C. J. De Feo et al.: Tryptophan Scanning of Copper Transporters A80; yCTR3: V55; and from Aller et al. 2004, hCTR1: G167, G171; yCTR3: G202, G206). However, using a singular probe, Trp, our comparative study also revealed signiﬁcant differences between the transporters. Most notably, steric constraints clustered toward the extracel- lular ends of TM1 and TM2 in yCTR3, while they were distributed more evenly along the length of both TM segments in the case of hCTR1 (Figs. 1, 4, 5).This more even distribution resulted in a larger sensitivity toward steric bulk on the intracellular side of the membrane, which was not observed in yCTR3. What gives credence to the belief that these observations were meaningful was that constraints appeared to be coupled within each transporter. That is, the higher degree of sensitivity toward the bulk of the Trp side chain at the intracellular end of TM1 in hCTR1 was accompanied by a higher degree of Trp intolerance at the spatially close intracel- lular end of TM2 (Figs. 3, 5). A similar correlation was observed for the extracellular ends of TM1 and TM2 in yCTR3 (Figs. 4, 5). It therefore seems likely that the observed differences reﬂected individual structural and/or functional adaptations within transporter subfamilies. A second example moderating a ‘‘one structure ﬁts all’’ model of CTR proteins originated from the behavior of Trp mutants with substitutions in the functionally important MetxxxMet motif at the extracellular end of TM2. Abol- ishing function in both transporters, the reasons for this outcome seemed to differ. In hCTR1, replacement with Trp of either methionine was structurally tolerated, while the equivalent mutations in yCTR3 resulted in a transporter that displayed abnormal trafﬁcking and thus was likely to be structurally compromised (Fig. 2). Consequently, the high conservation of the MetxxxMet motif among CTR proteins did not seem to translate into a strict and rigid conservation of structural context in yCTR3 and hCTR1. This is a simple ﬁnding. However, context-dependent structural differences between transporters, even in what is one of only three strictly conserved motifs within the family, can explain why the functional importance of these residues ranges from being essential to merely beneﬁcial under various experimental approaches (Eisses and Kaplan 2005; Puig et al. 2002). Related to the MetxxxMet motif and concerning mechanism, our low-resolution model suggests that the size of the apparent ‘‘pore’’ can be modulated by very small movements of TM2 or even changes in the rotamer con- formation of the Met side chains. Notably, such a ‘‘gating motion’’ may involve an ‘‘aromatic girdle’’ composed of Phe and Tyr residues (hCTR1 Phe153 & Tyr147) that appeared to facilitate packing of TM2 within the entrance to the pore. Consistent with this idea, substitution of Trp for Tyr147 in hCTR1 destabilized the protein so strongly that its expression could not be detected (Fig. 2b), while a 121 more conservative replacement by Phe or reduction of the side chain size to alanine yields transporters that transport copper at about half Vmax of WT hCTR1 (Eisses and Kaplan 2005). A third structural property that is put into perspective through our comparative study is the tight packing inter- face between TM1 and TM3. From our initial observations of the experimental structure it was clear that these TMs are packed very closely, which prompted us to propose that the conserved GlyxxxGly motif, found on TM3, might mediate this close helical apposition (De Feo et al. 2009). Our previous studies of TM3 indicated that the Gly resi- dues of the GlyxxxGly motif are critical for forming a functional and structurally intact transporter. Interestingly though, TM3 is more sensitive to steric bulk in yCTR3 than in hCTR1 (Aller et al. 2004). Intriguingly, the larger degree of steric constraint in TM3 of yCTR3 is mirrored by what we observed for TM1. Speciﬁcally, the TM1 component of the putative packing interface with TM3 was relatively insensitive to Trp in hCTR1 but not in yCTR3 (Fig. 5). By orienting TM1 such that Ala80 in hCTR1 and Val55 in yCTR3 pointed into the packing interface with the Gly- xxxGly motif on TM3, we observed that hCTR1 TM1 interface presented larger aromatic and hydrophobic resi- dues, F and V, while TM1 of yCTR3 presented smaller residues, S and C (Figs. 1, 5). This feature of our low- resolution model explained why introduction of steric bulk was more disruptive in the latter case and provided an alternative explanation for the previous observation that a double mutant C48SC51S was shown to be partially functional and structurally compromised (Pena et al. 2000). While many details of these interactions and their mecha- nistic contributions need to be established through future studies, our current study not only conﬁrmed the helix assignments proposed previously but extended them by demonstrating that TM1 serves the role of an adaptor that allowed the overall architecture of CTRs to be preserved throughout evolution. Finally, the Trp scan indicated that residues A70 and G71 for hCTR1 and A45, I49 and G50 for yCTR3 at the extra- cellular end of TM1 were sensitive to the introduction of steric bulk. At ﬁrst sight, these observations did not make much sense since these affected positions pointed away from the core of the trimer and into the lipid. However, when these mutations were considered in the context of the 2D crystal lattice of membrane-embedded hCTR1, the Trp-sensitive positions mapped to regions where hCTR1 trimers contacted each other on the lattice. While it is possible that these interactions are crystallization ‘‘artifacts,’’ it cannot cate- gorically be ruled out that interactions between CTR trimers are physiologically meaningful. For instance, lateral asso- ciation of two CTR trimers may allow coordinated transport of multiple copper ions, which may be key to effective 123