MCL-1 promiscuity and the structural resilience of its binding partners.

The allosteric protein MCL-1 and its natural inhibitors, the BH3-only proteins PUMA, BIM, and NOXA regulate apoptosis by interacting promiscuously within an entangled binding network. Little is known about the transient processes and dynamic conformational fluctuations that are the basis for the formation and stability of the MCL-1/BH3-only complex. In this study, we designed photoswitchable versions of MCL-1/PUMA and MCL-1/NOXA, and investigated the protein response after an ultrafast photo-perturbation with transient infrared spectroscopy. We observed partial α-helical unfolding in all cases, albeit on strongly varying timescales (1.6 ns for PUMA, 9.7 ns for the previously studied BIM, and 85 ns for NOXA). These differences are interpreted as a BH3-only-specific "structural resilience" to defy the perturbation while remaining in MCL-1's binding pocket. Thus, the presented insights could help to better understand the differences between PUMA, BIM, and NOXA, the promiscuity of MCL-1, in general, and the role of the proteins in the apoptotic network.

Signal Propagation Within the MCL-1/BIM Protein Complex.

The protein MCL-1 is a crucial factor in regulating apoptosis, the programmed cell death, and thus plays a major role in numerous cancer types. The allosteric protein MCL-1 is naturally moderated by the BH3-only peptide BIM, which binds at its canonical binding groove. In its isolated form, BIM is disordered but assumes an α-helical shape when bound by MCL-1. The underlying binding mechanism (i.e., induced fit vs conformational selection), as well as the time scales of the signal cascade subsequent to binding, are not understood. Here, an artificially photoswitchable variant of the MCL-1/BIM complex was designed and investigated by transient infrared spectroscopy. By destabilizing the α-helix of BIM with a covalently linked azobenzene photoswitch, the dynamical response of the whole complex upon an ultrafast photo-perturbation was characterized. While the destabilized and partially unfolded BIM still binds to MCL-1, a step-like cascade of structural rearrangements of both, MCL-1 and BIM was detected, spanning a wide range of time scales from pico- to microseconds. The results indicate that BIM binds according to an induced fit mechanism, while the structural adaptations of MCL-1 may constitute an allosteric signal.