Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer. To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer). In both constructs, the GXXXG motif is necessary for TM-TM association. Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXG-VXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold. Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity. Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and ϳ18 Å in structural space), work in concert to control TM-TM affinity synergistically.After their biosynthesis and subsequent integration into a membrane, many transmembrane (TM) 1 helices associate with other pre-formed helices to form functional membrane protein domains (1). Specificity for a given helix-helix interaction is achieved through the appropriate presentation of complementary side chains, which serve as recognition elements between associating helices. The most highly studied, and apparently widespread, mode of association is mediated by the so-called GXXXG motif, which is known to act as a universal scaffold for the assembly of both TM helices (2-9) and soluble ␣-helices (10). The GXXXG motif, where two glycine residues are separated by any three amino acids on a helical framework, gives rise to a flat surface region on one face of the helix. This arrangement of Gly residues permits the close approach of interacting helices, whereupon extensive packing interactions take place between pairs of surrounding residues. It has been proposed that a portion of the interactive strength of GXXXGmediated associations may originate from inter-helix hydrogen bonds between C␣ hydrogens and carbonyl oxygen atoms on the adjacent helix (11).The glycophorin A transmembrane (GpA-TM) segment is a well characterized transmembrane helix dimer that associates with high affinity, principally by using a central GXXXG motif (3,12). The details of side chain-side chain packing for GpA are known in considerable detail, having been gleaned originally from extensive mutagenesis experiments (3), computer modeling (13, 14) and, subsequently, from a high-resolution structural analysis using nuclear magnetic resonance (NMR) for the GpA dimer in both detergent micelles (12) and lipid bilayers (15).Despite the high occurrence of the GXXXG motif in transmembrane helices (7), GpA-TM is the only GXXXG peptide dimer with a structure det...