Solution conformations of wild-type and mutated Bak BH3 peptides via dynamical conformational sampling and implication to their binding to antiapoptotic Bcl-2 proteins

The BH3 (Bcl-2 homology 3) domain of the Bcl-2 family of proteins plays a critical role in the regulation of programmed cell death, or apoptosis. A 16-residue peptide derived from the BH3 domain of the proapoptotic protein Bak potently binds to the antiapoptotic proteins Bcl-2 and Bcl-xL. While this Bak BH3 peptide adopts a well-defined helical conformation in the experimental NMR solution structure in complex with Bcl-xL, it does not adopt a stable helical conformation in water. Experimental structural determination of peptides without a stable and well-defined solution conformation remains a difficult task. In this paper, we investigated the solution conformations of this Bak BH3 peptide through extensive molecular dynamics (MD) simulations in water using a recently developed self-guided MD simulation (SGMD) method. Our simulations showed that the Bak peptide exhibits a partially formed helical structure with a fairly stable 6-residue helical segment at the N terminus and a less stable approximately 4-residue helical segment at the C terminus. Additionally, we also performed extensive SGMD simulations of two mutated Bak peptides, in which the arginine residue at position 5 is mutated to either an alanine or a glycine residue (R5A or R5G mutant) to investigate the influence of mutation on the solution conformations of the Bak peptide. We found that the R5G mutation greatly affects the solution conformations of the peptide, and the overall helix ratio decreases by a factor of 2 as compared to the wild-type Bak peptide. In contrast, the R5A mutation does not affect significantly the peptide solution conformations observed in the wild type. By use of a fluorescence-polarization-based binding assay, we quantitatively determined the binding affinities of these three Bak BH3 peptides to Bcl-xL protein. We found that the R5G and R5A mutants are 24 and 6 times less potent than the wild-type Bak peptide, respectively, suggesting that the helical ratio of the peptides is an important but not the only factor for their binding to Bcl-xL. Analysis of representative conformations of the R5A mutant suggested that the relatively stable helical segment close to the N terminus may greatly facilitate its binding to Bcl-xL.