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.
Highlights
Magnesium is the most abundant divalent cation (Mg2+) inside the cell, where it is mainly associated with the biological energy source adenosine triphosphate and other negatively charged molecules[1]
We employ custom developed state-of-the-art methodology, i.e. size-exclusion chromatography (SEC) coupled to SANS26,27 and match-out deuterated carrier systems for SANS28,29, and >100 kHz MAS nuclear magnetic resonance spectroscopy (NMR) in lipid bilayers[30,31]. Based on these data in conjunction with molecular simulations and modeling, we propose a model in which CorA is in a dynamic equilibrium between symmetric and asymmetric states, independent of bound Mg2+, but where an ensemble of conducting states is energetically more favourable for Mg2+-free CorA due to increased conformational dynamics resulting from the released electrostatic constraint
We extended the analysis to metadynamics simulations (MetaD) that allows for enhanced sampling of structural dynamics
Summary
Magnesium is the most abundant divalent cation (Mg2+) inside the cell, where it is mainly associated with the biological energy source adenosine triphosphate and other negatively charged molecules[1]. From these observations, the proposed model involves a sequential destabilisation of CorA upon Mg2+ removal, leading to a highly dynamic protein with shuffling protomers in the ICD, increasing the likelihood of pore dilation and wetting events[20,21]. High-speed atomic force microscopy (HS-AFM) data on densely packed CorA in lipid bilayers supported this model, but at the same time provided more insight to the dynamic interconversion of different states, including a fourth population of highly asymmetric CorA, not resolved by cryo-EM23 This population accounted for most observed conformations at low Mg2+ concentrations, supporting that CorA is a dynamic protein with a relatively flat energy landscape and, potentially, multiple open states. The cryo[95] EM and AFM experiments hint towards a highly dynamic ensemble of primarily asymmetric states at low Mg2+ concentrations, while the successful crystallisation of M1 site mutants suggests that the closed state is significantly present at these conditions
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