Context-dependent stabilizing interactions among solvent-exposed residues along the surface of a trimeric helix bundle.

Here we show that a solvent-exposed f-position (i.e., residue 14) within a well-characterized trimeric helix bundle can facilitate a stabilizing long-range synergistic interaction involving b-position Glu10 (i.e., i - 4 relative to residue 14) and c-position Lys18 (i.e., i + 4), depending the identity of residue 14. The extent of stabilization associated with the Glu10-Lys18 pair depends primarily on the presence of a side-chain hydrogen-bond donor at residue 14; the nonpolar or hydrophobic character of residue 14 plays a smaller but still significant role. Crystal structures and molecular dynamics simulations indicate that Glu10 and Lys18 do not interact directly with each other but suggest the possibility that the proximity of residue 14 with Lys18 allows Glu10 to interact favorably with nearby Lys7. Subsequent thermodynamic experiments confirm the important role of Lys7 in the large synergistic stabilization associated with the Glu10-Lys18 pair. Our results highlight the exquisite complexity and surprising long-range synergistic interactions among b-, c-, and f-position residues within helix bundles, suggesting new possibilities for engineering hyperstable helix bundles and emphasizing the need to consider carefully the impact of substitutions at these positions for application-specific purposes.