Dataset Information

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.

SUBMITTER: Ray S

PROVIDER: S-EPMC10185341 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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

Ray Shamayeeta S Berry Samuel P SP Wilson Eric A EA Zhang Casey H CH Shekhar Mrinal M Singharoy Abhishek A Gaudet Rachelle R

eLife 20230411

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 ...[more]

PMID: 37039477

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Project description:BACKGROUND AND AIMS: Iron deficiency is one of the most common nutritional disorders in plants, especially in fruit trees grown in calcareous soil. Malus baccata is widely used as an apple rootstock in north China and is highly resistant to low temperatures. There are few studies on iron absorption by this species at the molecular level. It is very important to understand the mechanism of iron uptake and transport in such woody plants. As a helpful tool, the aim of the present study was the cloning and functional analysis of NRAMP (natural resistance-associated macrophage protein) genes from the apple tree in relation to trafficking of micronutrients (Fe, Mn and Cd). METHODS: Reverse transcription-PCR (RT-PCR) combined with RACE (rapid amplification of cDNA ends) was adopted to isolate the full-length NRAMP1 cDNA. Southern blotting was used to test gene copy information, and northern blot was used to detect the gene's expression level. Complementation experiments using the yeast mutant strains DEY1453 and SLY8 were employed to confirm the iron- and manganese-transporting ability of NRAMP1 from apple, and inductively coupled plasma (ICP) spectrometry was used to measure Cd accumulation in yeast. NRAMP1-green fluorescent protein (GFP) fusion protein was used to determine the cellular localization in yeast. KEY RESULTS: A 2090 bp cDNA was isolated and named MbNRAMP1. It encodes a predicted polypeptide of 551 amino acids. MbNRAMP1 exists in the M. baccata genome as a single copy and was expressed mainly in roots. MbNRAMP1 rescued the phenotype of yeast mutant strains DEY1453 and SLY8, and also increased Cd2+ sensitivity and accumulation. MbNRAMP1 expression in yeast was largely influenced by iron status, and the expression pattern of MbNRAMP1-GFP varied with the environmental iron nutrition status. CONCLUSIONS: MbNRAMP1 encodes a functional metal transporter capable of mediating the distribution of ions as well as transport of the micronutrients, Fe and Mn, and the toxic metal, Cd.

Dataset Information

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

Publications

High-resolution structures with bound Mn<sup>2+</sup> and Cd<sup>2+</sup> map the metal import pathway in an Nramp transporter.

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