C(alpha)-trace model of the transmembrane domain of human copper transporter 1, motion and functional implications

Abstract

The trimeric human copper transporter 1 (hCTR1) is essential for copper uptake and is implicated in sensitivity to chemotherapy drugs. Using the cryoelectron microscopy (cryoEM) map of hCTR1 and evolutionary data, we constructed a Calpha-trace model of the membrane region. The model structure, supported by mutagenesis data, was used to investigate global dynamics through elastic network models. Identified as dominant hinge regions, hCTR1's MxxxM and GxxxG motifs were shown to have significant roles in functional movements characterized by the two slowest modes of motion. Both modes predicted significant changes at the wide cytoplasmic pore region; the slowest mode introduced a rotational motion around the pore central axis, whereas in the following mode the cytoplasmic parts of the helices approached and moved away from the pore center. In the most cooperative mode, the MxxxM motif in the extracellular narrow region remained static. The second mode of motion, however, predicted a cooperative rotational motion of this copper-binding motif, possibly reflecting activation at the pore's extracellular entrance. We suggest a molecular mechanism of copper transport in which this motif serves both as a gate and as a selectivity filter. We also suggest residues that are responsible for pH activation.

Fig. 1.

Evolutionary conservation profile of the hCTR1 full sequence and TM model. In both panels, the coloring is according to the evolutionary conservation scores calculated via the ConSurf server (, http://consurf.tau.ac.il), with turquoise-to-maroon signifying variable-to-conserved positions (shown by the color bar). (A) The predicted topology of hCTR1. The three TM segments are marked. (B) Intracellular view of the Cα-trace model of the TM domain hCTR1, fitted onto the cryoEM map (9) (mesh). Residues of the MxxxM and GxxxG motifs are shown as spheres, and the three TM helices are marked on one of the subunits.

Fig. 2.

Validation of the model structure via mutagenesis data. Mutagenesis data mapped on the hCTR1 model. Three different views are presented. Cα-atoms of residues for which mutational data are available are shown as spheres and colored according to the functional effect of the mutation, with red and green depicting positions sensitive and nonsensitive to substitutions, respectively. Positions in which mutations induced partial function are colored yellow.

Fig. 3.

Square displacements and cooperative dynamics of the most cooperative GNM mode (mode 1,2). (A) Square displacements of mode 1,2. Prominent hinges Met150 and Met154 are marked, as well as the three chains. (B) Cooperative dynamics in mode 1,2. The three TM helices and chains are marked. Red-to-blue (according to the color bar) indicates positive-to-negative correlation of the structural dynamics between each residue pair. TM1, TM3 and the C-terminus of TM2 within each subunit are positively correlated with the N-terminus of TM2 of one of the two neighboring subunits. (C) Mapping of cooperative dynamics on the model. The hCTR1 model is shown as ribbons and colored according to the correlation of TM1 of chain A (TM1A) with the other residues. TM helices that showed positive correlation with TM1A are marked. Cα atoms of residues of the MxxxM and GxxxG motifs, as well as Glu84 and His139, are shown as spheres.

Fig. 4.

The ANM mode matched to GNM mode 1,2 (Table S4 and Movie S1). The conformations predicted by the ANM mode associated with GNM mode 1,2 (Table S4) are colored white, with approximated hinges at the MxxxM motif region in gray. As in GNM mode 1,2, motion was divided into three separate structural elements, each consisting of TM1 and TM3 from the same subunit, along with the N-terminus of TM2 of an adjacent subunit (marked). Conformations of one such structural element are colored according to the direction of motion, ranging from yellow to green and back. (A) Cytoplasmic view. (B) Side view. (C) Extracellular view.

Fig. 5.

Functional and cooperative dynamics of the third GNM and corresponding ANM modes. (A) Square displacements of the third GNM mode; the hinge positions are indicated on chain A. (B) Cooperative dynamics in GNM mode 3, colored as in Fig. 3B. Interresidue correlations are shown on one subunit only; the same correlations were obtained for the other two. The boundaries of the TM helices are marked. The cytoplasmic and the extracellular regions of the helices show opposite correlations. (C) The corresponding ANM mode (Table S4, Movie S2, and Movie S3). The conformations are colored according to the cooperative dynamics of GNM mode 3 and shown in three different views. The arrows mark the directions of the motions of the cytoplasmic (blue) and extracellular (red) helix regions.

Fig. 6.

Transport mechanism. In the proposed mechanism the copper ions are transported one at a time, and the TM region of hCTR alternates between four conformations. The three subunits are shown in red, blue, and gray, with residues of interest depicted in dark gray and the copper ion in yellow. Step 1: In the basal state, ions do not bind to the TM domain, the Met154 triad serves as an external gate and (unprotonated) His139 does not interact with Glu84. Our model structure is suggested to depict the basal conformation, because it was computed from a cryoEM map determined at pH 7.4 (9), a condition in which hCTR1 exhibits low activity according to Lee et al. (8). Step 2: Activation resulting, for example, from pH shift or ion binding, involves conformation change at the cytoplasmic end, along with a rotational movement at the extracellular end. A copper ion binds to the Met154 triad, and (protonated) His139 interacts with Glu84, stabilizing the conformation. Step 3: Following another conformational change at the extracellular end, the copper ion is passed on to the Met150 triad and the Met154 closes the pore entrance, thereby preventing the entry of a second ion. Step 4: The copper ion passes freely through the polar pore. It is yet to be revealed how copper ions are passed through to the C-terminus and from there to the copper chaperones.