Evidence for crucial electrostatic interactions between Bcl-2 homology domains BH3 and BH4 in the anti-apoptotic Nr-13 protein

Nr-13 is an anti-apoptotic member of the Bcl-2 family previously shown to interact with Bax. The biological significance of this interaction was explored both in yeast and vertebrate cells and revealed that Nr-13 is able to counteract the pro-apoptotic activity of Bax. The Bax-interacting domain has been identified and corresponds to alpha-helices 5 and 6 in Nr-13. Site-directed mutagenesis has revealed that the N-terminal region of Nr-13 is essential for activity and corresponds to a genuine Bcl-2 homology domain (BH4). The modelling of Nr-13, based on its similarity with other Bcl-2 family proteins and energy minimization, suggests the possibility of electrostatic interactions between the two N-terminal-conserved domains BH4 and BH3. Disruption of these interactions severely affects Nr-13 anti-apoptotic activity. Together our results suggest that electrostatic interactions between BH4 and BH3 domains play a role in the control of activity of Nr-13 and a subset of Bcl-2 family members.