Aberrant transmembrane helix-helix interactions as a biophysical cause of human disease

Mutations that alter transmembrane (TM) helix-helix interactions can result in disease by compromising protein structure/function. Recent reports showed that TM-embedded polar residues play powerful roles in TM domain (TMD) folding by forming interhelical H-bonds (reviewed in (Partridge et al., 2002b)). Thus, mutations involving these species could result in disease by disrupting the pattern of native electrostatic links. To explore this possibility, we performed a statistical analysis of the phenotypic missense mutations listed in the human gene mutation database. We found that both polar to non-polar and non-polar to polar mutations are more often associated with disease in TMDs compared to soluble domains. This suggests that, in TMDs, both the disruption of native structure-stabilizing H-bonds and the formation of non-native structure-destabilizing H-bonds are frequent causes of human disease. To experimentally investigate these concepts, we developed a peptide-based system for the study of TM helix-helix interactions. This 'Lys-tagged' approach involves the addition of several Lys residues to both the N- and C-termini of a single TM helix. The resulting species are water-soluble, a property that greatly facilitates their purification and characterization. Furthermore, such peptides retain the ability to insert into membrane-mimetic environments to assume their native-like secondary and tertiary structures. Using such peptides, we showed that when applied to TM4 from the cystic fibrosis conductance regulator, the apolar to polar phenotypic mutation V232D induces the formation of multiple non-covalent oligomeric states through the formation of an interhelical H-bonding network. The powerful effect of this mutation is context-specific, since randomized sequences containing the analogous interhelical H-bonding components could not form similar oligomeric assemblies. Studies on the TM helix from myelin protein zero (PO) and its phenotypic mutation show that an apolar to polar mutation can similarly disrupt native state oligomeric states through mechanisms not involving interhelical H-bonds. We demonstrate that the native P0-TM helix forms a tetrameric bundle and that the phenotypic mutation G163R prevents this assembly through steric clash interactions. The research presented suggests that the proper packing of TM helices is vital to protein function and that TM-embedded mutations involving polar residues manifest their deleterious effects through a variety of mechanisms.