Volume 55, Issue 6, Pages 938-946 (September 2014) Bak Core and Latch Domains Separate during Activation, and Freed Core Domains Form Symmetric Homodimers  Jason M. Brouwer, Dana Westphal, Grant Dewson, Adeline Y. Robin, Rachel T. Uren, Ray Bartolo, Geoff V. Thompson, Peter M. Colman, Ruth M. Kluck, Peter E. Czabotar  Molecular Cell  Volume 55, Issue 6, Pages 938-946 (September 2014) DOI: 10.1016/j.molcel.2014.07.016 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 55, 938-946DOI: (10.1016/j.molcel.2014.07.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Bak Core/Latch Dimerization (A) BH3 peptides that dimerize Bak (ΔN22, ΔC25, ΔCys) as analyzed by gel filtration (S75 10/300). Bak protein was incubated with CHAPS alone (black) or with CHAPS and Bid BH3 (magenta), Bim BH3 (green), or Bak BH3 (blue) peptides. (B) BH3 peptides that do not dimerize Bak (ΔN22, ΔC25, ΔCys) as analyzed by gel filtration (S75 10/300). Bak protein was incubated with CHAPS alone (black) or with CHAPS and Noxa BH3 (orange), Bad BH3 (red), Bax BH3 (purple), or Bid BH3 I82A/I83A (light blue) peptides. (C) BH3-peptide alignment with h0–h4 positions highlighted in gray and mutations colored red. (D) Crystal structure of Bak (ΔN22, ΔC25, ΔCys) core/latch dimer. One polypeptide is colored to show the core domain (α1–α5, green) with its BH3 domain (α2, red) and the latch domain (α6–α8, purple). The partner polypeptide is gray. (E) Residues V142 and F150 (gray) on the α5 (green) and α6 (purple) helices of the crystal structure of Bak (2IMS) (Moldoveanu et al., 2006). The double-mutant V142C/F150C was disulfide linked to block core/latch dissociation. (F) Isolated mitochondrial fractions from bak−/−bax−/− DKO MEFs reconstituted with Bak variants were evaluated for cytochrome c release with or without CuPhe (western blots are a representation of three independent experiments). Samples were divided into supernatant and membrane fractions, separated by reducing SDS-PAGE, and blotted with anti-cytochrome c antibody. The V142C/F150C double mutant was functional and could release cytochrome c into the supernatant in response to tBid, unless first oxidized by CuPhe. This was also observed for the positive control WT Bak but was not observed in the single cysteine mutants (V142C or F150C). See also Figure S1. Molecular Cell 2014 55, 938-946DOI: (10.1016/j.molcel.2014.07.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Crystal Structure of Bak Core Domain Dimer (A) Two orthogonal views of the crystal structure of GFP-Bak(α2–α5) colored by chain. The structure reveals two GFP dimers connected by two Bak core domain dimers. (B) Crystal structure of one Bak core domain dimer. The BH3 domain (α2, red) occupies the hydrophobic groove (α3-α5, green) of the partner molecule. Residues subjected to linkage analysis in (D) are colored yellow; the dyad partner distances as measured between β-carbon atoms areas follows: R88, 9.3 Å; D84, 11.4 Å; and I80, 22.8 Å. (C) Crystal structure of the Bak core domain dimer from the side showing the surface rich in aromatic residues (Y108, F111, F119, Y136, and Y143, all colored gray). (D) Reducing SDS-PAGE western blot analysis of BMOE-linked full-length Bak (ΔCys) mutants I80C, D84C, and R88C, expressed in bak−/−bax−/− DKO MEF cells, before and after tBid activation (western blots are a representation of three independent experiments). Following tBid activation, but not before, D84C and R88C residues could be crosslinked in a dimer of Bak; this was not observed for I80C, despite retaining BH3:groove dimer formation as analyzed by blue native PAGE (Figure S2B). (E) Crystal structure of the core domain dimer showing the BH3 domain (red) of one molecule interacting with the surface (white) and cartoon (green) of the hydrophobic groove (α3-α5) of the other molecule. The five hydrophobic positions h0–h4 (T70/M71, V74, L78, I81 and I85, see Figure 1C) protrude into the hydrophobic groove. The residues D83 and R127 form a salt bridge. See also Figure S2. Molecular Cell 2014 55, 938-946DOI: (10.1016/j.molcel.2014.07.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 CuPhe Crosslinking of α2 and α3 in the Bak Core Domain Dimer (A) Crystal structure of the Bak core domain dimer showing the BH3 domain (red) of one polypeptide in the hydrophobic groove of the neighbor. Side chains of crosslinkable residues are colored gray. (B) Side-by-side packing of Bak core domain dimers (see Figure 2A) showing adjacent G72 and H99 residues (yellow). For linkage to occur between dimers, significant movement would be required in the direction of the arrows. (C) SDS-PAGE followed by western blot analysis of CuPhe-linked full-length Bak (ΔCys) mutants G72C, H99C, and G72C/H99C, expressed in bak−/−bax−/− DKO MEF cells (western blots are a representation of three independent experiments). These mutants have been previously reported to be functional in death assays (Dewson et al., 2008). (D) Blue native PAGE analysis of samples from (C). Molecular Cell 2014 55, 938-946DOI: (10.1016/j.molcel.2014.07.016) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Proposed Model for Bak Activation and Oligomerization Bak resides in the outer mitochondrial membrane in an inactive monomeric conformation (Griffiths et al., 1999) represented by its crystal structure (Moldoveanu et al., 2006). (1) Activator BH3 domains transiently bind the hydrophobic groove of Bak, as shown by structural (Moldoveanu et al., 2013) and biophysical (Leshchiner et al., 2013) studies. (2) This interaction induces conformational changes in Bak, resulting in the separation of the core and latch domains. (3a) In solution, C-terminally truncated Bak forms off-pathway core/latch domain-swapped dimers that are not likely to be physiologically relevant. (3b) When anchored in the mitochondrial membrane, this conformation change enables formation of BH3:groove dimers as characterized in biochemical studies by Dewson et al. (2008). (4) BH3:groove dimers further oligomerize through an unknown mechanism and initiate MOMP. Molecular Cell 2014 55, 938-946DOI: (10.1016/j.molcel.2014.07.016) Copyright © 2014 Elsevier Inc. Terms and Conditions