120 C. J. De Feo et al.: Tryptophan Scanning of Copper Transporters Fig. 4 Trp-sensitive positions on yCTR3 reveal a common core structure for CTRs. a, b A pdb of the helices in yCTR3 manually ﬁt into hCTR1’s electron density (see ‘‘Experimental Procedures’’ section). The color scheme is the same as in Fig. 3. a A view from the extracellular side of the membrane looking down into to the cytosol. b A view looking up through the pore from the intracellular side. c, d Comparison of the complementation phenotype of both HA- hCTR1 and yCTR3-GFP. Positions where an introduction of Trp yielded the same response/phenotype in both transporters are colored gray. Positions where the two proteins showed different phenotypes are colored orange. Black color and ribbon-only representation refer to positions that were not tested in this study. Differences were plotted using the pdb model of hCTR1 as a template. The views in c and d are the same as a and b, respectively, except only one CTR trimer is displayed for clarity on CTR proteins. Similarly, the lack of an in vitro system presents additional challenges toward detailed structure– function studies. Facing these shortfalls, two major options exist: defer work until a high-resolution structure becomes available or attempt to generate a low-resolution structural framework to guide the formulation of testable hypotheses. We decided to pursue the latter and to combine results from our previous structural and biochemical work with the results of an extensive, comparative Trp scan. No doubt, mapping of phenotypic responses (functional vs. nonfunc- tional) onto generic a-helices, which in turn were mapped onto the 7A˚ structure of hCTR1 to form a low-resolution 3D trimer model, is limited in what it can accomplish. It clearly cannot provide understanding of what governs CTR1 folding, oligomerization or trafﬁcking. However, use of a comparative approach should minimize the num- ber of ‘‘unintelligible’’ results and therefore should identify regions of the transporters that are likely to be important for helix–helix packing and/or function. Moreover, if constructed using a minimal set of azimuthal constraints, then the low-resolution trimer model either would or would not be consistent with the observed Trp-scanning data. If the model were not consistent, it would need to be revised. However, if mapping of the Trp-scan data were to return credible patterns, as it did, then this work would provide a comprehensive, experimental validation of the helix 123 Fig. 5 TM1 is an adaptor helix in CTRs. Side-by-side comparison of Trp sensitivity within monomers of hCTR1 (a) and yCTR3 (b). Only the helical backbone is shown. Positions where introduction of steric bulk resulted in a phenotype are marked magenta. Positions plotted in black were of residues that were not tested in this study. Data for TM3 of both CTRs were taken from our earlier study (Aller et al., 2004). In the case of hCTR1, phenotypic responses for glycines of GlyxxxGly are of substitutions signiﬁcantly smaller than Trp. Double arrow indicates the possible helix-packing contact between TM1 and TM3 (see text). Helices are labeled according to their identity in the structure, and intra- as well as extracellular ends are indicated. The ﬁgure visualizes that sensitivity to steric bulk was correlated and, in the case of yCTR3, most sensitive positions clustered at the extracellular side of the membrane assignment that we put forth in the interpretation of our 7 A˚ density map of hCTR1 (De Feo et al. 2009). Even more, a consolidated model would yield further evidence that copper transport occurs along the central pore, clarify the question of how similar the structures of different CTR proteins are, identify residues that may warrant further structure–function studies and provide a framework for the generation of testable mechanistic hypotheses. These rea- sons were compelling enough to carry out the Trp scan and to generate the ﬁrst experimentally validated low-resolu- tion model of CTR trimers. Previous work in several laboratories has shown that the basic membrane topology appears conserved throughout the CTR family of transporters (Eisses and Kaplan 2002; Klomp et al. 2003; Pena et al. 2000; Petris 2004; Puig et al. 2002; Puig and Thiele 2002). Despite similar mechanistic features, however, only three sequence motifs are highly conserved throughout the family, raising the question of how strictly the structures of CTR proteins are conserved (Aller et al. 2004; Eisses and Kaplan 2002; Pena et al. 2000; Puig et al. 2002). Our comparative Trp-scan data are consistent with the notion that the membrane-embedded domains of CTR proteins share a common overall structure. For instance, a signif- icant number of positions where introduction of steric bulk interfered with CTR function corresponded to resi- dues that line the putative copper-permeable pore in both transporters (hCTR1: S146, M150, M154; yCTR3: S181, M185, M189) or pointed into packing interfaces (hCTR1: