Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation
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Evaluation Summary:
Magnesium is an essential metal that is involved in vital biological processes. Using a model system, the authors discovered the presence of a conformational equilibrium between different unligated states that may explain the mechanism of magnesium transport. The combination of several different approach support the hypothesis that molecular motion is involved in the transport mechanism of Mg2+ ions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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 Mg 2+ 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 Mg 2+ -bound closed structure nor the Mg 2+ -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 Mg 2+ . We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg 2+ but that conducting states become more populated in Mg 2+ -free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.
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Evaluation Summary:
Magnesium is an essential metal that is involved in vital biological processes. Using a model system, the authors discovered the presence of a conformational equilibrium between different unligated states that may explain the mechanism of magnesium transport. The combination of several different approach support the hypothesis that molecular motion is involved in the transport mechanism of Mg2+ ions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer 1 (Public Review):
Magnesium is an essential ion that is involved in several biological processes. How Mg2+ ions are transported in the cell is still unclear. The authors chose an excellent model system, CorA, a prototype of Mg2+ transport in cells. CorA has been previously crystallized in a Mg2+ bound state. It forms symmetric pentamers that are quite rigid. However, this well-resolved structure did not offer any mechanistic insights into the ion transfer process. The authors surmised that a possible explanation is the interconversion of CorA between different conformations (competent and incompetent for transport). Therefore, they studied CorA both in the unligated and ligated form using small-angle neutron scattering (SANS) in concert with molecular dynamics simulations (MD) and MAS (fast spinning) solid-state NMR …
Reviewer 1 (Public Review):
Magnesium is an essential ion that is involved in several biological processes. How Mg2+ ions are transported in the cell is still unclear. The authors chose an excellent model system, CorA, a prototype of Mg2+ transport in cells. CorA has been previously crystallized in a Mg2+ bound state. It forms symmetric pentamers that are quite rigid. However, this well-resolved structure did not offer any mechanistic insights into the ion transfer process. The authors surmised that a possible explanation is the interconversion of CorA between different conformations (competent and incompetent for transport). Therefore, they studied CorA both in the unligated and ligated form using small-angle neutron scattering (SANS) in concert with molecular dynamics simulations (MD) and MAS (fast spinning) solid-state NMR spectroscopy. SANS data suggest a spread of conformations that ranges from the symmetric arrangement observed in the crystal structure to various asymmetric topologies that are thought to be dynamically active conformations. MD simulations identified several relative conformational minima that make up for a quite ragged energy landscape for CorA. The simulations and SANS data suggest that the interconversion between different conformational states may represent a driving force for ion transport. Intriguingly, MAS solid-state NMR data do not show any significant changes in the protein fingerprint upon adding Mg2+ ions. The authors explain the lack of chemical shift differences is probably due to the fast/intermediate interconversion of the conformations in the NMR time scale. T1rho relaxation measurements support this hypothesis. The derived residue-specific R1rho values indicate a global increase of the conformational dynamics moving from the ligated to the unligated state of CorA, supporting the hypothesis that the transport process may be dynamically driven.
Overall, the research is well-executed, and the spectroscopic data are in agreement with the MD simulations. The latter explains how the unligated state of CorA is challenging to crystallize. The paper is of broad interest, and the combination of these biophysical techniques is compelling. Prior to publication, the authors need to address the following points:
A) The paper is centered on the hypothesis that ion transport is dynamically driven. The authors have carried out extensive MD simulations to sample the different conformations of CorA. However, there are no indications in this paper of how the actual transport mechanism can occur. What happens to the ion-binding site during the interconversion between different states?
B) Most of the discussion is quite speculative (and long). The paper would benefit from shortening the discussion and may focus more on the finding of the current manuscript. For instance, it is not clear to this reviewer whether asymmetric conformations are partially competent for ion transport. Also, what is the evidence of the existence of a deep minimum for a well-defined open state (fig 6)? Is it possible that these partially open states are sufficient to justify transport? Again, a figure, or better, the analysis of what happens to the binding site would probably explain the basis for ion transport.
C) The fitting with a mono-exponential function for the T1rho data points (F306) seems not to accurately report the decay of the signal (see panel D in Fig 5). Is it due to the presence of multiple states? Or is it due to the poor S/N in the spectra? Indeed, this reviewer understands the challenges for these kinds of systems, but the errors in the measurements at short delay times are quite large. -
Reviewer 2 (Public Review):
This work reports on the mechanisms of Mg2+ transport by CorA proteins. These pentameric channels form symmetric structures when Mg2+ is bound in the pore, whereas the structures of open channels have not been solved yet, and only models have been proposed. The authors used a combination of small-angle neutron scattering (SANS), 1H-detected solid-state nuclear magnetic resonance (NMR) spectroscopy, and negative stain electron microscopy (EM), together with molecular dynamics simulations, to understand structural and dynamic basis of Mg2+ ion conduction. The main conclusions of this elegant study are: i) conducting states are symmetry-broken structures; ii) in the absence of Mg2+, the channel is conformationally flexible adopting different symmetry-broken pentamer structures that are in dynamic equilibrium; …
Reviewer 2 (Public Review):
This work reports on the mechanisms of Mg2+ transport by CorA proteins. These pentameric channels form symmetric structures when Mg2+ is bound in the pore, whereas the structures of open channels have not been solved yet, and only models have been proposed. The authors used a combination of small-angle neutron scattering (SANS), 1H-detected solid-state nuclear magnetic resonance (NMR) spectroscopy, and negative stain electron microscopy (EM), together with molecular dynamics simulations, to understand structural and dynamic basis of Mg2+ ion conduction. The main conclusions of this elegant study are: i) conducting states are symmetry-broken structures; ii) in the absence of Mg2+, the channel is conformationally flexible adopting different symmetry-broken pentamer structures that are in dynamic equilibrium; iii) dynamic equilibria between different symmetric and asymmetric states are present with or without Mg2+, but the conducting structures are more populated in the ion-free channel; iv) the overall energy landscape is complex with multiple intermediate states; the populations of the limiting states, symmetric closed state and asymmetric open state, are tuned by the presence/concentration of regulatory ions. These conclusions are supported by rigorous experimental data and computations. Specifically, SANS and NMR data indicate that there are no major structural changes induced by Mg2+; SANS curves are consistent with an ensemble of different states that are overall asymmetric; the NMR peak intensities and relaxation results indicate that Mg2+ renders the backbone more flexible; the negatively stained EM images reveal the presence of asymmetric pentameric structures among the most abundant particles. In my opinion, this work is a tour de force and technically superb. The mechanistic insights gained from this study are very significant for understanding CorA function. The tight coupling of structural and dynamic degrees of freedom uncovered thanks to the multi-pronged integrative approach is a beautiful illustration of how function is tuned by motions. Unique information gained from the MAS NMR experiments gives insights into the CorA residues undergoing local motions.
The only weakness is that the proposed structural model is of limited resolution so far, but getting atomic-level structures associated with the multiple states is totally beyond the scope of this study.
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