Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter
- Harvard University, United States
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.
Article and author information
Author details
Christina M Zimanyi
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
© 2019, Bozzi 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.
Metrics
-
- 3,733
- views
-
- 591
- downloads
-
- 58
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter
eLife 8:e41124.
https://doi.org/10.7554/eLife.41124
Further reading
-
- Biochemistry and Chemical Biology
- Genetics and Genomics
RNA binding proteins (RBPs) containing intrinsically disordered regions (IDRs) are present in diverse molecular complexes where they function as dynamic regulators. Their characteristics promote liquid-liquid phase separation (LLPS) and the formation of membraneless organelles such as stress granules and nucleoli. IDR-RBPs are particularly relevant in the nervous system and their dysfunction is associated with neurodegenerative diseases and brain tumor development. Serpine1 mRNA-binding protein 1 (SERBP1) is a unique member of this group, being mostly disordered and lacking canonical RNA-binding domains. We defined SERBP1’s interactome, uncovered novel roles in splicing, cell division and ribosomal biogenesis, and showed its participation in pathological stress granules and Tau aggregates in Alzheimer’s brains. SERBP1 preferentially interacts with other G-quadruplex (G4) binders, implicated in different stages of gene expression, suggesting that G4 binding is a critical component of SERBP1 function in different settings. Similarly, we identified important associations between SERBP1 and PARP1/polyADP-ribosylation (PARylation). SERBP1 interacts with PARP1 and its associated factors and influences PARylation. Moreover, protein complexes in which SERBP1 participates contain mostly PARylated proteins and PAR binders. Based on these results, we propose a feedback regulatory model in which SERBP1 influences PARP1 function and PARylation, while PARylation modulates SERBP1 functions and participation in regulatory complexes.
-
- Biochemistry and Chemical Biology
Missense mutations in the amyloid precursor protein (APP) and presenilin-1 (PSEN1) cause early-onset familial Alzheimer’s disease (FAD) and alter proteolytic production of secreted 38-to-43-residue amyloid β-peptides (Aβ) by the PSEN1-containing γ-secretase complex, ostensibly supporting the amyloid hypothesis of pathogenesis. However, proteolysis of APP substrate by γ-secretase is processive, involving initial endoproteolysis to produce long Aβ peptides of 48 or 49 residues followed by carboxypeptidase trimming in mostly tripeptide increments. We recently reported evidence that FAD mutations in APP and PSEN1 cause deficiencies in early steps in processive proteolysis of APP substrate C99 and that this results from stalled γ-secretase enzyme-substrate and/or enzyme-intermediate complexes. These stalled complexes triggered synaptic degeneration in a Caenorhabditis elegans model of FAD independently of Aβ production. Here, we conducted full quantitative analysis of all proteolytic events on APP substrate by γ-secretase with six additional PSEN1 FAD mutations and found that all six are deficient in multiple processing steps. However, only one of these (F386S) was deficient in certain trimming steps but not in endoproteolysis. Fluorescence lifetime imaging microscopy in intact cells revealed that all six PSEN1 FAD mutations lead to stalled γ-secretase enzyme-substrate/intermediate complexes. The F386S mutation, however, does so only in Aβ-rich regions of the cells, not in C99-rich regions, consistent with the deficiencies of this mutant enzyme only in trimming of Aβ intermediates. These findings provide further evidence that FAD mutations lead to stalled and stabilized γ-secretase enzyme-substrate and/or enzyme-intermediate complexes and are consistent with the stalled process rather than the products of γ-secretase proteolysis as the pathogenic trigger.
