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.
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).
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.
- Rachelle Gaudet
- Abhishek Singharoy
- Samuel P Berry
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Merritt Maduke, Stanford University School of Medicine, United States
© 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.
Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the Saccharomyces cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.
Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized ‘codes’ that are read by specialized regions (reader domains) in chromatin-associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP: histone PTM] specificities, and thus decipher the histone code and guide epigenetic therapies. However, this has largely been done using the reductive approach of isolated reader domains and histone peptides, which cannot account for any higher-order factors. Here, we show that the [BPTF PHD finger and bromodomain: histone PTM] interaction is dependent on nucleosome context. The tandem reader selectively associates with nucleosomal H3K4me3 and H3K14ac or H3K18ac, a combinatorial engagement that despite being in cis is not predicted by peptides. This in vitro specificity of the BPTF tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. We propose that regulatable histone tail accessibility and its impact on the binding potential of reader domains necessitates we refine the ‘histone code’ concept and interrogate it at the nucleosome level.