indicated with dotted lines. The aspartate residue is conserved in Beclin 1 and the equivalent of the arginine residue is a lysine. Note that the important contact of Arg¹³⁹ of Bcl-xL with an aspartate residue in Beclin 1 (conserved in all BH3 domain–Bcl-xL complexes) is missing in the SOUL peptide.