The identification of the homodimer interface provides insight into the nature of the interactions that stabilize the EBC5-16 dimer. Transmembrane helix homodimerization is typically mediated by van der Waals interactions and various types of hydrogen bonds. Although Ser25 lies in the homodimer interface of EBC5-16 and its side-chain has hydrogen bonding potential, it does not appear to increase dimerization of EBC5-16 via interhelical hydrogen bonding. Substitution of the Ser25 to alanine,10-Deacetylbaccatin III which cannot hydrogen bond, does not affect the activity of EBC5-16. Furthermore, in the preferred model of the EBC5-16 homodimer, the serine side-chain hydrogen bonds with the polypeptide backbone on the same helix. Thus, the small sidechains of serine and alanine at position 25 appear to allow the helices to approach one another more closely and form more favorable packing contacts. In contrast, replacement of serine with several large hydrophilic amino acids capable of hydrogen bonding abolished activity. We also note that the orientation of several of the other side-chains in the interface is markedly different in the EBC5-16 model compared to TC2-3. This side-chain rearrangement may also contribute to more optimal packing of the helices and the formation of additional van der Waals contacts that stabilize the dimer. Similarly, in other systems, van der Waals interactions can make a significant contribution to the tight packing of Tazarotene transmembrane dimers, and conservative amino acid substitutions at such tightlypacked positions can affect the ability of a transmembrane protein to dimerize. Two glycine residues and the proline are predicted to lie in the EBC5-16 homodimer interface and are required for EBC5-16 activity. Although glycine and proline can be helix-disrupting in soluble proteins, this does not appear to be the case for EBC5-16. Glycine is readily accommodated in helices in hydrophobic environments. Notably, a GxxxG motif is present in.30% of all transmembrane domains and facilitates dimerization by permitting the close approach of transmembrane helices, providing a relatively flat surface for tight interhelical packing interactions and allowing larger neighboring side-chains to participate in favorable van der Waals interactions. b-branched residues adjacent to these glycine residues in GxxxG motifs, such as isoleucine, valine, and threonine, are also important for homodimerization of transmembrane helices, including the GpA transmembrane domain.