C. J. De Feo et al.: Tryptophan Scanning of Copper Transporters apposition with TM1. TM1 was then positioned to align its Trp-sensitive positions with the tight packing interface with TM3. The helices for the monomer were then rotated ±120 and manually placed in the remaining electron densities to ﬁll in the trimer. Finally, the helices for the trimer were rotated 180 and manually placed within the densities for the adjacent hCTR1 molecule in the crystal lattice. The helices for yCTR3 were manually aligned with those of hCTR1. All visualizations were performed using Chimera (Pettersen et al. 2004). Results Trp Mutations throughout TM1 and TM2 Confer Nonviability in Growth Complementation for both Yeast and Human CTR The lack of extensive sequence identity between different CTR transporter subfamilies has been an impediment to their study because it precluded rational structure–func- tion studies (De Feo et al. 2007; Dumay et al. 2006; Puig and Thiele 2002). We therefore sought to obtain an unbiased map of structurally and/or functionally important residues in the membrane-embedded domain of CTR proteins with a focus on those regions that had not been studied extensively in the past. Speciﬁcally, we extended our previous study of TM3 (Aller et al. 2004) and sub- jected the TM1 and TM2 helices to Trp-scanning analy- sis. We cannot rule out that this analysis missed a few residues at the ends of some TM segments because the precise boundaries of each TM helix have not been established experimentally. Nevertheless, our analysis covered those parts of each segment that were deemed the most important for forming the core of the membrane- embedded domain. We chose this approach because the steric bulk of Trp tends to identify helix–helix interac- tions (Choe et al. 1995; Irizarry et al. 2002; Monks et al. 1999), which seemed useful for the validation of our previous helix assignment in the density map of human CTR1 (De Feo et al. 2009). As for any scanning approach, results are descriptive and may be difﬁcult to explain without a high-resolution structure. To compen- sate for this shortcoming, we chose to analyze TM1 and TM2 from two distantly related CTR proteins: human CTR1 and yeast CTR3 (yCTR3). These two transporters share only 35% sequence identity within the two TM segments and are widely believed—yet not proven—to have nearly identical structures. If the latter were true, then corresponding residues should yield similar results. Similarly, if differences were observed, then these dif- ferences may reﬂect differences in the structures (and/or 117 dynamics) of these two transporters, which would be important to consider when interpreting the results of biochemical studies. To broadly assess the functionality of mutant CTRs, we used genetic complementation of yeast that lacked high-afﬁnity copper uptake transporters (Dctr1,3; Aller et al. 2004) because alternative assays, such as uptake measurements of the short-lived isotope Cu64, would have been impractical at the scale of this study, which evalu- ated [70 mutant CTR proteins. For simplicity, mutants were deemed nonfunctional if they failed to restore growth of the Dctr1,3 strain on nonfermentable carbon sources in the presence of the copper-speciﬁc chelator BCS. For this classiﬁcation, we did not differentiate between failure that was caused by a structural defect and that due to substitution of functionally important residues while otherwise maintaining the structural integrity of the trimeric transporter. Using this criterion, multiple posi- tions within TM1 and TM2 were found to be sensitive to the presence of steric bulk in both transporters (Fig. 1).Insertion of the bulky Trp into TM1 resulted in nonfunctional phenotypes for seven out of 19 positions tested for both the GFP-tagged yCTR3 (yCTR3-GFP) and the HA-tagged hCTR1 (HA-hCTR1). In TM2, introduc- tion of Trp yielded nonfunctional transporters for ﬁve out of 17 positions in yCTR3-GFP and seven out of 17 for HA-hCTR1. Thus, the total number of positions where introduction of Trp had a negative impact on the struc- ture/function of the transporter was similar in both pro- teins and for both TM segments. This ﬁnding supported the belief that the transporters share a common overall structure. However, the results also suggested that the structures of the transporters are not identical since the distribution of positions that were sensitive toward steric bulk differed between the two transporters. In yCTR3 most of the positions were clustered close to the predicted extracellular ends of TM1 and TM2, while the constraints were more evenly spread along the helices in the case of hCTR1. These differences did not appear to be caused by the presence of the GFP or HA tags since the tagged but otherwise unmutated constructs (yCTR3:GFP and HA- hCTR1) were fully functional in the complementation assay. Interestingly, one of the ‘‘homologous’’ positions where Trp negatively affected the structure/function in both transporters was located around the midpoint on TM1 (yCTR3 Val55, hCTR1 Ala80), where it comes into close contact with TM3 in the hCTR1 structure. Aware of the limitations of the approach, the scanning results nevertheless suggested that Val55 (yCTR3) and Ala80 (hCTR1) might contribute to the TM1:TM3 packing interface in both transporters (see double arrows in Fig. 5). 123