Real time dynamics of Gating-Related conformational changes in CorA

Abstract

CorA, a divalent-selective channel in the metal ion transport superfamily, is the major Mg2+-influx pathway in prokaryotes. CorA structures in closed (Mg2+-bound), and open (Mg2+-free) states, together with functional data showed that Mg2+-influx inhibits further Mg2+-uptake completing a regulatory feedback loop. While the closed state structure is a symmetric pentamer, the open state displayed unexpected asymmetric architectures. Using high-speed atomic force microscopy (HS-AFM), we explored the Mg2+-dependent gating transition of single CorA channels: HS-AFM movies during Mg2+-depletion experiments revealed the channel’s transition from a stable Mg2+-bound state over a highly mobile and dynamic state with fluctuating subunits to asymmetric structures with varying degree of protrusion heights from the membrane. Our data shows that at Mg2+-concentration below Kd, CorA adopts a dynamic (putatively open) state of multiple conformations that imply structural rearrangements through hinge-bending in TM1. We discuss how these structural dynamics define the functional behavior of this ligand-dependent channel.

Mg2+-dependent conformational equilibria in CorA: an integrated view on transport regulation

“…However, a single asymmetric model as derived from NMA (Figure 4B) is neither compatible with a single set of peaks in NMR (Figure 2C and Figure 2E) nor the substantial experimental evidence for a nearly symmetric, closed state in presence of Mg 2+ . Likely, CorA adopts multiple different states 20,23 , and according to our data does so both with and without Mg 2+ bound. In this case, the SANS data would represent the number weighted average of different states that must be overall asymmetric.…”

“…Recent coarse grained MD simulations revealed the residue level details of how a complex interaction network involving asymmetric movements of ICD monomers ultimately leads to a conducting state upon removal of Mg 2+ 22 . 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-EM 23 . Interestingly, this population accounted for most observed conformations at low Mg 2+ concentrations, supporting that CorA is a dynamic protein with a relatively flat energy landscape and, potentially, multiple open states.…”

Asymmetric CorA Gating Mechanism as Observed by Molecular Dynamics Simulations

“…The asymmetric models that we obtained from CG MD simulations are consistent with HS-AFM data. 15 The asymmetric movement of the cytoplasmic domain is also in line with the previous structural and experimental data derived from the low resolution (∼21 Å) cryo-EM structure of MjCorA obtained in low magnesium conditions and functional and biophysical (EPR, fluorescence) studies. 43 These conformational changes result in destabilization of the interaction of the acidic-rich loop region formed by α5 and α6 helices (the CorA elbow) with the α7 helix.…”

“…These highly dynamic conformational changes of individual monomers in our simulations are consistent with recent studies on protein structural rearrangements caused by the decrease in magnesium concentration in real-time 15 and as seen in asymmetric cryo-EM structures. 14 However, it is important to emphasize that the resolution of these cryo-EM structures and HS-AFM data is limited and cannot give a precise picture of the ensemble of multiple intermediate structures and detailed changes in the conformation of the hydrophobic pore region at the molecular level.…”

“… 14 Such asymmetric movements of the cytoplasmic domains were also observed recently with real-time high-speed atomic force microscopy (HS-AFM). 15 Apart from the proposed iris-like and asymmetry collapse mechanisms, the helix rotation mechanism was proposed based on mutagenesis studies and whole-cell transport assays, 5 where rotation of the α7 helix segment replaces apolar residues with polar ones inside the pore hence allowing the ion movement ( Figure 1 C). Interestingly, recent crystallographic structures of one of the CorA Mg 2+ binding site mutants ( Figure 1 C, M1 binding site mutation) showed a limited effect of magnesium ions on the large conformational rearrangements and transport activity.…”