Insights into the molecular structure of copper uptake in eukaryotes

Members of the copper uptake transporter family (CTR) from yeast, plants and mammals, including human, are required for cellular uptake of the essential metal copper. Based on biochemical data, CTRs have three transmembrane domains and have been shown to oligomerize in the membrane. Among individual members of the family there is little amino acid sequence identity, raising questions as to how these proteins adopt a common fold, oligomerize and participate in copper transport. This work entails a two pronged approached to probe the structure-function relationships of two members of the CTR family - human CTR1 and yeast CTR3. The first half of this work employs site directed mutagenesis, tryptophan scanning, genetic complementation, subcellular localization, chemical crosslinking, and the yeast unfolded protein response to demonstrate that at least half of the third transmembrane domain (TM3) plays a vital role in CTR structure and function. Two functionally distinct faces of TM3 were identified. One face bears a highly conserved Gly-x-x-x-Gly (GG4) motif, which was shown to be essential for CTR oligomerization. Moreover, steric constraints reach past the GG4-motif itself, including amino acid residues that are not conserved throughout the CTR family. A second face of TM3 contains three amino acid positions that when mutated to tryptophan cause predominantly abnormal localization but are still partially functional in growth complementation experiments. These mutations cluster on the face opposite to the GG4-bearing face of TM3 where they may mediate interactions with the remaining two transmembrane domains. Taken together, the data presented here support TM3 as being buried within trimeric CTR where it plays an essential role in CTR assembly.; The second part of this work initiates the first direct structural analysis of this family of proteins. Overexpression and purification protocols of human CTR1 were developed to allow two-dimensional crystallization of the protein in the presence of purified phospholipids. The projection structure obtained for hCTR1 using electron crystallography of two-dimensional protein crystals in a native phospholipid bilayer was solved to 6 A resolution. This first glimpse of the architecture of human CTR1 reveals a highly symmetrical trimer that is less than 40 A wide. Notably, the center threefold axis of each trimer forms a region of very low electron density likely to be involved in copper translocation. The formation of the putative pore for metal ions at the interface of three identical subunits deviates from the structural design of typical transporter proteins and reveals that copper uptake transporters have a novel architecture that is structurally more closely related to channel proteins.