Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation
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
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.
Metrics
-
- 1,288
- views
-
- 227
- downloads
-
- 17
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
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)
Further reading
-
- Structural Biology and Molecular Biophysics
The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G-protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.
-
- Immunology and Inflammation
- Structural Biology and Molecular Biophysics
Increasing evidence suggests that mechanical load on the αβ T-cell receptor (TCR) is crucial for recognizing the antigenic peptide-bound major histocompatibility complex (pMHC) molecule. Our recent all-atom molecular dynamics (MD) simulations revealed that the inter-domain motion of the TCR is responsible for the load-induced catch bond behavior of the TCR-pMHC complex and peptide discrimination (Chang-Gonzalez et al., 2024). To further examine the generality of the mechanism, we perform all-atom MD simulations of the B7 TCR under different conditions for comparison with our previous simulations of the A6 TCR. The two TCRs recognize the same pMHC and have similar interfaces with pMHC in crystal structures. We find that the B7 TCR-pMHC interface stabilizes under ∼15 pN load using a conserved dynamic allostery mechanism that involves the asymmetric motion of the TCR chassis. However, despite forming comparable contacts with pMHC as A6 in the crystal structure, B7 has fewer high-occupancy contacts with pMHC and exhibits higher mechanical compliance during the simulation. These results indicate that the dynamic allostery common to the TCRαβ chassis can amplify slight differences in interfacial contacts into distinctive mechanical responses and nuanced biological outcomes.