Three-dimensional structure of the human copper transporter hCTR1

Abstract

Copper uptake proteins (CTRs), mediate cellular acquisition of the essential metal copper in all eukaryotes. Here, we report the structure of the human CTR1 protein solved by electron crystallography to an in plane resolution of 7 A. Reminiscent of the design of traditional ion channels, trimeric hCTR1 creates a pore that stretches across the membrane bilayer at the interface between the subunits. Assignment of the helices identifies the second transmembrane helix as the key element lining the pore, and reveals how functionally important residues on this helix could participate in Cu(I)-coordination during transport. Aligned with and sealing both ends of the pore, extracellular and intracellular domains of hCTR1 appear to provide additional metal binding sites. Consistent with the existence of distinct metal binding sites, we demonstrate that hCTR1 stably binds 2 Cu(I)-ions through 3-coordinate Cu-S bonds, and that mutations in one of these putative binding sites results in a change of coordination chemistry.

Fig. 1.

Molecular organization of hCTR1. (A) Side view of a CTR1 dimer-of-trimers that constitutes the basic building block of the 2D-lattice (contoured at 2σ above mean). Solid black lines indicate the approximate boundaries of the hydrophobic core of the membrane, assuming a thickness of 30 Å. The orientation (topology) of the trimers in the 2 layers was derived from gold labeling of hCTR1 2D crystals (E and F) and immuno-precipitation experiments using WT and cysless hCTR1 (Fig. S3). (B) Slab of a longitudinal section through the middle of one trimer molecule illustrating the shape of the membrane-spanning pore. Red double-headed arrows mark the narrowest (≈8 Å, side chains neglected) and widest part (≈23 Å, side chains neglected) of the pore. Areas marked red correspond to extramembraneous densities that cap the pore at its ends. (C and D) 10 Å wide cross-sectional slabs taken from the extracellular (C), and intracellular (D) end of the membrane domain illustrate how the 9 TMs (three from each subunit) form the pore. (E and F) Representative examples of ImmunoGold labeling experiments demonstrating that the extracellular hemagglutinin epitope (HA) of the recombinant HA-hCTR1^N15Q is accessible on the surface of the 2D lattice (E) whereas an epitope contained in the intracellular C terminus of hCTR1 is accessible only along the edges of crystals (F).

Fig. 2.

Helix assignment. (A) Side view of a HA-hCTR1^N15Q trimer (contoured at 3σ above mean) reveals a direct connectivity (red asterisks) between 2 TM-segments on the extracellular side of the membrane. TM segments are labeled with the number of the corresponding TM segments in the sequence (1 = TM1, 2 = TM2, 3 = TM3). Assignments are shown for 2 subunits (TM123 and TM1′2′3′ respectively). (B) Top view looking down the 3-fold axis as seen from the extracellular side. The putative assignment of the subunit boundary (red triangle) and the short loop (red asterisk) connecting TM2 and 3 are shown. (C–E) Cross-sections through the membrane embedded domain. (E) Extracellular side. (D) Middle of the bilayer. (E) Intracellular side. Numbering of helices is as described for A. (F) Helical wheel plot of TM2. Nonpolar residues are colored gray. Black coloring indicates residues that are polar or possess metal binding properties. Residues His-139 and Met-154 are explicitly labeled because they were individually replaced by cysteine for disulfide cross-linking experiments. (G) Disulfide cross-linking of recombinant, purified HA-hCTR1^N15Q(WT), HA-hCTR1^{N15Q,C161A,C189A} (Cysless), HA-hCTR1^{N15Q,C161A,C189A,M154C} (M154C), and HA-hCTR1^{N15Q,C161A,C189A,H139C} (H139C). Proteins were incubated in the presence of copper phenanthroline (Cu-P) to facilitate disulfide bridge formation, separated and blotted. Ovals to the right indicate the mobility of hCTR1 oligomeric species in SDS. Similar results were obtained without use of Cu-P. Dimer formation of WT is through disulfide bridge formation between cysteines in the C-terminal HCH-sequence of 2 subunits of the hCTR1 trimer (19, 37). White separation between lanes indicates that some data were taken from separate gels.

Fig. 3.

Copper binding by hCTR1. (A and B) Fourier transform and EXAFS (Upper Inset) for WT HA-hCTR1^N15Q (A) and cysless hCTR1 (B). Black lines represent experimental data, simulations are shown in red. The appearance of a shoulder (arrow in B) indicates the involvement of Cu-N(His)coordination. Blue Insets are absorption edges in the region 20 keV below to 20 keV above the edge jump. The weak shoulder at 8.985 keV on the edges is typical of 3-coordination. Parameters used to simulate the data are given in Table S1. Only the best fit is shown for the cysless mutant (see Fig. S8 for comparison with fit for one 3-coordinate Cu–S(Met) site and one 3-coordinate Cu-N(His)-site). (C) Copper binding data obtained by independent ICP-OES experiments for WT-hCTR1 (i) and cysless-hCTR1 (ii) respectively. P, protein. Concentrations are micromolar. (D) Scheme illustrating how 3 subunits of the timer construct an appropriate geometry for copper coordination through symmetry related sulfur-bearing residues. (E) Diagram illustrating the putative trajectory of copper through the pore, and how alignment of external copper binding sites with the central pore would provide a series of checkpoints to ensure regulated copper movement through hCTR1. “MBD”s refer to the multiple metal binding residues found in the N-terminal domain, and MPM, M, and HCH refer to the amino acid residues that are involved in the formation of defined and highly conserved putative metal binding sites as discussed in the text.