Selectivity and promiscuity in protein:protein interactions controlling apoptosis.
Peter Colman, Doug Fairlie, Erinna Lee, Peter Czabotar, David Huang


The Bcl-2 protein family plays a central role in controlling the commitment of cells to programmed cell death (apoptosis). Five known members of the immediate family in mammals are pro-survival molecules, whose over-expression leads to resistance to death stimuli. Nine distantly related mammalian family members, the so-called BH3-only proteins, contain just one of the four Bcl-2 Homology domains, and these family members are pro-apoptotic. The BH3 domain can adopt an ?-helical structure that inserts into a cognate groove in the pro-survival molecules, antagonising their function.

We have explored the interactions between eight mammalian BH3-only proteins and the five mammalian Bcl-2 family proteins and discovered unexpected patterns of selectivity and promiscuity, though in no case is the selectivity so tight as to be restricted to a single binding partner. Our results generally suggest that the expansion of the families from two BH3-only proteins and one pro-survival protein in C. Elegans represents more than simply the acquisition of redundancy. An engineered variant of one of the selective BH3-only proteins (Noxa) converts it into a more promiscuous binder with increased killing activity. The Noxa variant has a unique binding profile, not mimicked by any of the naturally occurring BH3-only proteins, and has proven to be a useful reagent for teasing out functional differences between the pro-survival family members.

We have further characterised these interactions using scanning, random and targeted mutagenesis in phage display. The consequences of introducing particular mutations in the BH3 ligand are highly dependent on the particular pro-survival family binding partner. Killing assays indicate a threshold Kd for function. Our results point to design strategies for ligands capable of binding individual Bcl-2 family members, and may inform the search for organic ligands capable of selectively activating the apoptotic pathway, which is dysfunctional in certain tumours.