EPR Data Support the Existence of a Symmetric BH3-in-Groove Homodimer in Oligomeric BAK

Abstract

The BAX or BAK oligomeric pore formation in the mitochondrial outer membrane is a critical step in apoptosis, yet their structures are not clearly understood. Czabotar et al. (Cell 2013, 152, 519) reported a crystal structure of a water-soluble tetramer of the BAX fragment (helices α2-α5) fused to the green-fluorescent protein (GFP), in which the α2-α3 extended helices and the α5 helix, respectively, were juxtaposed to each other, in an anti-parallel orientation, forming a symmetric “BH3-in-groove homodimer (BGH).” We have constructed a GFP-BAK fusion protein using the α2-α5 helices of mouse BAK, designated as GFP-BAKα2-α5, which also forms a soluble tetramer. To determine whether the BGH exists in the BAK oligomers in the membrane or not, we spin labeled the C-terminal hexahistidine-tagged soluble form of mouse BAK (helices α1-α8) at residues 84, 122, 128 and 135 and the corresponding residues in GFP-BAKα2-α5. We then compared the continuous wave (CW) EPR spectra of the spin-labeled residues from the tetrameric GFP-BAKα2-α5 with those from the oligomeric BAK in membrane. Spin labeled residue 122R1, located in the loop interconnecting helices α4 and α5 in the homology model of the GFP-BAKα2-α5 tetramer, displayed a mobile lineshape. The corresponding residue in the oligomeric BAK also had a remarkably similar lineshape, indicating that the two residues are in similar structural environments. Residues 84R1, 128R1 and 135R1, located at the anti-parallel helical interfaces in the BGH also had remarkably similar immobile lineshapes both in the GFP-BAKα2-α5 tetramer and in the oligomeric BAK in membrane, further strengthening the above conclusion. The intra-dimer distances between 84R1 spin label pairs in the GFP-BAKα2-α5 and the oligomeric BAK, determined by the double electron electron resonance (DEER) method, also support this interpretation.