The BH3-only members of the Bcl-2 protein family play a central role in the process that leads to programmed cell death or apoptosis. Their effect is due to inhibition through binding of their BH3 domain in the hydrophobic cleft of the anti-apoptotic members of the Bcl-2 family, such as Bcl-2, Bcl-xL or MCL-1. Indeed, one of the criteria to include a protein in this group is a demonstration of its interaction with one of the pro-survival proteins of the Bcl-2 family, in addition to showing that they have a cellular death-inducing activity. On the basis of these criteria, since the discovery of the first member of the family, BIK [44
], many other members have been added to the list [9
]. In many cases, the proteins seem to have additional functions besides their role in inducing cellular death. Although a very large number of complexes of BH3 peptides with pro-survival proteins of the Bcl-2 family are available [12
], only one NMR structure of an intact BH3-only protein is known, BID [46
Two different mutually non-excluding functions have been attributed to SOUL: haem transport [19
] and a role in apoptosis as a BH3-only protein [20
]. We have used NMR, SPR, UV spectroscopy and crystallography to explore both functions and, in addition, we have determined the three-dimensional structure of the protein. Human SOUL is a monomer with a fold which is quite different from that of the other BH3-only protein whose three-dimensional structure is known, BID, which is similar to Bcl-xL. BID contains eight α-helices: two central hydrophobic helices surrounded by six amphipathic helices with their hydrophilic face exposed to the solvent. The SOUL molecule is similar to murine p22HBP [33
The interaction of human SOUL with haemin was explored by titrating the latter with the former and following the UV spectrum, using BSA as a control. In our experiments, we made sure that the protein used did not contain any traces of the histidine tag used for purification and that no imidazole, residual from the purification in the affinity columns, was present in the samples. We did not find any evidence of an interaction between haemin and SOUL. This result was confirmed by the 15
H HSQC NMR spectra of 15
N-labelled SOUL in the presence of increasing amounts of haemin. We do not have an explanation as to why our results appear to be at variance with those reported for mouse SOUL [19
]. The latter is reported to be a dimer in the absence of haem and a hexamer in the bound state, which is different from both the results of the present study and work on the related p22HBP proteins. It is also worth noting that the histidine residue that Sato et al. [19
] found to be essential for haem binding does not seem to be involved in haem binding to p22HBP, which appears to bind this moiety through hydrophobic interactions and not metal co-ordination.
The interaction of a peptide spanning residues 147–172 of human SOUL with human Bcl-xL was also studied with 15N-1H HSQC NMR spectroscopy using two different forms of human Bcl-xL, the entire molecule lacking only the hydrophobic transmembrane domain after amino acid 209 and another truncated form lacking also amino acids 27–82 (Δ27–82). The results were comparable and indicated in both cases that there is an interaction of the 26-amino-acid SOUL peptide with Bcl-xL with a dissociation constant estimated to be 40–50 μM. These results were confirmed by SPR measurements and by preparing co-crystals of the complex and solving their three-dimensional structure. The new crystal structure revealed swapping of the first helix of the Bcl-xL dimer, a phenomenon associated with the presence of the Δ27–82 truncation. The amino acids participating in the interaction of the BH3 domain and protein were identified. The interactions are mostly hydrophobic, but a significant number of specific charged residue contacts were also found to be present.
When the SPR experiments performed to detect the interaction of the SOUL BH3 domain peptide were repeated using the entire SOUL molecule instead, no interaction was detected between ligand and analyte. This result might be explained by the fact that our findings predict a very drastic conformational change in the protein molecule to allow the portion of polypeptide chain involved in the contacts to adopt the conformation required for the contacts to be established. The last eight amino acids of the BH3 domain are not in a helical conformation in intact SOUL and, in addition, side chains that are important for the interaction point towards the interior of the molecule and are not available on the protein surface. Conformational changes in the anti-apoptotic members of the Bcl-2 family upon interaction with BH3 domains have been described [38
], as well as changes in the interacting BH3 domains [41
]. In addition, it has been shown that Bim, Bad and Bmf have intrinsically unstructured BH3 domains that undergo a localized conformational change upon binding to pro-survival Bcl-2 targets [48
]. No important changes were found in Bcl-xL, but the changes in the BH3 domain are remarkable and are sufficient to explain why no interaction is observed with the intact SOUL molecule. These drastic modifications might require conditions that have not yet been found, but that should be explored further given the importance of this interaction in the functionality of the two proteins.
The mechanism of activation of BID, the prototype of BH3-only proteins, involves cleavage by caspase 8 in a region with the sequence LQTDG [46
]. The second amino acid in the sequence can be an glutamate residue and the last can be any amino acid other than phenylalanine, glutamate, glutamine, lysine or arginine. This cleavage site is not present in SOUL, although the similar sequence LESDV spans residues 123–127 and is exposed to the solvent in the molecule, in the loop connecting strand D to E. However, an experiment with caspase 8, using human BID as a control, revealed that SOUL is not a substrate of this enzyme (results not shown).
The role of SOUL in inducing apoptosis has been documented, but up to now there has been no information at the molecular level on the mechanism through which this function is accomplished. We have shown that its BH3 domain interacts with a pro-survival member of the Bcl-2 family and thus we have provided new evidence that SOUL behaves like a novel member of the expanding family of BH3-only proteins.
Two very important questions remain unanswered: (i) the nature of the molecular alteration that intact SOUL must undergo for the interaction to take place, and (ii) the precise specificity of the interaction of the SOUL BH3 domain with different members of the Bcl-2 family.