Oligomerization and analysis of the dimer interface of the human ABC half transporter ABCG2

Human ABCG2 is a 655 amino acid protein belonging to the ATP binding cassette (ABC) transporter superfamily. ABCG2 is expressed at the plasma membrane of cells where it functions to export compounds from the cell into the extracellular environment. Most ABC transporters have two cytosolic nucleotide binding domains (NBD) where ATP, the energy source for powering the transport cycle, is bound and hydrolyzed. They also have two transmembrane domains (TMD) that span the bilayer and form the environment for substrates to be recognized and transported. ABCG2 is a half transporter because it only has one NBD and one TMD. Half transporters must heterodimerize with a partner or homodimerize with themselves in order to form a functional transport complex. No heterodimer partner has been found for ABCG2, and when expressed alone in a recombinant system it is functional. These data suggest that the minimal functional unit of ABCG2 is a homodimer. In this dissertation, we investigated the oligmerization pattern of ABCG2 and probed which segments of the transporter contribute to the oligomer interface. Using chimeric dimer constructs of ABCG2, we showed that ABCG2 is able to function in the dimer state and, unlike other related proteins, ABCG2 did not need a flexible linker between the two monomers. Furthermore, using the chimeric dimers we found that ABCG2 required two functional ATP sites in order to transport the usual array of substrates. We explored the various parts of the transporter and found that the factors mediating dimerization are complex. Cross-linking analysis revealed that there are probable interactions in the nucleotide binding domain, the transmembrane domain, and the extracellular loop. We found that there are residues in the nucleotide binding domain that contribute to substrate specificity, in particular V186 and E190. Secondly, a common glycine-containing dimerization motif found in the first transmembrane segment of ABCG2 could be mutated (the glycines changed to alanines), without interfering with dimer formation as observed by cross-linking analysis. We expanded upon this work to show that disruption of this motif, when performed in conjunction with additional mutations, had a larger impact on activity and therefore possibly dimer formation. Finally, mutagenesis of cysteine residues in the extracellular domain of ABCG2 led to changes in activity and conformation, suggesting a role for the extracellular loop in proper assembly of the proteins. Overall, our findings contribute to the understanding of the oligmer state of ABCG2, and provide information about some of the specific interactions that occur in the dimer interface