Modeling backbone flexibility to achieve sequence diversity: the design of novel alpha-helical ligands for Bcl-xL

Computational protein design can be used to select sequences that are compatible with a fixed-backbone template. This strategy has been used in numerous instances to engineer novel proteins. However, the fixed-backbone assumption severely restricts the sequence space that is accessible via design. For challenging problems, such as the design of functional proteins, this may not be acceptable. Here, we present a method for introducing backbone flexibility into protein design calculations and apply it to the design of diverse helical BH3 ligands that bind to the anti-apoptotic protein Bcl-xL, a member of the Bcl-2 protein family. We demonstrate how normal mode analysis can be used to sample different BH3 backbones, and show that this leads to a larger and more diverse set of low-energy solutions than can be achieved using a native high-resolution Bcl-xL complex crystal structure as a template. We tested several of the designed solutions experimentally and found that this approach worked well when normal mode calculations were used to deform a native BH3 helix structure, but less well when they were used to deform an idealized helix. A subsequent round of design and testing identified a likely source of the problem as inadequate sampling of the helix pitch. In all, we tested 17 designed BH3 peptide sequences, including several point mutants. Of these, eight bound well to Bcl-xL and four others showed weak but detectable binding. The successful designs showed a diversity of sequences that would have been difficult or impossible to achieve using only a fixed backbone. Thus, introducing backbone flexibility via normal mode analysis effectively broadened the set of sequences identified by computational design, and provided insight into positions important for binding Bcl-xL.