C. J. De Feo et al.: Tryptophan Scanning of Copper Transporters 119 Fig. 3 Visualization of Trp-mutation phenotypes in the context of the hCTR1 3D structure. Straight a-helical segments were modiﬁed to create a pdb of hCTR1 helices that ﬁt into the EM map of hCTR1 (see ‘‘Experimental Procedures’’ section for more detail) to help visualize the Trp-mutation phenotypes obtained in this study. a The electron density (gray mesh) of two hCTR1 trimers making 2D crystal lattice contacts (*3r). b The pdb of the helices representing hCTR1 (shown as ribbons) after ﬁtting into the 3D hCTR1 density map (gray surface, *3.5r). Positions that were tested in this study and where introduc- tion of Trp had no phenotypic effect are colored gray. Positions where introduction of Trp caused a phenotype are colored magenta, with side chains shown (b). Positions that were either not tested or part of our previous study are labeled in black. The position of the GlyxxxGly motif in TM3 is indicated by a yellow arrow. Black arrow points to the Met residues of the MetxxxMet motif on TM2. c– e Different surface views of the trimer model: c looking down through the pore from the extracellular side, d a side view and e a view looking up through the pore from the intracellular side. The coloring scheme is the same in b except mutated residues are displayed by surface representation and all else are kept in ribbon the register of the helices, we used the close apposition of TM1 and TM3 in the region of the GlyxxxGly motif and the proximity of the end of TM2 to its extracellular con- nectivity with TM3 (De Feo et al. 2009). After assembly of this frame, which was based on only one constraint to ﬁx the azimuthal orientation of each helix, we mapped the positions identiﬁed by the Trp scan onto the scaffold without making any further adjustments because the inherent inaccuracy of the experimental density map pre- cludes generation of an accurate model. The rationale was that if the observed outcomes of the scan were ‘‘random,’’ then mapping of both functional and nonfunctional phe- notypes onto the known low-resolution structure of hCTR1 should appear just as ‘‘random.’’ However, this was not what was observed. Instead, the majority of the Trp-sen- sitive positions pointed into the pore or into packing interfaces between helices (Figs. 3, 4).Previously reported data for the scan of TM3 of yCTR3 were fully consistent with this pattern in that the sterically constrained helix face bearing the GlyxxxGly motif composed the tight interface with TM1, while less constrained positions resided in the looser interface with TM2, leaving most of the nonsensitive positions lipid-exposed, as was predicted (data not shown; Aller et al. 2004). Mapping of Trp-sensitive positions on TM1 revealed a region at its extracellular end where the constraints pointed toward the lipid-exposed face of the molecule (hCTR1: A70, G71; yCTR3: A45, I49, G50). This result was unexpected and puzzling at ﬁrst. However, when put into the context of the crystallographic infor- mation that is available from 2D crystals of membrane- embedded hCTR1, the Trp-sensitive residues at the extra- cellular end of TM1 corresponded to positions that partake in interactions between neighboring hCTR1 trimers in the 2D lattices (Figs. 3, 4) (De Feo et al. 2009). The fact that hCTR1 and yCTR3 were both sensitive toward steric bulk in this region suggested that some aspect of copper uptake through CTRs may involve interactions between CTR tri- mers or possibly other functionally important membrane proteins localized at the plasma membrane with CTRs. This is a novel ﬁnding and will be pursued in future studies. Discussion Copper is essential for life. Yet, exactly how cells acquire this essential metal ion and how platinum-based chemo- therapeutics manage to hijack the transporters for copper uptake remain largely unknown. The biggest impediment to the study of the mechanisms of copper and cisplatin uptake is the lack of high-resolution structural information 123