Bcl-2 a chief modulator of apoptosis. It is an anti-apoptotic protein and is directly involved in avoidance of apoptosis through its actions of abrogating the formation of the permeability transition pore. This is achieved through its interaction with Bax and Bak proteins that in the absence of Bcl-2 are free to form homo-dimers and subsequently homo-oligomers that essentially compose the permeability transition pores, leading to the release of proteins such as cytochrome C that correspond to the induction of the typical apoptosis associated caspase cascade.

 

Structure

For the structural analysis, there was no empirically determined protein structure available on the RCSB PDB but only the protein sequence on NCBI. The predicted structure using this sequence and ‘Swissmodel’ exhibits a long loop region between helices 1 and 2. However, the model is still missing the important transmembrane domain and we therefore decided to evaluate the structure using the empirically determined (X-ray crystallography) PDB File 1G5M which has been useful in Bcl-2 analysis since its structure has been resolved by Petros et al. in 2001.

 

The animations and images we created show that Bcl-2 consists of 7 alpha helices and adapts a globular structure. The centre of the molecule consists of two mostly hydrophobic alpha helices and is surrounded by 4 amphipathic alpha helices. This creates a sterically stable protein. Using the results from our BLAST analysis we located the regions conserved not only between family members but across isoforms in different organisms - the BH domains 1-4 - and noted where the missing transmembrane domain is ought to be. Furthermore, we located these conserved regions in our structure-predicted Bcl-2 model with the long loop and showed the similarity between our two models through a short MSA in Figure 3. The active site itself has been localised using the descriptions given by Petros et al. (2001)   and has been included in the animations.

 

Protein interaction

In the presence of Bcl-2, and in line with the structural analysis described of this protein, the hydrophobic BH3-binding groove (contributed to by alpha helices 2, 3, 4, 5 and 7) is able to interact with the BH3 domains of Bax and Bak proteins to prevent their dimeric association that would typically occur via this same interaction. The nature of this interaction is as follows: the BH3 domain of Bax/Bak inserts into the hydrophobic groove as an amphipathic alpha-helix and makes multiple hydrophobic interactions, permitting a tight association that varies slightly between different isoforms of Bcl-2 and different executioner proteins.

 

This mechanism of interaction between Bcl-2 and the BH3 domain of other proteins is of particular interest as a therapeutic target. Defective Bcl-2 signalling plays a fundamental role in a plethora of cancers due to its importance in preventing apoptosis. Therapeutic agents called BH3 mimetics have been formulated to mimic the function of the BH3 domain to allow apoptosis to be induced because Bcl-2 can no longer inhibit the dimerisation of Bak and Bax as described above.

 

Bcl-2 can also be neutralised using this mechanism by a host of BH3 only proteins that are implicated within the intrinsic pathway of apoptosis such as Puma, Noxa and Bim. All of the proteins mentioned are members of the Bcl-2 family. Bcl-2 itself was the first protein to be discovered and hence is the namesake of the family. Members are defined by exhibiting at least one Bcl-2 homology domains.

 

Representative proteins and family members

We analysed these Bcl-2 family members using multiple sequence alignments (MSA). The MSAs revealed regions of high homology within the Bcl-2 family as well as between organisms and their respective Bcl-2 proteins. The domains located correspond to the BH domains of which there are up to 4 in proteins such as Bcl-2 and, additionally, a C-terminal transmembrane domain that permits Bcl-2 localisation to the outer mitochondrial membrane. This was further compounded by BLAST search analysis which showed that both the alpha and beta human isoforms of Bcl-2 have the same conserved domains of BH1-4, and belong to the Bcl-2_like superfamily.

 

Through analysis of these MSAs, it is clear that the evolution of the Bcl-2 protein has marked some interesting changes. Less evolutionarily developed organisms such as C. elegans and D. melanogaster have their BH4 domain closer to the other BH domains in the protein. In M. musculus and H. sapiens, the BH4 domain is closer to the N-terminus. It is unknown how this benefited the function of BCL-2. The evolutionary divergence of the Bcl-2 protein in the 4 species studied can be seen in the cladogram under "Evolution and Homologues".

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References:

[1] Petros AM, Medek A, Nettesheim DG, et al. Solution structure of the antiapoptotic protein bcl-2. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(6):3012-3017. doi:10.1073/pnas.041619798. [PubMed]