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

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.

Structural and functional properties of a plant NRAMP-related aluminum transporter

“…The observed structure resembles an inward-occluded state of the classical SLC11 transporter DraNRAMP (PDBID: 8E60, DraNRAMP occ ), where most of the structure retains a conformation observed in the inward-facing structure of the same protein (PDBID: 6D9W, DraNRAMP inw ) except for a movement of the intracellular part of α-helix 1 (α1a), which has rearranged to close the access path to the ion binding site ( Figure 3 , Figure 3—figure supplement 1A, B ; Bozzi et al, 2019b ; Ray et al, 2023 ). This resemblance is reflected in an RMSD of Cα atoms of 1.57 Å upon a superposition of SiNRAT with DraNRAMP occ and 1.74 Å with DraNRAMP inw , whereas the RMSD of 2.71 Å compared to the outward-facing conformation of the same protein (PDBID: 6BU5, DraNRAMP out ) is considerably larger ( Figure 3C , Figure 3—figure supplement 1C ).…”

“…In both structures, the water-filled paths to the metal ion binding site are sealed from both sides of the membrane and bound ions would thus be segregated from their aqueous environment, although the occlusion toward the intracellular solution is more pronounced in DraNRAMP occ ( Figures 3C , 4B and C , Figure 3—figure supplement 1B, D ). In its binding site, the interacting transition metal ion in DraNRAMP occ is predominantly enclosed by protein residues except for one contact with a small pocket harboring trapped water molecules ( Ray et al, 2023 ). The side chains of Asp 56 and Asn 59 on α1, in conjunction with the backbone carbonyls of Ala 53 and Ala 227, tightly surround the bound Mn 2+ .…”

“…The SiNRAT structure has shed light into the basis for the altered selectivity of a transporter, whose evolutionary distance to classical NRAMPs is much smaller than to NRMTs ( Lu et al, 2018 ; Ramanadane et al, 2022 ). Although, for technical reasons, our structure did not contain trivalent ions, we assigned their presumable binding region based on the structural equivalence to the transition metal ion transporters DraNRAMP and ScaDMT and the Mg 2+ transporter EleNRMT ( Ehrnstorfer et al, 2014 ; Ramanadane et al, 2022 ; Ray et al, 2023 ). Residual density at the supposed location in the SiNRAT map provides further evidence for this assignment.…”

They all rock: A systematic comparison of conformational movements in LeuT-fold transporters

“…BetP, SERT, and Mhp1 have Ooc, while MntH has Ioc, and KCC1 and LAT1 have no available occluded state. Because several of these proteins have been extensively investigated, these results suggest that some transporters have only one or no occluded state as a well-populated intermediate 10,14 . In addition to the seven proteins listed above, AdiC structures are available for both outward conformations (Oop and Ooc), and SGLT structures for both inward conformations (Ioc and Iop).…”

“…For each protein for which at least one structure has been determined, we chose one representative structure (generally the one with highest resolution) for each available conformation (Table 3). We also added a structure of human KCC1 determined in an additional conformation 13 after we built our list and updated structures of Deinococcus radiodurans MntH to ones recently determined at higher resolution 14 . Nine proteins have structures available in multiple conformations (Table 3 and Table S3).…”