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

  1. Karthik Ramanadane
  2. Márton Liziczai
  3. Dragana Markovic
  4. Monique S Straub
  5. Gian T Rosalen
  6. Anto Udovcic
  7. Raimund Dutzler
  8. Cristina Manatschal

  • Curated by eLife

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    eLife assessment

    This study provides valuable insight into the diversity within the NRAMP superfamily of transporters. Evidence of divalent metal ion transport and the structure (obtained without added metal ions) are convincing. However, molecular insight into Al3+ recognition and transport is incomplete, and the work would be strengthened by the determination of a metal-bound structure or additional experiments (such as molecular dynamics simulations or quantitative Al3+ binding/transport assays) to support the proposed Al3+ binding site. The work will be of interest to structural biologists and biophysicists studying NRAMP transporters.

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Abstract

The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe 2+ and Mn 2+ across all kingdoms of life. Despite the strong conservation of the family, two of its branches have evolved a distinct substrate preference with one mediating Mg 2+ uptake in prokaryotes and another the transport of Al 3+ into plant cells. Our previous work on the SLC11 transporter from Eggerthella lenta revealed the basis for its Mg 2+ selectivity (Ramanadane et al., 2022). Here, we have addressed the structural and functional properties of a putative Al 3+ transporter from Setaria italica . We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al 3+ and Ga 3+ , which are both presumable substrates. Its cryo-electron microscopy (cryo-EM) structure displays an occluded conformation that is closer to an inward- than an outward-facing state, with a binding site that is remodeled to accommodate the increased charge density of its transported substrate.

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  1. Version published to 10.7554/elife.85641 on eLife
  2. eLife

    eLife assessment

    This study provides valuable insight into the diversity within the NRAMP superfamily of transporters. Evidence of divalent metal ion transport and the structure (obtained without added metal ions) are convincing. However, molecular insight into Al3+ recognition and transport is incomplete, and the work would be strengthened by the determination of a metal-bound structure or additional experiments (such as molecular dynamics simulations or quantitative Al3+ binding/transport assays) to support the proposed Al3+ binding site. The work will be of interest to structural biologists and biophysicists studying NRAMP transporters.

  3. eLife

    Reviewer #1 (Public Review):

    Members of the SLC11/NRAMP family of transporters permit the movement of transition metals across cell membranes in all kingdoms of life. The current study builds off previous structural and mechanistic work on the SLC11/NRAMP family of transporters by Manatschal and colleagues reported in eLife; the current study presents a cryo-EM structure of a plant aluminum (Al3+) transporter that combats aluminum toxicity in soil. The structure was not determined in the presence of added metal ions, so the paper also employs a variety of established functional assays to test the effects of mutating suggested binding site residues. One notable result is the identification of a mutation (S68A) that maintains divalent transport but disrupts trivalent binding/transport. Strengths of the manuscript include the extensive legwork required to identify a combination of plant homologue, cameloid nanobody, and amphipol that is required to provide homogenous protein and interpretable cryo-EM data. The cryo-EM maps are reliable with low orientation bias and clear features. In addition, the authors perform a number of biochemical and transport assays with divalent metals to bolster their structural model.

  4. eLife

    Reviewer #2 (Public Review):

    In this work, the authors aimed to understand the ion selectivity mechanism of a plant NRAMP-related aluminum transporter by structural and biochemical characterizations.

    The authors successfully identified SiNRAT as a promising candidate for structural and biochemical analyses, showed that SiNRAT transport various divalent cations as well as binding to trivalent cations, determined the cryo-EM structure of SiNRAT, and performed structure-based mutational analysis to identify a potential binding site for metal ions. Unfortunately, the authors failed to show direct evidence of Al3- transport, due to technical problems. Furthermore, the structure of SiNRAT in complex with Al3+ was also not shown.

    Despite such weakness, the structural comparison with other NRAMP members with different ion selectivity properties together with the extensive biochemical analyses would support the statement by the authors on a mechanism of ion selectivity for Al3+.

    In the discussion section, the authors posed an important question. Considering the weak ion selectivity of SiNRAT over divalent cations, it is still unclear how NRAT proteins can function as an Al3+ transporter in a physiological condition where other divalent cations are also abundant. This would be an important question to be addressed in the related research field in the future.

    The methods section is well written and the atomic coordinates and EM map file will be available to the community.

  5. eLife

    **Reviewer #3 (Public Review):
    **
    This paper addresses the structure and mechanism of a presumed Al3+ transporter from the NRAMP superfamily from the plant Setaria italica. This protein belongs to a small clade of NRAMPs, termed NRATs that are postulated to protect plants from Al3+ which is both toxic and prevalent in soil. The NRAT clade is characterized by the substitution of key amino acids at the substrate binding site which has been shown to coordinate either Mg2+ in NRMTs or Mn2+ in classical NRAMP transporters. Evidence for Al3+ transport comes from a previous study utilizing heterologous expression in yeast; this study concluded that NRAT1 from rice (Oryza sativa) is highly specific for Al3+ over Mn2+, Fe2+, Cd2+, Mg2+ which have been shown to be transported by homologs in other clades of the NRAMP family. The current study screened the expression of five homologues of NRAT1, choosing SiNRAT for structural and functional analysis. Unlike previous work on NRAT1, SiNRAT readily transported Mn2+, and experiments with Ca2+ and Mg2+ indicate that these ions are likely also transported. Unlike classical NRAMPs, Mn2+ transport appears to be passive and not coupled to proton transport. Although technical limitations precluded direct measurement of Al3+ transport, ITC measurements provided qualitative evidence for binding in the uM range. A cryo-EM structure is presented, showing an occluded conformation similar to the recent high-resolution X-ray structure of a classical NRAMP bound to Mn2+. The structure of SiNRAT does not show bound ions, but allows comparison of the substrate binding pocket and shows the disposition of key amino acids that distinguish the NRAT clade. Finally, mutagenesis was used to evaluate the role of four of these residues, thus concluding that Ser68 plays a role in coordinating Al3+ as well as its analog Ga3+. Thus, although the transport data with Mn2+ are rigorous, interactions of the putative substrate, Al3+, are only addressed in a qualitative way. The cryo-EM structure is similarly rigorous but provides only modest insight into substrate specificity. Furthermore, the discussion of proton coupling - or the lack thereof - is very speculative. Thus, although new information on this novel clade of NRAMP transporters will be welcomed by specialists in this field, the paper is likely to have only a modest impact beyond this cohort.

  6. Version published to 10.1101/2022.12.21.521437v1 on bioRxiv