Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation
- University of Copenhagen, Denmark
- UMR 5280, CNRS, University of Lyon, France
- University of Queensland, Australia
- Stockholm University, Sweden
- Australian Nuclear Science and Technology Organization, Australia
- Institut Laue-Langevin, France
- University of Oxford, United Kingdom
Abstract
The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.
Data availability
SANS data have been deposited in SASBDB under IDs SASDM42, SASDM52, SASDM62, SASDM72.EM data have been uploaded to the Electron Microscopy Data Bank under IDs EMD-13326 and EMD-13327.Activity (fluorescence) data have been uploaded to GitHub at https://github.com/Niels-Bohr-Institute-XNS-StructBiophys/CorAData/.The Metadynamics simulations have been uploaded to GitHub at https://github.com/KULL-Centre/papers/tree/main/2021/CorA-Johansen-et-al.NMR data have been deposited in Biological Magnetic Resonance Data Bank under ID 50959.
Article and author information
Author details
Nicolai Tidemand Johansen
Martin Cramer Pedersen
Kresten Lindorff-Larsen
Funding
Lundbeckfonden (R155-2015-2666)
- Kresten Lindorff-Larsen
- Lise Arleth
European Commission (INFRAIA-01-2018-2019 GA 871037 (iNext Discovery))
- Tamim Darwish
- Tobias Schubeis
- Guido Pintacuda
Villum Fonden (35955)
- Nicolai Tidemand Johansen
- Tobias Schubeis
- Guido Pintacuda
ERC: European Union's Horizon 2020 research and innovation programme (ERC-2015-CoG GA 648974)
- Guido Pintacuda
Novo Nordisk Fonden (NNF15OC0016670)
- Lise Arleth
Biotechnology and Biological Sciences Research Council (BB/R00126X/1)
- Mark SP Sansom
Biotechnology and Biological Sciences Research Council (BB/N000145/1)
- Mark SP Sansom
Engineering and Physical Sciences Research Council (EP/R004722/1)
- Mark SP Sansom
Engineering and Physical Sciences Research Council (EP/R029407/1)
- Mark SP Sansom
Engineering and Physical Sciences Research Council (EP/V010948/1)
- Mark SP Sansom
Wellcome Trust (208361/Z/17/Z)
- Mark SP Sansom
National Collaborative Research Infrastructure Strategy (N/A)
- Tamim Darwish
- Mark SP Sansom
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Johansen 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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Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation
eLife 11:e71887.
https://doi.org/10.7554/eLife.71887
Further reading
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- Biochemistry and Chemical Biology
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Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes.
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- Structural Biology and Molecular Biophysics
SCARF1 (scavenger receptor class F member 1, SREC-1 or SR-F1) is a type I transmembrane protein that recognizes multiple endogenous and exogenous ligands such as modified low-density lipoproteins (LDLs) and is important for maintaining homeostasis and immunity. But the structural information and the mechanisms of ligand recognition of SCARF1 are largely unavailable. Here, we solve the crystal structures of the N-terminal fragments of human SCARF1, which show that SCARF1 forms homodimers and its epidermal growth factor (EGF)-like domains adopt a long-curved conformation. Then, we examine the interactions of SCARF1 with lipoproteins and are able to identify a region on SCARF1 for recognizing modified LDLs. The mutagenesis data show that the positively charged residues in the region are crucial for the interaction of SCARF1 with modified LDLs, which is confirmed by making chimeric molecules of SCARF1 and SCARF2. In addition, teichoic acids, a cell wall polymer expressed on the surface of gram-positive bacteria, are able to inhibit the interactions of modified LDLs with SCARF1, suggesting the ligand binding sites of SCARF1 might be shared for some of its scavenging targets. Overall, these results provide mechanistic insights into SCARF1 and its interactions with the ligands, which are important for understanding its physiological roles in homeostasis and the related diseases.
