Figure 3. Coordination sphere changes across the Mn²⁺ transport cycle of DraNramp.

(A) Structures of the orthosteric metal-binding site in six conformations reveal the differences in coordination geometry and illustrate that the bound Mn²⁺ is more hydrated in the outward-open and inward-open states than the occluded state. In the occluded structure of metal-free WT DraNramp a density we have assigned as water replaces Mn²⁺. Y54 in TM1a progressively moves to open the inner vestibule in the transition from outward to inward open, shown by black curved arrows. (B) TM1 and TM6 from a superposition of the three Mn²⁺-bound structures in panel a illustrate the swing of the Y54 sidechain as sticks. The view is rotated 180° along the vertical axis from Figure 2C. (C) Initial Mn²⁺ uptake rates for DraNramp variants Y54A and Y54F at membrane potentials ranging from ΔΨ=0 to −120 mV (n=2–3; each data point is on the scatter plot and black bars are the mean values). The Mn²⁺ concentration was 750 μM, and the pH was 7 on both sides of the membrane. Y54A nearly abolishes transport whereas Y54F has near-wildtype initial transport rates. Corresponding time traces are plotted in Figure 1—figure supplement 4. (D) ITC measurements of G223W (left; one-site binding model with fixed n=1) and M230A (right; two-site sequential binding model) binding to Mn²⁺. One isotherm is shown of two measured, and the listed K_d values are the average ± SEM (see Appendix 1 for ITC analysis).

Figure 3—figure supplement 1. Structure and Mn²⁺-binding site architecture of D296A•Mn²⁺.

(A) Cartoon representation of the inward-open structure of D296A•Mn²⁺, which is nearly identical to the M230A•Mn²⁺ inward-open structure (Cα RMSD of 0.38 Å). (B) Coordination sphere of the orthosteric Mn²⁺ ion in the D296A structure is nearly the same as in M230A Mn²⁺-bound structure except for the sulfur of M230 replacing a water seen in M230A. We do not observe a bound water to complete the coordination sphere of D296A, likely because the resolution of the structure is lower (2.52 Å for M230A•Mn²⁺ vs. 3.12 Å for D296A•Mn²⁺), otherwise the structures are analogous. The peak from the anomalous difference Fourier map (magenta mesh; 4.5σ) calculated from a D296A•Mn²⁺ crystal confirms a bound Mn²⁺ in this inward-open state. (C) 2F_o-F_c map (gray mesh; 1σ) of the orthosteric site of D296A•Mn²⁺.

Figure 3—figure supplement 2. 2F_o-F_c maps (gray mesh; 1σ) of the Mn²⁺-coordination sphere at the orthosteric site across different conformations of the Mn²⁺ transport cycle of DraNramp.

Figure 3—figure supplement 3. Sequence logos highlighting that Y54 in TM1a is 80% conserved in all Nramps (3762 sequences), 100% conserved in bacterial clades A and C, but replaced by a phenylalanine in clade B.

Eukaryotic Nramps have either tyrosine or phenylalanine at the corresponding position. Residue coloring is based on the ‘chemistry’ coloring scheme of WebLogo (Crooks et al., 2004). Clades are as defined in Figure 1—figure supplement 3B.