1.A.56 The Copper Transporter (Ctr) Family

Copper (Cu⁺) transporters of the Ctr (also called the CopT) family are sequence diverse eukaryotic proteins that function by a channel mechanism.  Bioinformatic analyses of sequenced Ctr proteins (Dumay et al., 2006) revealed multiple paralogues in single organisms, and these may be either closely or distantly related to each other. Protein phylogeny generally correlates with organismal source and protein size with proteins of each cluster being derived from a specific eukaryotic kingdom and exhibiting characteristic domain arrangements. Some homologues exhibit repeats of the basic 3 TMS unit. Regions of conserved hydrophobicity and amphipathicity suggest functional roles, particularly for putative TMSs 2 and 3 which possess a nearly fully conserved M X₃ M motif in putative TMS2. This motif may comprise the transmembrane Cu⁺ binding site in oligomeric channels that take up Cu⁺ by a passive, membrane potential-dependent mechanism (Dumay et al., 2006). De Feo et al. (2010) have presented a mechanistic model in which copper transport occurs along the center of the trimer. Abrusci et al. 2021 proposed a tilt and shift mechanism of the helices surrounding the ion binding cavity as the working principle of the reported conformational changes in complete transporters.

Six proteins, one from Arabidopsis thaliana (CopT1, 169 amino acyl residues), one from humans (hCTR1, 190 residues), three from Saccharomyces cerevisiae (Ctr1p, 406 residues; Ctr2p, 189 residues; and Ctr3p, 241 residues) and one from Candida albicans (Ctr1p, 251 residues) have been cloned, sequenced and expressed in mutant S. cerevisiae (Dancis et al., 1994a,b; Marvin et al., 2004). They have multiple homologues in Schizosaccharomyces pombe, C. elegans, and Drosophilia melanogaster. A few of these proteins exhibit 2 or 2 1/2 repeat units of about 124 residues each. A differential subcellular localization of two copper transporters from the COPT family suggests distinct roles in copper homeostasis in the moss, Physcomitrium patens (Rosas-Santiago et al. 2021).

The Ctr6 (148 aa) protein of Schizosaccharomyces pombe is an integral membrane protein, the synthesis of which is induced by copper limitation (Bellemare et al., 2002). It can trimerize and harbors a putative copper-binding M-XC-XM-XM motif in its N-terminus that is essential for function. The physiological function of Ctr6 is to mobilize stored copper from the vacuole to the cytosol (Bellemare et al., 2002). The Ctr1 homologue of S. cerevisiae transports Cu⁺ and the platinium anticancer drug, cisplatin. A Ctr1 allele defective in copper transport enhanced cellular cisplatin accumulation. N-terminal methionine-rich motifs were reported to be dispensable for copper transport but critical for cisplatin uptake (Sinani et al., 2007). The Ctr1 multimeric complex may thus use distinct mechanisms for copper and cisplatin transport.

The H. sapiens, A. thaliana, S. cerevisiae and T. parva proteins have 3 putative transmembrane α-helical spanners and display N-terminal hydrophilic sequences homologous to the methionine and histidine-rich copper binding domains of various copper binding proteins. Human CTR1 has its N-terminus extracellular and its C-terminus intracellular (Eisses and Kaplan, 2002). It transports copper, which induces internalization and degradation, presumably as a copper protective mechanism. The chemotherapeutic drug, cisplatin (cis-diamminedichloroplatinum [DDP]), stabilizes formation of a homotrimeric complex of human Ctr1 (Guo et al., 2004). It is not clear that all of these proteins are localized to the plasma membrane, but the majority of the evidence implicates them in Cu⁺ uptake.

The homotrimeric Ctr1 of humans appears to bind Cu⁺ in the channel, altering transport rates. The hydrophilic N- and C-termini are non-essential for transport but play a role in delivering Cu⁺ to the transport pathway. A model suggests that a Cu⁺ binding site, close to the intracellular exit site, undergoes a conformational change that is rate limiting for transport (Eisses and Kaplan, 2005). Complexation with Cu⁺ induces a change in the secondary structure of the Cys/Trp motif, which results in the peptide embedding in the lipid bilayer (Okada et al. 2018).

Cu⁺ is taken up via an energy (ATP) independent process, and the trimeric complex probably uses a channel-type mechanism (Nose et al., 2006). For example, human high affinity (~3 μM) Ctr1, a homotrimer, takes up Cu⁺ across the plasma membrane by an energy-independent mechanism that is stimulated by extracellular acidic pH and high K⁺ concentrations (Lee et al., 2002). The mouse Ctr1 transporter is essential for copper homeostasis and embryonic development (Andrews, 2001; Kuo et al., 2001; Lee et al., 2001). The Arabidopsis CopT1 protein plays a role in root elongation and pollen development, revealing a role for copper acquisition in these processes (Sancenón et al., 2004).

In S. pombe, two proteins, Ctr4 and Ctr5, together comprise a heteromeric Cu⁺ uptake transporter. Both proteins exhibit regions of strong sequence similarity with Ctr3 of S. cerevisiae. They exhibit 2 and 3 putative TMSs, respectively, and are coregulated by copper and the Cuf1 transcription factor. They have been shown to physically interact to yield the transporter (Zhou and Thiele, 2001). The fact that Ctr proteins have only 3 TMS per polypeptide chain argues for a channel-type mechanism (Nose et al., 2006).

Distantly related to the proteins described above, two proteins, Ctr1p of Saccharomyces cerevisiae and Ctr1p of Candida albicans were initally characterized as high affinity Cu⁺ uptake permeases. Ctr1p of S. cerevisiae possesses N-terminal repeats rich in serine and methionine that might be involved in copper binding. This Ctr1p includes a repeated M-XXM motif that occurs 11 times. The shorter C. albicans homologue has an MXM motif that occurs 6 times, and an MXMXM motif that occurs twice. Two or three putative transmembrane α-helical spanners in the S. cerevisiae homologue (residues 153-173 and either 251-271 or 241-261 and 262-281) and a C-terminal region that is O-glycosylated characterize the protein. The C. albicans protein also has these 3 TMSs. Ctr1p of S. cerevisiae is probably present in the membrane as an oligomer. The synthesis of Ctr1p is induced by copper deficiency and repressed by copper excess. The protein transports Cu⁺, but not Cu²⁺ or any other metal ion. The N-terminal repeat sequences noted above are found in a number of proteins including putative DNA binding proteins.

Wu et al., 2009 have provided evidence that in response to excess copper, yeast Ctr1-mediated copper transport is rapidly blocked in a C terminus-dependent mechanism associated with direct binding of copper. They suggest that conformational changes in the cytosolic tail of yeast Ctr1 allows copper sensing within this domain and leads to the inhibition of Ctr1-mediated copper transport. This regulatory mechanism may allow yeast cells to maintain homeostatic levels of copper.

Electron crystallography with human Ctr1 revealed the 6 Å resolution structure (Aller and Unger, 2006; De Feo et al., 2007). It is trimeric with 9 TMSs, three per subunit. It has radial symmetry like other ion channels such as K⁺ and gap junction channels. A region of low density at the center of the trimer suggests that the pore is along the center of the 3-fold axis of the trimer (De Feo et al., 2007).

De Feo et al., 2009 reported an additional structure of the human CTR1 protein, solved by electron crystallography to an in plane resolution of 7 A. Trimeric hCTR1 forms a pore that stretches across the membrane bilayer at the interface between the subunits. The second transmembrane helix is probably the key element lining the pore. How functionally important residues on this helix participate in Cu(I)-coordination during transport was suggested. Aligned with and sealing both ends of the pore, extracellular and intracellular domains appear to provide additional metal binding sites. Consistent with the existence of distinct metal binding sites, hCTR1 stably binds 2 Cu(I)-ions through 3-coordinate Cu-S bonds. In a minireview, Kaplan and Lutsenko (2009) have discussed the molecular mechanisms by which copper enters and exits animal cells.

Yang et al. (2012) determined the 3-d structure and oligomerization of the transmembrane domains (TMDs) of hCtr1 using solution-state NMR spectroscopy. TMS1 forms an α-helical structure from Gly67 to Glu84 and is dimerized by close packing of its C-terminal helix; TMS2 forms an α-helical structure from Leu134 to Thr155 and is self-associated as a trimer by the hydrophobic contact of TMS2 monomers; TMS3 adopts a discontinuous helix structure, known as 'α-helix-coiled segment-α-helix', and is dimerized by the interaction between the N-terminal helices. The motif GxxxG in TMS3 is partially unstructured as a linker between helices. The flexible linker of TMS3 may serve as a gating adapter to mediate a pore on and off switch. The differences in the structure and aggregation of the TMS peptides may be related to their different roles in channel formation and transport function (Yang et al., 2012).

The trimeric hCtr1 transports copper and silver with TMS 2 lining the central pore, and the MXXXM motif in the C-terminal end of TMD2 being important for metal binding and function. A trimer of the isolated hCtr1-TMD2 forms in SDS micelles which binds Ag⁺ in a stoichiometry of 3:2 for peptide:Ag^{+ (Dong et al. 2015)}.  The N-terminal, extracellular regions of eukaryotic high affinity copper transport (Ctr) proteins vary in composition of the Cu⁺ binding amino acids: methionine, histidine, and cysteine. To examine why certain amino acids are exploited over others in Ctrs from different organisms, the relative Cu⁺ binding affinities and the dependence of binding on pH were examined for 3 peptides of the sequence MG(2)XG(2)MK, where X is either Met, His, or Cys. Cu⁺ affinity was examined (Rubino et al., 2010; 2011). The relative affinities of the peptides with Cu⁺ proved to be Cys > His > Met at pH 7.4 but Cys > Met > His at pH 4.5. Ligand geometry and metric parameters were determined with X-ray absorption spectroscopy.

A comprehensive phylogeny and a molecular structure analysis of the Ctr (COPT) family in plants and animals has been presented with an emphasis on the copper transporters in Populus trichocarpa (PtCOPT). Structural analyses of PtCOPTs showed that most have 3 TMSs, with an exception of PtCOPT4 (2 TMSs). Tandem and segmental duplications probably contributed to the expansion and evolution, and promoter analyses showed that the function of PtCOPTs is related to Cu and Fe transport. The genes are expressed at high levels in roots and leaves. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that the expression of PtCOPT genes were induced not only in limited and excessive Cu, Fe, zinc (Zn) and manganese (Mn) stress, but also in lead (Pb), and cadmium (Cd) stress (Zhang et al. 2015).

The generalized transport reaction catalyzed by proteins of the Ctr family is:

Cu⁺ (out) → Cu⁺ (in)