Structural and mechanistic basis of proton-coupled metal ion transport in the SLC11/NRAMP family

Secondary active transporters of the SLC11/NRAMP family catalyse the uptake of iron and manganese into cells. These proteins are highly conserved across all kingdoms of life and thus likely share a common transport mechanism. Here we describe the structural and functional properties of the prokaryotic SLC11 transporter EcoDMT. Its crystal structure reveals a previously unknown outward-facing state of the protein family. In proteoliposomes EcoDMT mediates proton-coupled uptake of manganese at low micromolar concentrations. Mutants of residues in the transition-metal ion-binding site severely affect transport, whereas a mutation of a conserved histidine located near this site results in metal ion transport that appears uncoupled to proton transport. Combined with previous results, our study defines the conformational changes underlying transition-metal ion transport in the SLC11 family and it provides molecular insight to its coupling to protons.

(a) Experimental electron density (calculated at 3.6 Å with solvent flattened MAD phases and contoured at 1σ, blue mesh) is shown superimposed on α-helices 1 and 6 of the refined model. (b) 2Fo-Fc electron density of the same region is shown superimposed on the refined model. (c) Stereo representation of the ion-binding site viewed from the extracellular side with 2Fo-Fc electron density superimposed. (b,c), Sharpened electron density (b=120) was calculated with model phases at 3.3 Å, contoured at 1σ and is shown as cyan mesh. (d) Anomalous difference electron density (calculated at 3.6 Å and contoured at 6σ, magenta mesh) from data of crystals containing Se-Met derivatized WT is shown superimposed on a Cα-trace of EcoDMT. Methionine side chains are displayed as sticks. (e) Anomalous difference electron density from data of crystals containing Se-Met derivatized point mutants in the vicinity of introduced methionine positions (calculated at 4.5 Å and contoured at 4.5σ, magenta mesh). The refined WT structure is shown as sticks.

Supplementary Figure 4
Structural features of ScaDMT and EcoDMT and reconstitution efficiency of EcoDMT ion-binding site mutants.
(a) Transition-metal ion coordination within the ion-binding site of ScaDMT. The protein is shown as Cα trace, selected main chain and side chain atoms as sticks. Interactions with the Mn 2+ ion (green sphere) are indicated by dashed lines. (b) Stereo representation of EcoDMT viewed from the extracellular side. The protein is shown as sticks with the molecular surface superimposed as grey mesh. A modeled Mn 2+ ion is shown as green sphere. (c) Ribbon representation of ScaDMT with the modeled helix α1, which was not present in the crystallized construct, shown in black. The corresponding helices from the superimposed structures of LeuT (PDB ID 3TT3) and vSGLT (PDB ID 3DH4) are shown for comparison in red and blue respectively. (d) Stereo representation of ScaDMT (containing helix α1 in a modeled conformation) viewed from the intracellular side. The protein is shown as sticks, the molecular surface is superimposed as grey mesh. A crystallographically defined Mn 2+ ion is shown as green sphere. (e) Substrate-binding sites of EcoDMT (left) and ScaDMT (right). Proteins are shown as sticks. Mn 2+ (green sphere) in the EcoDMT structure was modeled, in ScaDMT it is placed at the experimentally defined position. Binding-site residues in EcoDMT are labeled. In (a,e) * indicates a carbonyl group of the backbone of α-helix 6a that is involved in Mn 2+ interactions in ScaDMT. (f) SDS-PAGE gel of EcoDMT (*) extracted with the detergent DM from equivalent amounts of proteoliposomes shows a comparable reconstitution efficiency for WT and the metal ion-binding site mutants D51A, N54A and M234A.

Supplementary Figure 5
Structure and reconstitution efficiency of potential proton acceptor mutants of EcoDMT.
(a) Left: Superposition of a Cα-trace of WT (grey) and the mutant E129Q (beige). Right: 2Fo-Fc electron density calculated at 3.6 Å superimposed on the refined E129Q structure. (b) Left: Superposition of a Cα-trace of WT (grey) and the mutant E129A (beige). Right: 2Fo-Fc electron density calculated at 3.9 Å superimposed on the refined E129A structure. (c) Left: Superposition of a Cα-trace of WT (grey) and the mutant H236A (beige). Right: 2Fo-Fc electron density calculated at 3.7 Å superimposed on the refined H236A structure. (a-c) Sharpened 2Fo-Fc Electron densities (b=120, contoured at 1σ and shown as cyan mesh) were calculated with phases from the refined models. In (a-c), an asterisk indicates the mutated side chain. (d) SDS-PAGE gel of EcoDMT (*) extracted with the detergent DM from equivalent amounts of proteoliposomes shows a similar reconstitution efficiency for WT and mutants of potential proton acceptors.