Combination of Cα–H Hydrogen Bonds and van der Waals Packing Modulates the Stability of GxxxG-Mediated Dimers in Membranes

[Image: see text] The GxxxG motif is frequently found at the dimerization interface of a transmembrane structural motif called GAS(right), which is characterized by a short interhelical distance and a right-handed crossing angle between the helices. In GAS(right) dimers, such as glycophorin A (GpA), BNIP3, and members of the ErbB family, the backbones of the helices are in contact, and they invariably display networks of 4 to 8 weak hydrogen bonds between Cα–H carbon donors and carbonyl acceptors on opposing helices (Cα–H···O=C hydrogen bonds). These networks of weak hydrogen bonds at the helix–helix interface are presumably stabilizing, but their energetic contribution to dimerization has yet to be determined experimentally. Here, we present a computational and experimental structure-based analysis of GAS(right) dimers of different predicted stabilities, which show that a combination of van der Waals packing and Cα–H hydrogen bonding predicts the experimental trend of dimerization propensities. This finding provides experimental support for the hypothesis that the networks of Cα–H hydrogen bonds are major contributors to the free energy of association of GxxxG-mediated dimers. The structural comparison between groups of GAS(right) dimers of different stabilities reveals distinct sequence as well as conformational preferences. Stability correlates with shorter interhelical distances, narrower crossing angles, better packing, and the formation of larger networks of Cα–H hydrogen bonds. The identification of these structural rules provides insight on how nature could modulate stability in GAS(right) and finely tune dimerization to support biological function.