Effects of mutation of tyrosine 110 (Y110) on the ability of BAK to form multimers following DNA damage. (A) PyMol generated model of BAK structure using PBD file 2IMS. Locations on the α4 helix of Y108 (red), together with S117 (blue) and Y110 (red) where the side chains of these residues face the hydrophobic surface groove (green), are indicated. Both the BH3 domain and BH1 encompassing α2-α5 helices (residue 70–145) constitutes the hydrophobic surface groove. (B) BAK multimerization following DNA damage was analysed in HCT116-BAK cells as described in . HCT116 DKO cells were reconstituted with the WT BAK, Y110F or Y110E BAK proteins. Mitochondria were isolated from cells expressing WT, Y110F and Y110E in ±UV 10mJ/cm2 for 8 hrs. 100 μg of mitochondria were cross-linked with either BMH (top panel) or BMOE (bottom panel). BAK was detected by western blot using a rabbit anti BAK monoclonal (abcam, Y164). The input was the 5% of mitochondrial extract used in the cross-linking studies to ensure equal loading (middle panel). Non cross-linked BAK runs as a monomer (M) and also as an intra-molecularly linked monomer (Mx). BAK dimers (D) and trimers (T) and higher order structures are indicated. Image is representative of 3 independent experiments. (C) Similar multimerization experiment to (B) was performed with HCT116 cells expressing WT BAK, Y110F or Y110E proteins ± 50 μM etoposide treatment for 8 hrs. Mitochondrial proteins were cross-linked with BMOE as described previously and detected as above. Note that BMOE generates only dimer forms of BAK.