Anti-apoptotic BCL-2 regulation by changes in dynamics of its long unstructured loop

Abstract

BCL-2, a key protein in inhibiting apoptosis, has a 65-residue-long highly flexible loop domain (FLD) located on the opposite side of its ligand-binding groove. In vivo phosphorylation of the FLD enhances the affinity of BCL-2 for pro-apoptotic ligands, and consequently anti-apoptotic activity. However, it remains unknown as to how the faraway, unstructured FLD modulates the affinity. Here we investigate the protein-ligand interactions by fluorescence techniques and monitor protein dynamics by DEER and NMR spectroscopy tools. We show that phosphomimetic mutations on the FLD lead to a reduction in structural flexibility, hence promoting ligand access to the groove. The bound pro-apoptotic ligands can be displaced by the BCL-2-selective inhibitor ABT-199 efficiently, and thus released to trigger apoptosis. We show that changes in structural flexibility on an unstructured loop can activate an allosteric protein that is otherwise structurally inactive.

Single-molecule scale quantification reveals interactions underlying protein–protein interface: from forces to non-covalent bonds

“…Previously, methods based on Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), and pulsed ESR double electron–electron resonance (DEER) revealed structural flexibility and kinetic changes in Bcl-2 upon ligand binding. 12 We suppose that multiple interactions in the final bound state will lead to a restriction of the directional degrees of freedom between the bound interfaces and therefore be accompanied by a change in the mechanical properties of BAX/Bcl-2 complexes. Thus, we defined the stiffness properties of the complex in terms of the effective spring constant K eff = Δ F /Δ t , where F is the applied force over time (Fig.…”

“…11 Interestingly, important changes of the relative orientation of the Bcl-2 flexible loop domain (FLD) were observed when comparing the BAX unbound and bound structures. 11,12 In addition, it has been shown recently that the BAX and Bcl-2 interaction interface also involves novel interfacial interactions of the BH4 motif, 13 and transmembrane domain. 14 Overall, these conformational changes reflect further changes in the interaction positions, particularly the noncovalent bonds, which play a role in the regulation of PPIs.…”

Molecular and functional characterization of reversible‐sunitinib‐tolerance state in human renal cell carcinoma

“…A distinct ability to overcome cell death is a further trait of sunitinib tolerance. We observed a sunitinib dose‐dependent increase in the anti‐apoptotic protein BCL‐2, 46 while the pro‐apoptotic kinase p38 47 was stepwise downregulated. We found a heterogeneous apoptotic response (caspases 3/7) between 786‐O and Caki‐2 cell lines.…”

Domain-specific insight into the recognition of BH3-death motifs by the pro-survival Bcl-2 protein

“…1 and S5 , most prominent are the seven solvent-exposed and highly flexible glycines belonging to the natively disordered loop region that links the Bcl-2 homology BH4 and BH3 domains. This regulatory FLD (aa 34–90) stretches into the aqueous cellular interior where it can sense cytosol signals and become phosphorylated at multiple sites for controlling the activity of Bcl-2 ( 12 , 28 , 29 ). The FLD glycines at both ends (G33 and G83) are slightly more ordered (positive heteronuclear NOE values) presumably due to the proximity to the highly structured BH4 and BH3 domains.…”