The dimerization interface of the glycoprotein Ibβ transmembrane domain corresponds to polar residues within a leucine zipper motif

Experiments with the transmembrane (TM) domains of the glycoprotein (GP) Ib-IX complex have indicated that the associations between the TM domains of these subunits play an important role in the proper assembly of the complex. As a first step toward understanding these associations, we previously found that the Ib beta TM domain dimerized strongly in Escherichia coli cell membranes and led to Ib beta TM-CYTO (cytoplasmic domain) dimerization in the SDS-PAGE assay, while neither Ib alpha nor IX TM-CYTO was able to dimerize. In this study, we used the TOXCAT assay to probe the Ib beta TM domain dimerization interface by Ala-and Leu-scanning mutagenesis. Our results show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids. Mutating either one of polar residues Gln129 or His139 to Leu or Ala disrupted Ib beta TM dimerization dramatically, indicating that polar residues might form part of the leucine zipper-based dimerization interface. Furthermore, these specific mutational effects in the TOXCAT assay were confirmed in the thiol-disulfide exchange and SDS-PAGE assays. The computational modeling studies further revealed that the most likely leucine zipper interface involves hydrogen bonding of Gln129 and electrostatic interaction of the His139 side chain. Correlation of computer modeling results with experimental mutagenesis studies on the Ib beta TM domain may provide insights for understanding the role of the association of TM domains on the assembly of GP Ib-IX complex.