Decision letter: High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporter

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+ and Mn2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps. Editor's evaluation This manuscript provides fundamental new insight into protein conformational transitions underlying the transport mechanism of Nramps, an important and widespread transporter family that facilitates the uptake and movement of essential transition metals. Eight new crystallographic structures of the prokaryotic homolog DraNramp in a variety of ligand-bound and conformational states, along with companion molecular dynamics simulations and metal binding and transport assays, provide compelling evidence supporting most of the conclusions. These findings will be of broad interest to scientists studying transport mechanisms and ligand recognition. https://doi.org/10.7554/eLife.84006.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Transition metal ions like Mn2+ and Fe2+ are essential for various metabolic processes in all living cells and are usually required in low intracellular concentrations for optimal activity (Andrews, 2002; Bozzi and Gaudet, 2021). Excess or deficiency of transition metal ions leads to diseases (Bleackley and Macgillivray, 2011; Nies and Grass, 2009). For example, Fe2+ deficiency causes anemia and neurodegenerative diseases, whereas Fe2+ overload increases the risk of cancer by generating toxic reactive oxygen species (ROS) and mutations (Ekiz et al., 2005; Jung et al., 2015). Mn2+ overload in the brain is linked to neurological disorders and deficiency causes metabolic defects and impairs growth (Budinger et al., 2021; Pittman, 2005). Other transitions metals, like Cd2+ and Hg2+, are toxic and their accumulation affects health by disrupting the physiological levels of essential metals or displacing them in enzyme active sites, thus inhibiting the proteins, or changing their activity (Andrews, 2002; Lin et al., 2009). Cells and organisms have evolved strategies to maintain metal ion homeostasis via highly regulated transport and storage processes (Bleackley and Macgillivray, 2011; Cellier and Gros, 2004; Nies and Grass, 2009). Natural resistance-associated macrophage proteins (Nramps) are ubiquitous importers of Fe2+ and Mn2+ across cellular membranes into the cytosol (Bozzi and Gaudet, 2021; Cellier and Gros, 2004; Nevo and Nelson, 2006). In humans, Nramp1 extrudes essential metals from phagosomes of macrophages to aid in killing engulfed pathogens, and Nramp2 (DMT1) is expressed at low levels in the endosomes of all nucleated cells and imports Mn2+ and Fe2+ into the cytosol (Pujol-Giménez et al., 2017; Skamene et al., 1998; Vidal et al., 1993). Plant and fungal Nramps aid in Fe2+ and Mn2+ uptake and trafficking, and bacterial Nramps are involved in the acquisition of Mn2+, an essential nutrient (Bozzi and Gaudet, 2021). In addition to the physiological substrates Fe2+ and Mn2+, Nramps can also transport toxic metals like Cd2+ and Hg2+ but exclude the abundant alkaline earth metals like Mg2+ and Ca2+ (Bozzi and Gaudet, 2021). Recent bacterial Nramp structures reveal a LeuT fold, three stable conformations (outward-open, occluded, and inward-open), and identify the metal-binding site residues, including conserved aspartate, asparagine, and methionine residues (Bozzi et al., 2016b; Bozzi et al., 2019b; Ehrnstorfer et al., 2014; Ehrnstorfer et al., 2017). The metal-binding methionine is essential to select against alkaline earth metals (Bozzi et al., 2016a). This finding is corroborated by the fact that a bacterial Nramp homolog which lacks a metal-binding methionine, NRMT (Nramp-related Mg2+ transporter), can transport Mg2+ (Ramanadane et al., 2022). However, little is known about whether the canonical Nramps can mechanistically distinguish between their physiological substrates (Fe2+ and Mn2+) from non-essential ones like Cd2+ within their broad spectrum of transition metal substrates. Functional studies on Deinococcus radiodurans (Dra)Nramp revealed that Mn2+ and Cd2+ transport differ in their dependence on pH, proton flux, and membrane potential (Bozzi et al., 2019a; Bozzi et al., 2019b). However, we lack high-resolution structural information on binding of different metals to explain these differences. We present high-resolution structures of DraNramp in three conformations in both Mn2+-bound and metal-free states, providing the first molecular map of the entire Mn2+ transport cycle. The structures along with molecular simulations reveal that Nramps achieve alternate access during transport by adopting distinct Mn2+-coordination spheres in different conformations. These different conformations are also supported by dynamic rearrangements of key polar-residue networks that gate the inner and outer vestibules. This Mn2+ transport cycle also informs on the transport of Fe2+, the other common physiological Nramp substrate, because Fe2+ and Mn2+ have similar coordination preferences and chemical properties (Bozzi and Gaudet, 2021; Davidsson et al., 1989; Kawabata, 2019; Liu et al., 2021). Comparisons with an additional high-resolution structure of DraNramp bound to a non-physiological substrate, Cd2+, and complementary binding and transport measurements and mutational analyses, suggest that Nramps can distinguish physiological from toxic substrates through thermodynamic differences in the conformational landscape of the transport cycle. Results DraNramp transports a mostly dehydrated Mn2+ ion To visualize how the metal substrate is coordinated in Nramps, we determined crystal structures of DraNramp using lipid-mesophase based techniques (Supplementary file 1a). We obtained a structure of wildtype (WT) DraNramp in an occluded state bound to Mn2+ at 2.38 Å by soaking crystals with Mn2+ (WT•Mn2+; Table 1, Figure 1A–B). We resolved a comparable structure using co-crystallization with Mn2+ and the inward-locking mutation A47W (A47W•Mn2+; Supplementary file 1b; Cα RMSD=0.47 Å; all pairwise RMSD values listed in Supplementary file 1c; Bozzi et al., 2016b). The similarity of both structures, including a nearly identical Mn2+-coordination sphere (Figure 1B, Figure 1—figure supplement 1A), suggests that the observations we make based on these two structures are robust. Both structures superimpose best with the published occluded metal-free G45R structure (Bozzi et al., 2019b). Although the inner vestibule is occluded in both structures, WT•Mn2+ and A47W•Mn2+ differ in their TM1a position, with WT•Mn2+ nearly identical to G45R whereas the A47W•Mn2+ TM1a is displaced within the inner vestibule, likely to accommodate the bulky tryptophan sidechain. Therefore, we generally used the WT•Mn2+ structure for analysis of the occluded state. As in the metal-free G45R, the Mn2+-bound occluded structures have a completely sealed outer vestibule and a partially closed inner vestibule, with the Mn2+ occluded from bulk solvent (Figure 1A). Figure 1 with 4 supplements see all Download asset Open asset The occluded structure of DraNramp reveals a largely dehydrated Mn2+-coordination sphere. (A) Cartoon representation of WT•Mn2+ in an occluded state. Anomalous signal confirmed the presence of Mn2+ in both the orthosteric metal-binding site and an additional site at the mouth of the external vestibule (Figure 1—figure supplement 1A) which is less conserved across the Nramp family (Figure 1—figure supplement 3). TM1 and TM6 are labeled. (B) Detail of the orthosteric metal-binding site of WT•Mn2+ where D56, N59, M230, and the pseudo-symmetrically related carbonyls of A53 and A227 coordinate the Mn2+ ion (Figure 1—figure supplement 1B). A water molecule completes the six-ligand coordination sphere. Coordinating residues are shown as sticks, and coordinating distances are indicated in Å. (C) ITC measurement of the affinity of WT DraNramp for Mn2+. Top graph shows heat absorbed upon injection of Mn2+ solution to the protein solution. Bottom graph shows the fit of the integrated and corrected heat to a binding isotherm. The data show an endothermic mode of binding and fits best with a two-site sequential binding model. The figure shows one of three measurements and the average Kd values ± SEM (Kd1=190±30 µM, Kd2=1970±520 µM; see Appendix 1). Based on ITC experiments comparing Mn2+ binding to WT or DraNramp constructs with mutations at the external site (Figure 1—figure supplement 2A), we assigned Kd1 to the orthosteric site. In all figures, unless otherwise noted, TMs 1, 5, 6, and 10 are pale yellow, TMs 2, 7, and 11 gray, TMs 3, 4, 8, and 9 light blue, and Mn2+ atoms are magenta spheres. Figure 1—source data 1 Multiple sequence alignment of 6172 Nramp homologs. https://cdn.elifesciences.org/articles/84006/elife-84006-fig1-data1-v2.zip Download elife-84006-fig1-data1-v2.zip Figure 1—source data 2 Maximum likelihood phylogenetic tree of Nramp homologs built with RAxML-NG. https://cdn.elifesciences.org/articles/84006/elife-84006-fig1-data2-v2.zip Download elife-84006-fig1-data2-v2.zip Figure 1—source data 3 Raw data of metal ion uptake into proteoliposomes measured at four ΔΨ values for each DraNramp construct. https://cdn.elifesciences.org/articles/84006/elife-84006-fig1-data3-v2.xlsx Download elife-84006-fig1-data3-v2.xlsx Table 1 Data collection and refinement statistics for four new DraNramp structures. StructureConformationBound metal ion substratePDB IDWTsoakOccluded none8E5VWT•Mn2+OccludedMn2+8E60M230A•Mn2+Inward openMn2+8E6IWT•Cd2+Inward openCd2+8E6MData CollectionBeamlineGMCA 23IDBGMCA 23IDBGMCA 23IDBNECAT 24IDCWavelength (Å)1.0331.0331.0330.984Resolution range (Å)41.23–2.36 (2.44–2.36)41.28–2.38 (2.46–2.38)45.32–2.52 (2.61–2.52)45.54–2.48 (2.57–2.48)Space groupP 2 21 21P 2 21 21P 2 21 21P 2 21 21Unit cell (a, b, c)58.95, 71.04, 98.7759.08, 71.10, 98.7558.67, 71.35, 98.5959.14, 71.37, 99.05Unit cell (α, β, γ)90, 90, 9090, 90, 9090, 90, 9090, 90, 90Number of crystals1131Total reflections58744 (5928)57472 (5733)146077 (14913)76829 (7275)Unique reflections17477 (1718)16468 (1646)14548 (1427)15351 (1507)Redundancy3.4 (3.4)3.5 (3.5)10.0 (10.4)5.0 (4.8)Completeness (%)98.71 (98.85)95.11 (96.92)99.90 (99.79)99.03 (99.47)Mean I/σ (I)8.89 (0.97)8.92 (0.89)8.47 (0.75)9.90 (1.12)Wilson B-factor49.9750.5554.2949.52Rmerge0.106 (1.292)0.109 (1.241)0.269 (2.475)0.158 (1.626)Rmeas0.127 (1.511)0.127 (1.447)0.284 (2.603)0.178 (1.831)Rpim0.067 (0.759)0.063 (0.722)0.090 (0.800)0.078 (0.816)CC1/20.99 (0.37)0.99 (0.39)0.98 (0.34)0.99 (0.34)RefinementResolution range (Å)41.23–2.36 (2.44–2.36)41.28–2.38 (2.46–2.38)45.32–2.52 (2.61–2.52)45.54–2.48 (2.57–2.48)No. reflections17441 (1714)16438 (1636)14547 (1425)15291 (1507)No. reflections in Rfree1743 (171)1642 (164)1454 (143)1530 (151)Rwork0.217 (0.340)0.207 (0.316)0.225 (0.313)0.202 (0.319)Rfree0.245 (0.350)0.259 (0.358)0.266 (0.349)0.250 (0.354)Number of atoms3449338534513321Protein2945293329342905Ligand443405448362Water61476954Protein Residues392393392388Ramachandran plotFavored (%)98.4698.4798.2198.96Allowed (%)1.541.531.791.04Outliers (%)0000Rotamer outliers (%)0.331.001.011.01Clashscore8.258.977.155.57RMS (bonds)0.0020.0020.0020.002RMS (angles)0.430.460.430.46Average B-factor65.1264.9866.6164.82Protein63.3663.2365.0462.68Ligand77.9978.4877.7083.11Water56.3458.2461.3357.45No. of TLS groups9833 Values in parentheses are for highest-resolution shell. Data for M230A•Mn2+ merge reflections from three crystals. Data for the other structures were obtained from a single crystal. See Supplementary file 1a for details on soaking or co-crystallization procedures for bound metal ion substrates. Anomalous difference Fourier maps confirmed presence of Mn2+ at the canonical, orthosteric Nramp metal-binding site between the unwound regions of TM1 and TM6 (Supplementary file 1d, Figure 1—figure supplement 1A). In WT•Mn2+, the Mn2+ is coordinated by conserved residues D56, N59, and M230, and backbone carbonyls of A53 and A227 (Figure 1A, Supplementary file 1e). A53 and A227 are pseudosymmetrically related in the inverted repeats of the LeuT fold of DraNramp (Figure 1—figure supplement 1B). The coordination sphere is completed by a water bridging Mn2+ with Q378, a residue previously proposed to directly coordinate Mn2+ in the occluded state (Bozzi et al., 2019b). This yields a coordination number of 6, typical for Mn2+, and a largely dehydrated metal-binding site with a distorted octahedral Mn2+-coordination geometry (Supplementary file 1f), as often observed in other Mn2+-protein complexes (Barber-Zucker et al., 2017; Couñago et al., 2014; Dudev and Lim, 2014). At the mouth of the outer vestibule, an additional Mn2+ bridges D296 and D369 at the N termini of extracellular helix 2 (EH2) and TM10, respectively (Figure 1—figure supplement 1A). We denote this site as the ‘external site’ and the canonical substrate-binding site as the ‘orthosteric site’. Corroborating the structures, isothermal titration calorimetry (ITC) measurements reveal an endothermic mode of binding and are best fitted with a two-site model for WT (Kd1=190±30 µM, Kd2=1970±520 µM; Figure 1C) and A47W (Kd1=125±5 µM, Kd2=2450±650 µM; Figure 1—figure supplement 2A; all Kd values are in Supplementary file 1g; see Appendix 1 for a description of our ITC data analyses). To determine the affinity of the orthosteric site, we mutated the external-site aspartates. The D296A and D369A substitutions have little impact on Mn2+ transport (Figure 1—figure supplement 2B). The D296A and D369A variants each bind one Mn2+ with Kd=370±30 µM and 420±30 µM respectively (Figure 1—figure supplement 2A), which is closest to Kd1 of WT. Hence, the affinity for Mn2+ at the orthosteric site is higher than at the external site. A metal ion is present at the external site in all inward-open and occluded metal-bound DraNramp structures, but not outward-open structures, as opening the outer vestibule separates D296 and D369 and disrupts the site (Figure 1—figure supplement 3A). D296 and D369 are not conserved across Nramps, but they are more conserved within bacterial clade A, and there is a general abundance of acidic residues in the corresponding loop regions across all clades (Figure 1—figure supplement 3B–C). At present, our results provide little evidence of a biological role for this previously unidentified external site; perhaps the concentration of acidic residues at the mouth of the outer vestibule (Figure 1—figure supplement 3D) provides electrostatic attraction for metal cations. Snapshots of the complete Mn2+ transport cycle by DraNramp We also determined high-resolution DraNramp structures in metal-free occluded (WT) and Mn2+-bound inward-open (M230A•Mn2+) states and re-refined a Mn2+-bound outward-open conformation (G223W•Mn2+; Table 1, Supplementary file 1a and b). Along with the published structures of outward-open metal-free G223W and inward-open metal-free ‘Patch’ (which has a patch of mutations in intracellular loops) (Bozzi et al., 2016b; Bozzi et al., 2019b), these new structures allow us to map the entire Mn2+ transport cycle to three major conformations, each in Mn2+-bound and metal-free states (Figure 2A–B). By ordering and comparing these six structures, we outline a molecular mechanism by which metal substrate binds, induces conformational change, and is released. Figure 2 with 2 supplements see all Download asset Open asset Structures in new conformations complete the Mn2+ transport cycle by DraNramp. (A) Schematic of the conformational states that DraNramp traverses to import Mn2+. The mobile and stationary parts are pale yellow and light blue, respectively. (B) Corresponding structures of DraNramp, showing TMs 1 and 6 in green, TMs 5 and 10 in pale yellow, and TMs 2, 7, and 11 gray. Stationary TMs 3, 4, 8, and 9 are omitted to highlight the key motions in the mobile parts. Mn2+ ions are magenta. Black arrows indicate the key motions in TMs 1a and 6a detailed in panel (C), and TMs 5 and 10 detailed in Figure 2—figure supplement 1. Full structures and the electron density for TMs 1 and 6 are illustrated in Figure 2—figure supplement 2. (C) Pairwise superpositions of whole Mn2+-bound structures highlight the motions of TMs 1 and 6. Conformations are indicated at the bottom. The distance between residues 46 and 240 in TMs 1a and 6b, indicated for the green structures, increases from 5.9 Å to 13.8 Å from outward open to inward open. The large angular motions of TM1a and TM6b are also indicated. (D) Plots of B-factor by residue for the TM1 region (residues 40–70) and the TM6 region. The B-factors are highest for the inward-open state in which the interaction between TMs 1a and 6b (both in the inner leaflet) is broken. TMs 1, 5, 6, and 10 move the most as the Mn2+-binding site accessibility switches from outward to inward across the conformations (Figure 2B–C, Figure 2—figure supplement 1), as also highlighted in previous studies (Bozzi and Gaudet, 2021; Bozzi et al., 2019b; Ehrnstorfer et al., 2017). As Mn2+ binds to the outward-open state, TM10 tilts toward TM1b, the upper half of TM5 toward TM7, and TM6a toward TM11 to seal the outer vestibule and yield an occluded state. The lower half of TM5 also moves away from TM1a, allowing it to swing upward to open the inner vestibule in the following transition. This swing of TM1a is the only noteworthy difference between the Mn2+-bound occluded and inward-open states, allowing release of the Mn2+ into the inner vestibule. TM1a swings to a similar angle in the new inward-open M230A•Mn2+ as in the previous low-resolution inward-open metal-free structure (Bozzi et al., 2016b), and structures of the homologous Eggerthella lenta Nramp-related magnesium transporter (EleNRMT), LeuT, and serotonin transporter (Coleman et al., 2019; Krishnamurthy and Gouaux, 2012; Ramanadane et al., 2022). Thus, most of the structural reorganization in Nramps occurs in the shift from outward open to occluded. The three metal-free DraNramp conformations are similar to their corresponding Mn2+-bound structures, suggesting that once Mn2+ is released, the conformational transitions are reversed, including passing through an occluded metal-free intermediate, to reach the outward-open metal-free conformation ready to accept Mn2+ (Figure 2A–B, Figure 2—figure supplements 1 and 2A). The substrate-binding TM1 and TM6 are well-resolved in our structures (Figure 2—figure supplement 2B) and pairwise superpositions reveal how their motions contribute to the conformational changes across the transport cycle (Figure 2C). TM6a tilts 22° and the unwound region of TM6 becomes more helical as it moves toward the Mn2+ to close the outer gate. The central unwound regions of TM1 and TM6 are closest in the occluded state, resulting in an almost dehydrated Mn2+-coordination sphere. Finally, the inner vestibule opens when TM1a tilts upward by 32°, increasing the distance between TM1a and TM6b by ~8 Å (Figure 2C). TM6b is largely static relative to the protein core, although in the inward-open structure it has high B-factors (Figure 2D), indicating that the interaction with TM1a stabilizes TM6b to close the inner vestibule. Different conformations have distinct Mn2+ coordinations Our Nramp structures provide snapshots of the complete Mn2+-coordination sphere geometries in each conformation (Figure 3). Two new structures of DraNramp point-mutants reveal the coordination of Mn2+ in the inward-open state: M230A•Mn2+ and D296A•Mn2+ (Cα RMSD of 0.42 Å). The Mn2+ is in the same location of the orthosteric site as in the occluded state, as confirmed by anomalous diffraction for D296A•Mn2+ (Supplementary file 1d, Figure 3—figure supplement 1). As in the occluded state, the Mn2+ binds D56, N59, and the A227 carbonyl, with a water replacing M230 in M230A•Mn2+ (Figure 3A, Supplementary file 1e). However, with TM1a displaced, the A53 carbonyl no longer coordinates Mn2+; instead, the Y54 carbonyl approaches Mn2+ at a longer distance of 3.1 Å. Two more waters, one bound to Q378 and another from the inner vestibule, complete a seven-coordination sphere resembling a pentagonal bipyramidal geometry with substantial distortion (Supplementary file 1f). Seven coordination is infrequent but found in Mn2+-coordinating proteins like MntR (Chen and He, 2008; Glasfeld et al., 2003). Our inward-open structures provide the first evidence that Y54 participates in the Mn2+ transport cycle. Figure 3 with 3 supplements see all Download asset Open asset 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 Kd values are the average ± SEM (see Appendix 1 for ITC analysis). The new occluded and inward-open Mn2+-bound structures have a monodentate coordination of D56 with Mn2+. For consistency, we reinterpreted the outward-open G223W•Mn2+ map (PDB ID: 6BU5) with a monodentate coordination of D56 with Mn2+ instead of previously modeled bidentate interaction (Bozzi et al., 2019b); the local and global model statistics are very similar to the original structure (Supplementary file 1e). Re-refined G223W•Mn2+ has six Mn2+-coordinating ligands: D56, N59, M230, carbonyl of A53 and two waters (Figure 3A, Supplementary file 1e); the overall geometry resembles a distorted octahedron as in the occluded structure (Supplementary file 1f). The coordination spheres in all conformations of the transport cycle are well defined as confirmed by the 2Fo-Fc maps of the closeup snapshots of their Mn2+-bound orthosteric site (Figure 3—figure supplement 1C, Figure 5—figure supplement 2). Comparing the three Mn2+-bound conformations (Figure 3A), the carbonyls of A53 in TM1a and A227 in TM6b alternately coordinate Mn2+ in the outward- and inward-open structures respectively, and both residues interact with Mn2+ in the occluded structure. The pseudosymmetrically related A53 and A227 may thus act as hinges altering the Mn2+-coordination sphere as TM1 and TM6 move in turn to open the gates during Mn2+ transport. Furthermore, as DraNramp switches from outward- to inward-open, Y54 progressively swings downward, acting as a gate in concert with TM1a’s upward swing to open the inner vestibule and allow metal release (Figure 3A–B). Our inward-open structures also suggest that the Y54 carbonyl may participate in Mn2+ release through interaction with the metal ion (Figure 3A). All 3796 Nramp sequences in our alignment have either a tyrosine or a phenylalanine at this position and Y54 is completely conserved among bacterial clades A (including DraNramp) and C, while the position is 40% and 100% phenylalanine among eukaryotes and bacterial clade B, respectively (Figure 3—figure supplement 3). To evaluate the significance of Y54 in Mn2+ transport, we purified and reconstituted into proteoliposomes the Y54A and Y54F variants. While Y54F has near-wildtype Mn2+-transport activity, Y54A nearly eliminates Mn2+ transport (Figure 3C), indicating that an aromatic ring is essential for the gating motion required for transport. We also measured the Mn2+-binding affinity of the constructs that yielded inward- or outward- open structures, M230A and G223W, respectively. Mn2+ is present at the external site in the inward-open M230A•Mn2+ (Figure 1—figure supplement 3A), and consistently, the ITC data fit a two-site model (Kd1=215±65 µM assigned to the orthosteric site, Kd2=4600±1300 µM for the external site; Figure 3D and Appendix 1). The ITC data for G223W with Mn2+ fits only in a one-site model (Kd=440±15 µM; Figure 3D), which we assign to the orthosteric site because the opening of the outer vestibule displaces TM10, disrupting the external site (Figure 1—figure supplement 3A). Mn2+ binding does not significantly alter the three main DraNramp conformations To compare the metal-bound states to analogous metal-free states of the transport cycle, we determined two metal-free occluded structures of wildtype DraNramp at a higher resolution than the previously reported G45R structure (Bozzi et al., 2019b), which we refer to as WT (2.38 Å; Supplementary file 1b) and WTsoak (2.36 Å; Table 1). The crystal used for WTsoak was mock-soaked (with no metal in the soaking solution). WT and WTsoak are nearly identical, confirming that the soaking process does not influence the conformational state. We analyzed WTsoak, unless otherwise noted. In WTsoak, we observed density but no anomalous signal at the orthosteric site and modeled a water molecule at the position where Mn2+ sits in the occluded state (Figure 3A). WTsoak is nearly identical to WT•Mn2+, indicating that the occluded conformation is unchanged by the presence of metal ion substrate, and the metal-binding site is instead filled by ordered water molecules. We used previously reported inward-open ‘Patch’ and outward-open G223W metal-free structures for analysis of the Mn2+ transport cycle (Figures 2B and 3A; Bozzi et al., 2016b; Bozzi et al., 2019b). These structures, resolved at lower resolution than the ones described here, have no density at the orthosteric site. This is consistent with a more flexible organization of a metal-binding site open to bulk aqueous solvent. In contrast, metal-free WT has a more ordered orthosteric site, suggesting a stable occluded intermediate in the switch from inward- to outward-open. Polar networks latch the gates to achieve alternating access Vestibules providing access to the orthosteric site from the extracellular or intracellular side alternately open from the motions of DraNramp’s TMs 1, 5, 6, and 10, which form the outer and inner gates during Mn2+ transport (Bozzi et al., 2020; Bozzi et al., 2019b). To pinpoint protein features that enable these motions, we used our Mn2+-bound structures to identify interaction networks with the following attributes: (i) they contain conserved polar residues from at least one of the four mobile helices; (ii) they line the gates; and (iii) they rearrange between the three resolved protein conformations (Figure 4A). Figure 4 with 3 supplements see all Download asset Open asset Networks of polar residues lining the outer and inner vestibules rearrange through the conformational transitions needed for Mn2+ transport. (A) The Cα positions of residues in the Q378 (orange) and T228 (pink) networks lining the outer gate, R244 (cyan) and Q89 (blue) networks within the inner gate, and the H232 (orange) network coordinating with the proton pathway, are mapped on the occluded WT•Mn2+ structure. Metal-binding and proton pathway residues (Bozzi et al., 2019b) are represented as brown spheres. (B) The Q378 network forms as TM10 moves when DraNramp transitions from outward-open to occluded to close the outer vestibule. Water-mediated interactions form between Q378, D56, and T130. The other D56 carbonyl interacts directly with Mn2+. TM1 is transparent. (C) The T228 network forms with N275, N82, and T228 coordinating a water as TM6a moves to close the outer vestibule. TM1 is transparent. (D) In the R244 network, interactions between R244, E176, and D263 break as TM5