Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter

Abstract
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Abstract

Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms.

Data availability

Source data for the Nramp sequence alignment have been provided as Figure 2-source data 1. Structural coordinates and structure factors for each crystal structure have been deposited in the PDB under accession codes 6D9W, 6C3I, 6BU5, and 6D91. The unprocessed X-ray diffraction images have been deposited in the SBGrid Data Bank under accession codes 332, 333, 334, 567, 564, and 576. All other data generated or analyzed in this study are included in the manuscript.

The following data sets were generated

(2018) Crystal structure of the Deinococcus radiodurans Nramp/MntH divalent transition metal transporter G45R mutant in an inward occluded state
Protein Data Bank, 6C3I.

https://www.rcsb.org/structure/6C3I
(2017) Crystal structure of the Deinococcus radiodurans Nramp/MntH divalent transition metal transporter in the outward-open, manganese-bound conformation
Protein Data Bank, 6BU5.

https://www.rcsb.org/structure/6BU5
(2018) Crystal structure of the Deinococcus radiodurans Nramp/MntH divalent transition metal transporter in the outward-open, apo conformation
Protein Data Bank, 6D91.

https://www.rcsb.org/structure/6D91
1. Gaudet R
2. Bane LB
3. Weihofen WA
4. Singharoy A
5. Zimanyi CM
6. Bozzi AT
(2018) Crystal structure of Deinococcus radiodurans MntH, an Nramp-family transition metal transporter, in the inward-open apo state
Protein Data Bank, 6D9W.

https://www.rcsb.org/structure/6D9W
(2016) X-Ray Diffraction data from Deinococcus radiodurans Nramp/MntH divalent transition metal transporter mutant G45R, source of 6C3I structure
SBGrid Data Bank, 567.

https://data.sbgrid.org/dataset/567/
(2017) X-Ray Diffraction data from Deinococcus radiodurans Nramp/MntH divalent transition metal transporter in the outward-open, manganese-bound state, source of 6BU5 structure
SBGrid Data Bank, 564.

https://data.sbgrid.org/dataset/564
(2017) X-Ray Diffraction data from Deinococcus radiodurans Nramp/MntH divalent transition metal transporter mutant G223W, apo, source of 6D91 structure
SBGrid Data Bank, 576.

https://data.sbgrid.org/dataset/576/

The following previously published data sets were used

(2010) X-Ray Diffraction data from Deinococcus radiodurans MntH in complex with Fab, source of 5KTE structure
SBGrid Data Bank, 332.

https://data.sbgrid.org/dataset/332/
(2010) X-Ray Diffraction data from Deinococcus radiodurans MntH in complex with Fab, source of 5KTE structure
SBGrid Data Bank, 333.

https://data.sbgrid.org/dataset/333/
(2011) X-Ray Diffraction data from Deinococcus radiodurans MntH in complex with Fab, source of 5KTE structure
SBGrid Data Bank, 334.

https://data.sbgrid.org/dataset/334/

Article and author information

Author details

Aaron T Bozzi

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

Competing interests
The authors declare that no competing interests exist.
Christina M Zimanyi

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6782-507X
John M Nicoludis

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-3755-7844
Brandon K Lee

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

Competing interests
The authors declare that no competing interests exist.
Casey H Zhang

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

Competing interests
The authors declare that no competing interests exist.
Rachelle Gaudet

Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

For correspondence
gaudet@mcb.harvard.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-9177-054X

Funding

National Institute of General Medical Sciences (1R01GM120996)

Rachelle Gaudet

Jane Coffin Childs Memorial Fund for Medical Research

Christina M Zimanyi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

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.