High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporter
- Harvard University, United States
- Arizona State University, United States
- Broad Institute, United States
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.
Data availability
Atomic coordinates and structure factors for the crystal structures reported in this work have been deposited to the Protein Data Bank under accession numbers 8E5S (WT), 8E5V (WTsoak), 8E60 (WT•Mn2+), 8E6H (A47W•Mn2+), 8E6I (M230A•Mn2+), 8E6L (D296A•Mn2+), 8E6M (WT•Cd2+), and 8E6N (re-refined G223W•Mn2+). Corresponding X-ray diffraction images have been deposited to the SBGrid Data Bank under the respective accession numbers 962 (doi:10.15785/SBGRID/962), 963 (doi:10.15785/SBGRID/963), 964 (doi:10.15785/SBGRID/ 964), 966 (doi:10.15785/SBGRID/966), 967 (doi:10.15785/SBGRID/967), 968 (doi:10.15785/ SBGRID/968), 969 (doi:10.15785/SBGRID/969), and previously deposited 564 (doi:10.15785/ SBGRID/564). The multiple sequence alignment and phylogenetic tree have been provided as Figure 1-source data 1 and Figure 1-source data 2, respectively. All liposome-based transport data are provided in Figure 1-source data 3. Code for analysis of molecular dynamics data, as well as the raw data plotted in supplemental Figure 4-figure supplement 2 and Figure 4-figure supplement 3, can be found at https://github.com/samberry19/nramp-md (MIT license). Raw molecular dynamics trajectory files are available on Dryad (https://doi.org/10.5061/dryad.tx95x6b2b). Source files (origin files) of all ITC experiments are provided in Appendix 1-table 1-source data 1 (Mn2+ isotherms), Appendix 1-table 2-source data 1 (Cd2+ isotherms) and Figure 5-source data 1 (Mg2+ isotherms).
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Molecular dynamics simulations in: High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporterDryad Digital Repository, doi:10.5061/dryad.tx95x6b2b.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (R01GM120996)
- Rachelle Gaudet
National Science Foundation (MCB-1942763)
- Abhishek Singharoy
National Science Foundation (1764269)
- Samuel P Berry
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Ray et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporter
eLife 12:e84006.
https://doi.org/10.7554/eLife.84006
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