1. Structural Biology and Molecular Biophysics
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Real time dynamics of Gating-Related conformational changes in CorA

  1. Martina Rangl
  2. Nicolaus Schmandt
  3. Eduardo Perozo  Is a corresponding author
  4. Simon Scheuring  Is a corresponding author
  1. Weill Cornell Medical College, United States
  2. The University of Chicago, United States
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Cite this article as: eLife 2019;8:e47322 doi: 10.7554/eLife.47322

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.

Introduction

Magnesium (Mg2+) is a key divalent cation in biology. balaIt regulates and maintains numerous, physiological functions such as nucleic acid stability, muscle contraction, heart rate and vascular tone, neurotransmitter release, and serves as cofactor in a myriad of enzymatic reactions (Altura, 1991; de Baaij et al., 2015; Jahnen-Dechent and Ketteler, 2012; Romani, 2013; Ryan, 1991). Most importantly, it coordinates with ATP, and is thus crucial for energy production in mitochondria (Altura, 1991; Pilchova et al., 2017; Romani, 2011; Swaminathan, 2003; Yamanaka et al., 2016). In order to store Mg2+ in the mitochondrial lumen it is imported via Mrs2 (Kolisek et al., 2003) and Alr2 (Liu et al., 2002) ion channels that are closely related to CorA, the main Mg2+-importer in bacteria (Guskov and Eshaghi, 2012; Hmiel et al., 1986; Knoop et al., 2005; Schweyen and Froschauer, 2007). Although these Mg2+-transport proteins do not show much sequence conservation, they all share two trans-membrane domains (TMDs) with the signature motif Glycine-Methionine-Asparagine (GMN) at the extracellular loop (Knoop et al., 2005; Schweyen and Froschauer, 2007).

The crystal structure of CorA from Thermotoga maritima in its Mg2+-bound closed state (Eshaghi et al., 2006; Lunin et al., 2006; Payandeh and Pai, 2006) revealed a 5-fold symmetric homo-pentamer forming an ~11 nm funnel-like structure, with large intracellular domains and Mg2+-ions bound between the subunits (Eshaghi et al., 2006; Guskov et al., 2012; Lerche et al., 2017; Lunin et al., 2006; Payandeh and Pai, 2006). Through CorA, Mg2+-homeostasis is achieved by a negative feedback mechanism in which Mg2+ acts as both, charge carrier and ligand (Dalmas et al., 2014b). The binding of Mg2+ at the cytoplasmic subunit interfaces leads to channel closing and thereby limits further Mg2+-influx (Dalmas et al., 2014a; Dalmas et al., 2014b; Palombo et al., 2012; Pfoh et al., 2012; Schindl et al., 2007).

Determining the structure of CorA in its Mg2+-unbound (Apo) form is considered a fundamental step towards understanding its gating mechanism. Yet, the first CorA crystal structure in the absence of divalent cations showed little or no changes compared to the fully Mg2+-bound closed channel structure, with only a slight kink between TMDs and intracellular domains (Pfoh et al., 2012). In contrast, EPR spectroscopy indicated much larger structural rearrangements within the protein upon Mg2+-dissociation, suggestive of a more dramatic closed-to-open channel transition (Dalmas et al., 2010; Dalmas et al., 2014b). These results were confirmed by cryo-electron microscopy (cryo-EM), reporting surprising asymmetric rearrangements of the individual subunits within the homo-pentamer in Mg2+-free condition (Cleverley et al., 2015; Matthies et al., 2016). Based on ~7 Å cryo-EM structures (Matthies et al., 2016), a model was proposed in which Mg2+-unbinding leads to an increase in the inter-subunit conformational flexibility of CorA, thereby resulting in at least two asymmetric structures with, presumably, open gates (Matthies et al., 2016).

Despite progresses in the structural characterization of the Mg2+-free CorA structure(s), the transition from the closed (Mg2+-bound) state to the conductive unliganded conformation is still unclear. At 7.1 Å, the putative open CorA asymmetric cryo-EM structures were solved only from a relatively low number of particles (26,271 and 27,416 of 173,653 particles for open-I and open-II states, respectively) (Matthies et al., 2016), and due to its intrinsic averaging for structure determination, the cryo-EM data cannot inform about time-dependent behavior. We posit that these structures could be sampled from three different dynamic processes: they could represent two different open state conformations with significant state dwell-times (structurally resolved by cryo-EM); they could be intermediates along a conformational trajectory; or finally, they might be mere snapshots of a highly dynamic, fluctuating molecule without a long-lasting and well-defined high-resolution structure.

In order to elucidate the sequence of events underlying gating-related CorA conformational changes, we used high-speed atomic force microscopy (HS-AFM) (Ando et al., 2001; Ando et al., 2014) to capture CorA structural rearrangements during Mg2+-depletion experiments. HS-AFM is unique in its ability to concomitantly characterize molecular structures and dynamics under native-like conditions. In agreement with electrophysiological, spectroscopic and biophysical data (Dalmas et al., 2014b), we found that during Mg2+-depletion experiments individual CorA molecules lost pentameric symmetry and became highly dynamic at Mg2+-concentrations below ~2 mM. Under these conditions, the symmetric state is reversibly adopted by means of dynamic fluctuations among the individual subunits, indicative of spontaneous Mg2+-rebinding. However, in the absence of Mg2+, CorA transitions to an asymmetric state. Thus, the conformational energy landscape likely comprises two deep energy minima represented by the fully symmetric (Mg2+-bound) and the asymmetric (Mg2+-depleted) states interspersed by a wide plateau of conformational fluctuations of a highly flexible molecule, which likely represents the conductive state.

Results

Lipid composition determines reconstitution density and morphology

In recent years, HS-AFM has demonstrated its power to observe molecular mechanisms and structural dynamics of single molecules under physiological conditions with extraordinary detail (Chiaruttini et al., 2015; Kodera et al., 2010; Marchesi et al., 2018; Preiner et al., 2014; Rangl et al., 2016; Ruan et al., 2018; Ruan et al., 2017). Here, we have used HS-AFM to track the conformational changes of CorA as a result of Mg2+-concentration changes in real-time. For this, wild-type (wt) CorA was reconstituted at low lipid-to-protein ratios (LPRs between 0.2 and 0.4) in the presence of Mg2+. The resulting proteo-liposomes were adsorbed onto freshly cleaved mica under saturating Mg2+ (10 mM) condition and imaged at an acquisition rate of 1–2 frames s−1 with a resolution of 0.5 nm pixel−1. CorA-containing vesicles were generated from protein reconstitutions in POPC/POPG (3:1, w:w). When imaged by HS-AFM, vesicles spread on the mica support resulting in large membrane patches with densely packed CorA (Video 1). These patches mostly exposed the periplasmic face. Smaller crowded areas of molecules expose the intracellular side (Figure 1a, Video 1).

Figure 1 with 1 supplement see all
Sample morphology of CorA reconstitutions for HS-AFM.

(a) HS-AFM overview topograph of densely packed CorA in a POPC/POPG (3:1) lipid bilayer exposing the periplasmic side and a loosely packed protein area with diffusing molecules exposing the intracellular face (full color scale: 20 nm). Left: Height histogram of the HS-AFM image with two peaks representative of the mica and the CorA surface (∆Height (peak-peak): 12 nm (20,500 height values)). The dashed line indicates the position of the cross-section analysis shown in (b). (b) Profile of the membrane shown in a), including a cartoon (top) of the membrane in side view. The height profile (~12 nm) corresponds well to the all-image height analysis (a, left) and the CorA structure (Matthies et al., 2016). (c) High-resolution image (top) and cross-section analysis along dashed line (bottom) of the periplasmic face. The height and dimension of the periplasmic face is in good agreement with the structure (left), and the periodicity (~14 nm, n = 40) corresponds well with the diameter of the intracellular face spacing the molecules on the other side of the membrane (full color scale: 2 nm). (d) HS-AFM image of densely packed CorA embedded in a DOPC/DOPE/DOPS (4:5:1) membrane. This reconstitution resulted in two stacked membrane layers, both exposing the CorA intracellular face. The dashed line indicates the position of the cross-section analysis shown in (e). Left: Height histogram of the HS-AFM image with two peaks at ~12 nm and ~17 nm (32,500 height values), corresponding to the proteins in two stacked membranes (full color scale: 20 nm). (e) Section profile of the membrane shown in d), including a cartoon (top) of the membrane in side view. (f) High-resolution view and cross-section analysis along dashed line (bottom) of the CorA intracellular face revealing the individual subunits of the pentamers (full color scale: 3 nm). Inset: 5-fold symmetrized average of CorA. The dimensions of CorA observed with HS-AFM are in good agreement with the structure (left: PDB 3JCF). The structures in (c) and (f) are shown in ribbon (top) and surface (bottom) representations, respectively.

Video 1
CorA reconstituted in POPC/POPG liposomes.

In overview scans the proteoliposomes were opened by applying slightly increased loading forces, thereby revealing CorA membranes exposing both faces, the periplasmic face in crystalline packing and the intracellular side in crowded membrane areas. Video settings: Full scan size: 444 nm, 200 pixels, scan rate: 1 frame s−1, full color range: 20 nm.

Cross-section analysis of the CorA membrane patches showed overall heights of ~12 nm (Figure 1b). High-resolution movies of the periplasmic domains revealed protrusions of 0.9 ± 0.4 nm in height and 3.4 ± 0.4 nm in diameter, and resolved a central indentation where the channel pore is located (Figure 1c). Despite the fast diffusion of CorA molecules within the clusters exposing the intracellular side, some high-resolution snapshots allowed analysis of the surface structure. The intracellular face protruded ~7 nm from the membrane (Figure 1—figure supplement 1a) with top-ring and outer diameters of ~6 nm and ~10 nm, respectively (Figure 1—figure supplement 1b,c; Video 2). These measurements are in good agreement with the molecular dimensions of CorA (Eshaghi et al., 2006; Lunin et al., 2006; Matthies et al., 2016).

Video 2
Four examples of high magnification movies of the intracellular side of CorA reconstituted in POPC/POPG.

Fast diffusing molecules with flower-shaped structures were observed. Video settings: Full scan sizes: 80–120 nm, 160–200 pixels, scan rates: 1–2 frame s−1, full color range: 5 nm.

However, it became clear that in order to study Mg2+-dependent conformational dynamics, more stably packed molecules were required (Müller et al., 1999; Ramadurai et al., 2010). We found that reconstituting the protein in DOPC/DOPE/DOPS (4:5:1, w:w:w) and adsorbed and imaged at lower pH 6.0 resulted in widespread surface coverage of CorA-crowded membranes with only slowly moving molecules. Moreover, under these conditions densely packed CorA patches were stacked and exposed the intracellular face of the channel (Figure 1d,e; Video 3), thus providing an excellent experimental platform for studying CorA Mg2+-dependent conformational changes. High-resolution HS-AFM topographs displayed a ‘flower-shaped’ surface structure corresponding to the CorA intracellular face. This view allowed to resolve individual subunits in the pentamer with top-ring diameter of 5.0 ± 0.9 nm and a center-to-center distance of the molecules, that is outer diameter, of 10.9 ± 2.1 nm (Figure 1f).

Video 3
CorA reconstituted in DOPC/DOPE/DOPS 4:5:1.

The sample support was fully covered with CorA membranes exposing the intracellular face with areas of slowly moving molecules and membranes with densely packed channels stacked on top. Video settings: Full scan size: 200 nm, 200 pixels, scan rate: 1 frame s−1, full color range: 20 nm.

Mg2+-depletion induces large conformational changes of the intracellular face

We then monitored the structural changes of the channel upon Mg2+-depletion in real time at higher magnification, that is at scan sizes < 200 nm and pixel sampling of 0.5 nm pixel−1. First, we studied the periplasmic face of CorA under saturating Mg2+ (10 mM), in which all channels are expected to be in the closed conformation. Subsequently, membranes were imaged at reduced Mg2+-concentration near the reported apparent Kd for Mg2+ (~2 mM), and finally at 0 mM Mg2+ to focus on the dynamically open Apo form.

Mg2+-depletion was achieved by live injection of EDTA, while continuously monitoring CorA. Using the closed state as reference, no structural changes of the periplasmic face of CorA were observed at 10 mM Mg2+, at 2 mM Mg2+ (after ~10 min) and in absence of Mg2+ (after ~12 min) (Figure 2—figure supplement 1). At the present resolution (0.5 nm pixel−1) any putative changes in the conformation of the periplasmic face of the channel are beyond the current resolution limit of HS-AFM (although we cannot rule out that the structures of the small periplasmic loops might not change upon Mg2+-depletion). This is consistent with the ~7 Å cryo-EM structures, which show little or no Mg2+-dependent changes on the periplasmic face (Matthies et al., 2016; Pfoh et al., 2012).

In stark contrast, previous EPR (Dalmas et al., 2010; Dalmas et al., 2014b) and cryo-EM (Matthies et al., 2016) analyses have revealed dramatic conformational rearrangements of the intracellular domains (resulting in a loss of symmetry) in the nominal absence of Mg2+ (conditions favoring the functionally open state) (Figure 2a). First, to measure the CorA Mg2+-affinity (Pilchova et al., 2017) in our experimental set-up, we used a microfluidic system connected to a constant pressure and flow pump (Miyagi et al., 2016), with which we slowly exchanged the complete 10 mM Mg2+ measuring solution to a Mg2+-free buffer (containing additional 2 mM EDTA). Analysis of the distribution of symmetric and dynamically asymmetric CorA particles pointed to a Kd of ~2 mM Mg2+, which is in good agreement with the reported affinity (Figure 2—figure supplement 2). Next, we monitored the structural and dynamical transition of CorA upon Mg2+-depletion by pipetting defined amounts of EDTA into the measurement fluid cell to achieve the following equilibrium concentrations: 10 mM Mg2+, ~2 mM Mg2+ (~Kd), and 0 mM Mg2+ (putative).

Figure 2 with 2 supplements see all
CorA in presence and absence of Mg2+.

(a) Molecular surface representations of CorA structures in the Mg2+-bound (closed) and Mg2+-free (open) states (PDB: 3JCF and 3JCH) (Matthies et al., 2016). Top: intracellular view. Bottom: side view. The Mg2+-free (open) structure protrudes further (Δh) from the membrane. (b) HS-AFM images of a membrane patch with densely packed CorA exposing the intracellular face with corresponding cross-section analyses in 10 mM Mg2+ (left) and after ~20 min in absence of Mg2+ (right). The cross-section profiles (bottom) along the dashed lines demonstrate the height increase of ~1.5 nm of the same molecules in absence of Mg2+ compared to the topography height in presence of Mg2+.

Consistent with the Mg2+-liganded cryo-EM structure (Figure 2a, left), at saturating 10 mM Mg2+ condition CorA channels revealed a stable, flower-like 5-fold symmetric structure (Figure 2b, left). However, once Mg2+-concentrations dropped to ~2 mM and below, individual channels start to fluctuate between various structural states, occasionally assuming increased height and, after ~20 min in absence of Mg2+, adopting an ill-defined conformation with significantly increased protrusion height (Figure 2b, right). The Mg2+-dependent loss of symmetry and increased protrusion height are also consistent with expected structural features of the Mg2+-free (open) CorA structures, where individual subunits move towards the former 5-fold axis and thus stand taller (Matthies et al., 2016) (Figure 2a, bottom). The large, ~1.5 nm, protrusion height difference (ΔHeight) of such a conformation change presents a useful signature to detect and follow the conformational dynamics of individual channels in the membrane. However, these two ΔHeight-states must not be mistaken with functional or even structural states. They are merely a way to topographically discriminate between the closed 5-fold symmetric state and any other state where single subunits stand up, that is move towards the 5-fold axis and thus appear higher.

Height section kymographs of individual molecules (Figure 3a, top) over extended imaging periods (Figure 3a, middle) allowed the subsequent computational detection of single molecule dynamics based on the fluctuations in height differences (ΔHeight) as a function of time (Figure 3a, bottom): While a ΔHeight of ~1 nm represents a symmetric CorA structure with a pore in the center, larger ΔHeight values represent molecules with increased protrusion height. ΔHeight values < 1 nm were found for molecules of low height (putative closed state) where the central pore could not be resolved. Hence, the ΔHeight/time traces were idealized to represent two ΔHeight-states using the Step Transition and State Identification (STaSI) algorithm developed for single molecule experiments (Shuang et al., 2014), which we successfully adapted for the analysis of HS-AFM ΔHeight/time traces (Heath and Scheuring, 2018) (Figure 3a, red line).

CorA conformational changes and dynamics upon Mg2+-depletion.

(a) Single CorA molecule at indicated time points during Mg2+-depletion. Below: Section kymograph of the molecule and corresponding ΔHeight/time trace derived from the center area of the imaged CorA channel. The red line is a fitted idealized trace with two distinct ΔHeight-states. Right: Height histogram of the ΔHeight/time trace. (b) Time-lapse HS-AFM of membrane patches with densely packed CorA channels that expose the intracellular face during Mg2+-depletion experiments. Direction of Mg2+-depletion and time points are indicated above frames. Scale bars: 10 nm. Below: Percentage of CorA molecules with increased height (putatively open states) as a function of time. (a) and (b): Frames acquired in saturating 10 mM Mg2+-concentrations are indicated in green, at ~2 mM Mg2+ in blue (only tested in (a) and the bottom panel of (b)) and at complete Mg2+-depletion (0 mM Mg2+) in red. Depletion of Mg2+ was achieved by manual addition of EDTA. (c) Number of Δheight-transitions and associated dwell-times following Mg2+-depletion. Bars indicate the number of high-to-low (‘down’, turquois) and low-to-high (‘up’, red) events binned over a time window of 30 s. Turquois pentagons and red diamonds indicate average closing and opening event time-points of corresponding average dwell-time (right axis), respectively. These averages were calculated over a sliding window of 20 events along the time axis. Analysis included molecules from 2 experiments at 0 mM Mg2+ (shown in (b) top and bottom) and 30,500 HS-AFM images thereof. Right: Histogram of average dwell-times in the ‘high’ (red) and ‘low’ (turquois) states (where the high state represents/comprises all conformational states with elevated subunits).

We monitored molecular transitions from the Mg2+-bound to Mg2+-free states in time-lapse experiments. HS-AFM images of CorA membrane patches during these extended experiments reproducibly revealed the conformational changes of individual molecules over time (Figure 3b, Video 4, Video 5). While CorA maintained the 5-fold symmetric state at saturating Mg2+ condition (Figure 3b, green time stamps), the channels started switching dynamically between (at least) two conformational states at concentrations below ~2 mM Mg2+ (Figure 3b, blue and early red time stamps), and began populating strongly protruding asymmetric states as Mg2+-depletion progressed (Figure 3b, red time stamps). A cumulative height-based state-assignment of CorA as a function of Mg2+-depletion demonstrated that the number of channels in the putatively open state(s) gradually increased (Figure 3b, bottom). In contrast, the number of transitions (i.e. switching back and forth between the high (open) state(s) and the state of lower height) cumulated earlier during the titration experiment (Figure 3c). Thus, we are looking at a transition from the symmetric low (closed) to the asymmetric high (open) structures and a wide range of fluctuations between states at intermediate Mg2+-concentrations, or within the first minutes of complete Mg2+-depletion.

Video 4
CorA conformational changes upon Mg2+-depletion.

Within ~15 min most molecules transition from a 5-fold symmetric, Mg2+-bound, putatively closed, conformation to dynamically active, flexible structures that equilibrate into an asymmetric molecule of increased height, the putative ligand-free open state. Video settings: Full scan size: 60 × 40 nm, 0.4 nm/pixel, scan rate: 1.3 frame s−1, full color range: 5 nm.

Video 5
Monitoring the conformational changes upon Mg2+-depletion.

Within ~30 min, CorA molecules change from the stable 5-fold symmetric, putatively closed, conformation to a dynamic structure representing the apparent open conformation. Video settings: Full scan size: 100 nm, 200 pixels, scan rate: 1 frame s−1, full color range: 5 nm.

CorA state transition revealed a highly dynamic intermediate

In addition to the gradual state conversion during Mg2+-depletion, the number of transition events varied over time. At saturating Mg2+, all molecules occupied a stable conformation and the state interconversion activity is virtually zero (Figure 3b, green). About 5–8 min into the complete Mg2+-depletion experiment (at 0 mM Mg2+) the dynamics of the channels reach a maximum (Figure 3c), followed by another more stable, strongly protruding and asymmetric state, putatively representing an open conformation (Figure 3b,c, red). The time windows of these observations correlate well with electrophysiology experiments, in which the CorA-driven Mg2+-currents in Xenopus leavis oocytes decay within 15–20 min (Dalmas et al., 2010; Dalmas et al., 2014b). In addition to the number of transitions, the same trend is represented when analyzing the dwell-times of the ΔHeight-states in the nominal absence of Mg2+ (Figure 3c): Within the first 5 min in 0 mM Mg2+ the average time spent in the closed (low height) state is almost twice (~60 s) the averaged dwell-time in one of the putatively open (elevated height) conformations. After ~10 min in Mg2+-free condition, the situation is reversed, and more molecules show long dwell-times in the elevated state (Figure 3c, turquois pentagons and red diamonds). Thus, in this experiment, the CorA gating pathway initially favors the stable Mg2+-bound, low-height state by ~- 0.7kBT while elevated Mg2+-free (open) conformation(s) are favored when Mg2+ is depleted from all binding sites. In between, the channel is in a highly dynamic regime, probably reflecting Mg2+-unbinding and -rebinding events (Figure 3, Videos 4 and 5). We must highlight, however, that a total depletion of Mg2+ probably never occurs under physiological conditions, thus the fully Mg2+-depleted asymmetric stable states observed by cryo-EM and likely adopted here at the end of the depletion experiments might not be visited in the cell. More likely, the 5-fold symmetric closed state interchanges with a highly fluctuating molecule where single subunits dissociate from the quaternary structure of the cytoplasmic ensemble, opening the channel.

CorA fluctuates between several conformations in the open state

Following the general observation that the stable closed and open conformations are interconnected by a regime in which the channels are highly dynamic, we pursued a detailed structural examination of CorA in this intermediate dynamic stage at low Mg2+-concentrations. High-resolution HS-AFM image sequences of individual CorA channels revealed that CorA undergoes conformational rearrangements of the entire cytosolic Mg2+-sensor domain at rates beyond the bandwidth of the current measurements (<550 ms, the imaging rate of our videos). We also acquired movies at 250 ms frame acquisition and found that individual sequential frames displayed different apparently unrelated conformations, an indication that the conformational fluctuations are even faster (Figure 4d).

CorA adopts several highly dynamic conformations.

(a) Left: Surface representations of CorA cryo-EM structures in the high-resolution closed (PDB: 3JCF) and the two 7 Å resolution Mg2+-free open (PDBs 3JCH, 3JCG) conformations. Center: AFM topography simulations of the structures on the left. Right: Examples of HS-AFM frames of single CorA molecules in the symmetric closed (upper panel) and the asymmetric (putatively open) conformations (bottom panels). Full-frame size: 17.5 nm. Full z-scale: 2 nm. (b) High-resolution HS-AFM frames of an individual CorA channel upon Mg2+-depletion. The molecule fluctuates dynamically between several conformations. Time stamps are indicated. Full-frame size: 17.5 nm. Full z-scale: 2 nm. (c) HS-AFM image sequence of subsequent frames depicting CorA every 550 ms highlighting the structural flexibility of the molecule and the fast movements of the individual subunits. Full-frame size: 17.5 nm. Full z-scale: 2 nm. (d) HS-AFM image sequence depicting CorA every 250 ms. Full-frame size: 27 nm. Full z-scale: 2 nm.

Movie snapshots were classified into four conformational classes (Figure 4, Video 6): First, the fully-liganded 5-fold symmetric closed conformation (Eshaghi et al., 2006; Lunin et al., 2006; Matthies et al., 2016; Payandeh and Pai, 2006) (Figure 4a, top row, Figure 4b, 3.85 s, 7.15 s, 8,8 s and 14.3 s and Figure 4c, 1.1 s, 5.5 s and 6.05 s, Figures 4d, 0s, 0.75 s and 2.25 s). Second, a structure of reduced diameter with three asymmetrically distributed protrusions of increased height (Figure 4a, second row, Figure 4b, 9.35 s, Figure 4c, 0.55 s, 4.95 s, Figure 4d, 0.5 s), likely corresponding to the cryo-EM open-I state (Matthies et al., 2016). Third, a round- or star-shaped conformation with an elevated center resembling the cryo-EM open-II state where one subunit is displaced towards the channel axis (Matthies et al., 2016) (Figure 4a, third row, Figure 4b, 0s, 6.05 s, 7.7 s, 17.05 s and Figures 4c, 0s, 0.55 s, 2.75 s). However, given that our tentative assignment to open-I and open-II states only covers a fraction of all molecular observations, a fourth category was invoked to include the remaining range of structural variations, which we named open-+ states. This category includes asymmetric molecules of all kinds, even those without increased height (Figure 4a, fourth row, Figure 4b, 16.5 s, Figure 4d, 1.5 s, 1.75 s). It is clear that a key challenge in defining the conformational landscape of CorA will be the unbiased classification of discrete states in this highly flexible molecule, made evident by following a single CorA channel frame by frame (Figure 4c,d). Unfortunately, all our computational classification attempts failed likely due to the presence of protein-protein contacts with neighboring molecules and limited resolution due to the high mobility.

Video 6
High resolution HS-AFM movie of an individual CorA molecule switching between conformations in low Mg2+ concentration.

The molecule continuously switches between a subset of at least three states thought to represent conformations of the active CorA. Video settings: Full frame size: 17.5 nm, 35 pixels, scan rate: 2 frames s−1, full color range: 2 nm.

To work out a more detailed picture of the CorA-transitions we visually assigned these four different types of states to high-resolution HS-AFM movie sections recorded at different Mg2+-concentrations. In saturating Mg2+-concentrations (Figure 5a, green bars, Video 7), ~91% of the time molecules adopt the flower-shaped, 5-fold symmetric state, while the other states are barely populated. After Mg2+-reduction to 3 mM (Figure 5a, blue bars) and full Mg2+-depletion (Figure 5a, red bars, Video 8), the probability of finding CorA in any of the asymmetric states is reversed (Figures 5a, 3 classes on the right). Importantly, in 3 mM Mg2+ ~50% of the molecules pool in the open-+ (others) class, representative of a highly mobile and flexible molecule that switches fast between numerous sub-structures that could not be classified to any of the conformations described by cryo-EM. After full depletion, re-addition of Mg2+ could partially recover ~35% the symmetric closed-state structure (Figure 5a, yellow bars). Analysis of the probability of transitions from one structural state to the others (Figure 5b) demonstrated that under saturating Mg2+-concentrations the symmetric closed state is, as expected, stable. Once CorA starts adopting any of the asymmetric conformational states in Mg2+concentrations ~3 mM and lower, the probability to switch back to the symmetric state is only ~14%. This probability is further reduced to ~7% at 0 mM Mg2+.

Figure 5 with 1 supplement see all
CorA state occupancy and transition dynamics.

(a) State occurrence of 5-fold symmetric (assigned to Closed), dome-shaped (assigned to Open-II), elevated bean-shaped (assigned to Open-I) and other asymmetric CorAs (unassigned, Open +) at different Mg2+-concentrations: 10 mM Mg2+: green, 3 mM Mg2+: blue, 0 mM Mg2+: red, and after re-addition of 25 mM Mg2+: yellow. Bars represent the normalized percentages of state assignments of ~20 CorA molecules in ~80–100 frames, ie up to ~2400 molecular representations, for each Mg2+-condition. Error bars are standard error of mean (s.e.m.). Below, schematic representations of the various conformations. (b) CorA state transition-maps at 10 mM, 3 mM, 0 mM, Mg2+ and after subsequent re-addition of Mg2+ to 25 mM (from left to right). The schematic molecule on the left (rows) is the state in frame(n) and the schematic molecule on the top (columns) is the state in frame(n+1). Numbers are normalized percentages of the state transitions of the same experimental data as in (a). Color scale was adapted for each condition separately with a gradient from green (lowest occurrence of transition) over yellow and orange to red (highest occurrence of transition). Numbers in the center of boxes of 4 state transitions represent the sum of transitions between states with elevated subunits (blue dashed square) and between transitions of strongly elongated structures (red square). (c) CorA conformational transition model based on the HS-AFM observations. Within ~10 min of Mg2+-depletion, the 5-fold symmetric, fully Mg2+-liganded CorA transit into dynamically fluctuating molecules with flexible subunits until their conformation stabilizes in a Mg2+-free highly asymmetric structure with increased membrane protrusion height. Figure 5 - Information Supplement 1: Estimation of thermally activated TM1 helix motions We estimated the theoretical range of helical motion by considering that TM1 behaves like a flexible rod undergoing thermally excited motions. The helix (rod) is characterized by a specific persistence length LP that is related to the bending stiffness KS through LP=KSkBT. The basic description for the change in curvature between two points on the rod is given by t(s)s, with s being the arc length and  t a unit tangent vector at position (s). In an ideal system, the total elastic energy Eela of a particular conformation is given by the integral of the bending energies accumulated along a rod with contour length L: Eela= 0LKs2 ( t(s)s)2ds Assuming only circular curvatures along the rod, t(s)s=1r, where r is the radius of curvature. Using this basic description of an elastic polymer rod and considering the persistence length of protein α-helices Lp = 100 nm (as described in Choe and Sun, 2005) and a contour length L = 11 nm (length of TM1, see Supplementary Figure 4), we obtain Eela=LP kBT L2 r2 This equation thus estimates that the radius of curvature r = ~23 nm at 1 kBT. Helix bending of that range would result in ~2 nm movements at its end.

Video 7
CorA molecule cluster at saturating Mg2+ concentrations.

Molecules are found in the symmetric flower-shaped closed conformation more than 90% of the time. Video settings: Full frame size: 100 nm, 200 pixels, scan rate: 1.1 frames s−1, full color range: 5 nm.

Video 8
CorA molecules monitored just after complete Mg2+-depletion.

The majority of the molecules are in a highly dynamic state constantly switching between different structural states. Video settings: Full frame size: 100 nm, 200 pixels, scan rate: 1.8 frames s−1, full color range: 5 nm.

Interestingly, comparing 3 mM and 0 mM Mg2+-conditions gave a hint of the different Mg2+-loads between the various asymmetric states: while the number of transitions between elongated states (Figure 5b, red outline: open-I and open-+) is virtually identical in these two conditions, the transition into a state with elevated height is strongly favored at 0 mM Mg2+ (Figure 5b, dashed blue outline: open-II and open-I). We suggest that molecules with increased height where one subunit moves up close to the channel axis represent Mg2+-free states.

Discussion

On the basis of real-time HS-AFM imaging, we find that CorA Mg2+-dependent gating can be best described in three phases: Above the apparent Mg2+-affinity of the intracellular Mg2+-sensor sites (~2 mM Mg2+), the channel adopts a stable 5-fold symmetric state reminiscent of the high-resolution structures (phase 1). After several minutes of exposure to a Mg2+-free solution, CorA transitions into a set of asymmetric architectures, which are characterized by elevated cytoplasmic domains (phase 3). These structures are adopted through major conformational changes implicating hinge-bending of TM1 and reorientation of the cytoplasmic domains. This picture is wholly consistent with data derived from functional (electrophysiology), biochemical (crosslinking), biophysical (EPR, Fluorescence) and structural (cryo-EM) approaches. However, these results contrast with recent crystallographic data of CorA Mg2+ binding site mutants that reported limited or no structural changes compared to the closed wt channel, thereby challenging the hypothesis that Mg2+-depletion leads to large conformational changes and channel opening (Kowatz and Maguire, 2019). We argue, that the constrains of the crystal lattice on CorA limits the range of conformational changes that can be observed in X-ray structures. In contrast, HS-AFM imaging under physiological conditions shown here is more consistent with the single particle cryo-EM data reported in Cleverley et al. (2015); Matthies et al. (2016) and the EPR spectroscopic data (Dalmas et al., 2010; Dalmas et al., 2014b), that is when the Mg2+ is investigated under lattice-free conditions. These facts must be considered a critical factor in explaining these differences. Among multiple conformational states within phase 3, we identified CorA structures that resembled the low-resolution cryo-EM open-I and open-II structures. These phases 1 and 3 are characterized by structural stability or at least limitations by the structural freedom of CorA. Here, more work is required to identify and clearly distinguish between different sub-structures in the Mg2+-free elevated state of CorA. In contrast, at intermediate Mg2+-concentrations, and/or after short incubation times at 0 mM Mg2+, the channel reveals a highly mobile and fluctuating state (phase 2). Active subunits change and the entire molecule displays high structural variability, likely adopting many more conformations than the three states so far identified by cryo-EM. Indeed, while we can assign the fully 5-fold symmetric state with certainty, our assignment of molecules to state open-I and open-II must be considered as putative. Perhaps more importantly, at intermediate Mg2+-concentrations ~ 50% of the molecules could not be assigned and were pooled in a class of undefined quaternary structures (open-+). In this context, the details of the cryo-EM study become relevant: open-I and open-II only pooled 26,271 and 27,416 particles, respectively, of the total 173,653 particles, and thus a significant portion of the particles remained outside of these classes (Matthies et al., 2016). We note, however, that this initial assignment is ultimately constrained by the resolution limitations of both cryo-EM structural assignments and the present application of HS-AFM. We expect that given a larger number of particles to process (in cryo-EM) or improvements in the spatial and/or temporal resolution of HS-AFM, additional states are likely to emerge. We propose that the various CorA structural states and the associated subunit movements observed by HS-AFM may reflect variations of number and location of Mg2+-ions bound to the cytosolic domain (Figure 5c).

Among the main structural features of CorA is its long (~11 nm) first transmembrane helix (TM1), ranging all the way from the periplasmic face to the surface of the cytoplasmic Mg2+-sensor. TM1 is actually the only connection between the TM region and the cytoplasmic domain (Figure 5—figure supplement 1). Thus, it seems plausible that the large fluctuations of the cytoplasmic domains might be directly translated by TM1 into fluctuations within the channel pore. We suggest this mechanism as the basis for ion conductance through a particularly long (in comparison to other channels) ~55 Å pore, in which moving TM1s allow the asynchronous progression of ions through the pore. Considering the typical persistence length of an α-helix of ~100 nm (Choe and Sun, 2005), allowed us to estimate how much an 11 nm long thermally activated helix fluctuates (Figure 5 – Information Supplement 1). We find that at 1kBT TM1 could bend at its cytoplasmic end ~2 nm out of axis, in good agreement with observed bending of this helix in the open-I and open-II cryo-EM structures and the HS-AFM movies of subunit displacement. Thus, we propose that in the open state the pore is by far less narrow than assumed based on static structures. Indeed, constantly fluctuating TM1s would provide a considerable channel diameter towards the cytoplasmic side. This model would predict that amino acids close to the periplasmic side of the pore play the key role for gating. In agreement with this hypothesis, we find that the Mg2+-channel signature motif (GMN) is located right at the periplasmic end of the channel, whereas amino acids towards the cytoplasmic face of TM1 are less conserved (Figure 5—figure supplement 1).

The idea that there are multiple conductive conformations of CorA, or, as a paradigm shift, the conductive conformation of CorA is a fluctuating molecule, is favored by both, the subunit movement analysis and state transition analysis. In the long-term absence of Mg2+, CorA molecules adopt a rather stabilized asymmetric state resembling the putatively open state cryo-EM structures. However, a living cell under physiological conditions is unlikely to ever reach Mg2+-concentrations below ~1 mM. Thus we hypothesize that in cellula the physiologically relevant open state is a collection of fluctuating conformational states. Thus, the conformational energy landscape of CorA gating would consist of two deep energy minima representing the stable closed (symmetric) and putatively open (asymmetric) conformations connected by a wide plateau in which a variety of open conformations are adopted through permanent molecular fluctuations. The compelling utility of HS-AFM for the study of macromolecular dynamics at high spatio-temporal resolution and in native-like conditions is demonstrated, elucidating a process so far inaccessible to other structural or biophysical techniques. The present data set and our proposed mechanistic interpretation of the conformational dynamics of Mg2+-dependent CorA gating sets the stage for an unprecedented understanding of CorA as a ‘reverse’ polarity ligand gated ion channel with an unique gating mechanism.

Materials and methods

Protein purification

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CorA from T. maritima was expressed and purified as previously described (Dalmas et al., 2010). Briefly, the CorA-Pet15b vector was used to transform, then express CorA in E. coli BL21 DE3. After cell harvesting and disruption, membranes were collected by ultracentrifugation and gently solubilized. The sample was then cleared by ultracentrifugation and purified using cobalt high affinity chromatography column (Clontech Laboratories). The concentrated protein sample (AMICON 100 kDa cutoff membrane filters, EMD Millipore) was homogenized by gel filtration (Superdex 200 10/300 GL column, GE Healthcare Bio Sciences) and equilibrated in 50 mM HEPES, pH 7.3, 200 mM NaCl, 20 mM MgCl2, and 1 mM DDM).

Protein reconstitution

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For CorA reconstitution into liposomes, the protein was gently mixed with freshly prepared solubilized POPC-POPG (3:1) or DOPC-DOPE-DOPS (4:5:1) lipids (Avanti Polar Lipids) at low lipid to protein ratios (LPR) between 0.2–0.4 (w:w) at a total protein concentration of 1 mg/mL. After 4 hr equilibration, detergent was removed by addition of biobeads overnight.

Sample preparation for HS-AFM

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A 1.5 mm diameter muscovite mica sheet was glued on a HS-AFM glass rod sample support and mounted on a HS-AFM scanner. Reconstituted CorA membranes were adsorbed on freshly cleaved mica for ~5 min. Subsequently, the sample was rinsed with imaging buffer (50 mM MES, pH6.0, 200 mM NaCl) containing 10 mM Mg2+.

HS-AFM

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All experiments were performed using HS-AFM (Ando et al., 2001) (SS-NEX, Research Institute of Biomolecule Metrology Co.) operated in amplitude modulation mode, using ultra-short cantilevers (8 µm) with a nominal spring constant of ~0.15 N/m and a resonance frequency of ~600 kHz in liquid (USC, NanoWorld). Videos of CorA membranes were recorded with imaging rates of ~1–2 frames s−1 and at a resolution of 0.5 nm pixel−1. The energy input by the AFM tip (estimated to ~1.5 kBT, considering a 90% imaging amplitude of a 1 nm free amplitude)(Miyagi et al., 2016) was minimized by continuously adapting the drive and setpoint amplitude and optimizing the feedback parameters.

Structural titration experiments

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Monitoring the transition of CorA from Mg2+-saturated to low/no Mg2+-conditions was achieved by depleting Mg2+ by either adding EDTA into the measuring solution or alternatively by buffer solution exchange to Mg2+-free buffer using an integrated constant-pressure and constant-flow pump system (Miyagi et al., 2016). Experiments were performed with CorA membranes prepared from three different purifications and ~10 different reconstitutions. In total, about 50 transition experiments on different membranes patches were performed on different days (over ~30 days) using two different, but similar HS-AFM systems, and ~10 USC cantilevers. All recordings showed a clear structural transition from stable 5-fold symmetrical proteins to very dynamic molecules with increased height. About 20% of the recorded CorA transition movies were considered for further analysis.

Data analysis

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HS-AFM images were first-order flattened and contrast adjusted using laboratory-made routines in Igor Pro software (WaveMetrics). Videos were then drift corrected with respect to the membrane patch, or aligned on individual CorA molecules using an in-house developed analysis software plug-in for ImageJ (Fechner et al., 2009; Husain et al., 2012). Dimensions of the CorA were calculated by height histogram analysis (n = 20,500 height values) and cross section analysis (n = 25) for each condition. Estimation of dwell-times was based on CorA protrusion height: ΔHeight/time traces were generated by subtracting the minimum pixel value from the maximum pixel value in the 5 × 5 nm center area of the molecule. For the molecular height transition detection the ΔHeight traces were analyzed by a Step Transition and State Identification (STaSI) method in a MatLAB (MathWorks) routine (Heath and Scheuring, 2018; Shuang et al., 2014). StaSI indicated a minimum description length (MDL) for fitting the data with two ΔHeight states (where the increased height states comprises all conformational states with elevated topography). For in depth analysis, 25 molecules of two individual experiments were tracked over 900 and 1600 frames at different Mg2+ conditions, resulting in ~30,500 analyzed frames. Molecules in different height states and the corresponding dwell-times were binned either over 20 events along the time axis. Note, the transition analysis only discerned between states where subunits fluctuate between different height levels and did not classify between sub-states that exposed equivalent height levels to the putative closed (symmetric) state or the putatively open, activated (asymmetric and elevated) state. Such, the height/time traces did also not discriminate which of the open states was assumed. Notably however, our experiments showed that structures exposing increased protrusion heights are likely representatives of Mg2+-depleted molecules and thus detection of elevated height is a valuable fingerprint for the state transition. For conformational transition analysis membrane patches of ~20 CorA channels were imaged at Mg2+ concentrations of 10 mM, 3 mM, 0 mM, and after re-addition of 25 mM. Extracted CorA molecules from high-resolution movie sections (1,000–2000 molecular representations extracted from about 20 molecules for each Mg2+ condition) were manually assigned to different structural states of (C) symmetric flower-shaped, (O-1) asymmetric elevated, (O-2) dome-shaped, elevated and (O-+) all others. The significance of the occurrence of each conformation under the four tested Mg2+ concentrations was tested using a two-tailed students test.

References

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    The role of magnesium in clinical biochemistry: an overview
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    New Perspectives in Magnesium Research: Nutrition and Health
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    CorA-Mrs2-Alr1 Superfamily of Mg2+ Channel Proteins, New Perspectives in Magnesium Research: Nutrition and Health, Springer.
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    Magnesium metabolism and its disorders
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Decision letter

  1. Baron Chanda
    Reviewing Editor; University of Wisconsin-Madison, United States
  2. Richard W Aldrich
    Senior Editor; The University of Texas at Austin, United States
  3. Baron Chanda
    Reviewer; University of Wisconsin-Madison, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Mg2+bala is a critical element that is involved in various physiological processes. CorA is the bacterial ortholog of eukaryotic genes encoding a mitochondrial Mg2+ ion channel found in all eukaryotes. CorA function is negatively regulated by Mg2+. These channels are open when the binding site is unoccupied but they close in the presence of high Mg2+. Previous structural studies primarily using EPR and cryo-EM suggest that the channel undergoes a closed to open conformational switch in absence of Mg2+, which results in loss of five symmetry. Cryo-EM studies show that the open channel exists in at least two conformations which could either represent snapshots of a fluctuating channel or intermediates in the gating pathway. Recent crystallographic studies, however, suggest that the unbound channels may not undergo as much of a conformational change as suggested by the cryo-EM and EPR studies.

In this study, the authors have used high-speed AFM to monitor the time-dependent conformational changes as Mg2+ is removed from the channel. HS-AFM technique allows them to track conformational changes in a single CorA channel over time and correlate these changes with putative gating transitions. Functional experiments show that the gating process of the channel is relatively slow – currents decay over 15-20 minutes in electrophysiology experiments. Thus, the conformational changes are expected to be within the bandwidth of these measurements (500 ms per frame). Mg2+ depletion experiments show that the stable starting structure with five-fold symmetry becomes dynamic with time and ultimately settles to one or more asymmetric structures. One of the most interesting aspects of this study is that unlike most ion channels, the open channel conformation is both asymmetric and dynamic. Overall this is a technically work which advances the field both in terms of providing new information about the gating mechanism of CorA and demonstrating the power of HS-AFM to monitor single molecule structural transitions.

Decision letter after peer review:

Thank you for submitting your article "Real time dynamics of gating-related conformational changes in CorA" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Baron Chanda as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Essential revisions:

1) All of the reviewers are concerned about the two-state model assumption for data analysis. The authors themselves state that there are two open states and one closed state. The model fit shown in Figure 3A is clearly inadequate. The open conformation is quite heterogenous, correct? The kymograph shown really looks like 3 states to me (the third being at a lower height, near a Δ Height of 0). And if the Mg2+ bound state is the baseline, lowest-height state, why doesn't the "down" state have a Δ-Height of zero? The choice of 2 states affects the dwell time analysis (everything else in Figure 3), and without a better rationale, I don't see why this choice is valid. There are other possible reasons for poor fit. For instance, some of the conformational transitions in CorA is much faster than the sampling rate and this could result in aliasing. This is difficult to rule out but should be discussed.

Also the height histogram on the right side shows a very disperse distribution, only two not very distinct local maxima that were not taken as level thresholds (so why 2-state?). As this data depiction is crucial for all the analysis done in the rest of Figure 3 it needs to be clearly and convincingly communicated.

STaSI is a model-independent idealization algorithm but it tends to overfit data. Have the authors tried any of the other HHM based algorithms? Given that they are assuming a two-state model, it would make more sense to use HMM.

2) The reviewers are convinced that the CorA molecule becomes highly dynamic in the absence of Mg2+ and find it very interesting that this conformation is conducting. However, we all agree that the assignment of these highly dynamic structures to discrete states is restrictive and perhaps also subjective. It is not clear how the conformational states (closed, open I, open II) taken from EM work were assigned to the conformations recorded with high speed AFM. Was it done manually? What were the objective criteria? How many structures were taken? Figure 5B only gives percentages. Again, this is crucial for all the data analysis done in Figure 5. As the authors have stated, the classification of discrete state in this highly flexible molecule is a challenge. In Figure 5A, it is not clear whether the images shown below are idealized (expected) images or averaged images for each of the states. It should be stated clearly in the legend. The actual averaged image should be shown as an inset. In addition, in some instances, the state assignments are based on changes in the shape rather than height. If both heights and changes in shape are taken into consideration, it appears that the number of possible states will be much more and trying to bin these highly dynamic structures into a few discrete states becomes less meaningful.

3) My other main critique is that it is very difficult to understand how changes in Mg2+ concentration were achieved for each experiment, and why the experiments were designed the way that they were. It seems like two different methods were used to change Mg2+Mg2+ concentrations: either pipetting in EDTA, or slow perfusion with an EDTA-containing solution over 10's of minutes (Figure 2—figure supplement 2). (Subsection “Mg2+-depletion induces large conformational changes 1 of the intracellular face” paragraph two: it's unclear whether perfusion is performed with EDTA solution or not. This could be clarified in the main text so that the reader doesn't have to go to the supplemental figure legend).

I assume that adding EDTA nearly instantaneously removes bulk Mg2+, so that any slow kinetic component is due to channel kinetics. Whereas with perfusion, changes in Mg2+ concentration and the channel's conformational journey from "phase 1" to "phase 3" both occur on over a minutes-long time scale. Because of the slow kinetic component to channel dynamics, it's really important for the logic of the paper to be clear about which method of Mg2+ depletion was used in the data in Figures 2 through 5.

Do I understand correctly that many (all?) of the experiments in Figure 3 involved a ramp from high Mg2+ to low Mg2+ over about 14 minutes with another 12 minutes in Mg2+ free conditions (as shown in Figure 2—figure supplement 2)? In Figure 3, the sharp delineation of the green, blue, and red boxes seem to indicate Mg2+ concentration steps, and the legend refers to images collected at saturating, 2, and 0 mM Mg2+. This is confusing. The text goes back and forth between describing imaging at 2 mM (subsection “Mg2+-depletion induces large conformational changes 1 of the intracellular face” paragraph one and ~2 mM). According to my reading, Figure 5 then uses 3 discrete Mg2+ concentrations, but the use of the color-fade arrows seems to indicate a ramp.

I'm not clear what advantage ramping the Mg2+ concentrations provided. Given the amount of functional data already available for this channel, including a good idea of the Kd for Mg2+, I might have gone straight to imaging at discrete Mg2+ concentrations. Could the rationale be explained?

4) Abstract: "finally equilibrates to an asymmetric structure." Is the final state a single asymmetric structure or it is at least two conformations. Based on the state identification matrix in Figure 5, it would seem that at 0 Mg, there are more than one asymmetric structures, unlike the Mg bound state.

5) Abstract: – "putative open state adopts multiple conformations through hinge-bending motions". This sentence contradicts the previous sentence and it is not clear whether the data provided in this study show hinge-bending motion. The conformational changes are compatible with hinge-bending but there is no direct evidence here.

https://doi.org/10.7554/eLife.47322.sa1

Author response

Essential revisions:

1) All of the reviewers are concerned about the two-state model assumption for data analysis. The authors themselves state that there are two open states and one closed state. The model fit shown in Figure 3A is clearly inadequate. The open conformation is quite heterogenous, correct? The kymograph shown really looks like 3 states to me (the third being at a lower height, near a ΔHeight of 0). And if the Mg2+ bound state is the baseline, lowest-height state, why doesn't the "down" state have a Δ-Height of zero? The choice of 2 states affects the dwell time analysis (everything else in Figure 3), and without a better rationale, I don't see why this choice is valid.

CorA actually adopts many more than 2 structural states (indeed, that is the major point of this and previous manuscripts). As such, the analysis as presented in 3A is not based on explicit structural states nor determines dwell times directly associable with specific structural states. Let us first go over the analysis carried out in Figure 3 in some detail: Each molecule is analyzed as described in 3A and then the behaviors of all molecules are pooled in 3B and 3C.

Originally, we made a significant effort to track the movement of the individual subunits and make state assignments by transforming the CorA ring profiles into height cross-sections. Theses profiles were associated to the states described by cryo-EM. However, given the large variability within the molecules (lateral and rotational movements), combined to the temporal resolution limits of the experiment (and the bias from the polar transformation), it turned out to be impossible to determine states and/or individual subunit movements in any significant way.

Thus, as a subterfuge to define and assign a given structural state to each molecule at any instant, we simply tracked the height increase and fluctuations of CorA during Mg2+-depletion experiments. In light of the nature of the structural changes between the closed (Mg2+-bound) and many open (Mg2+-free) conformations, we are confident that height is a reliable measure to track the main closed-open transition. In other words, unable to define several states that could be integrated into a gating model as the reviewer suggests, we use a 2-state model with no other ambition than assigning OFF vs ON, where all of the poorly defined “open” state conformations are “pooled” as the ON state. This is clearly not ideal, but we argue that given the fact that the open state is unlikely to be defined in the classical, “single conformation” sense (as is one of the main conclusions of this work), this simple model is just intended to assess the kinetics of entry and exit of the closed state i.e. the symmetric state vs all other states.

How was this assessed: To determine CorA’s height changes we only used the center area of each molecule (5x5nm) and calculated the relative ΔHeight (the maximum pixel value minus the minimum pixel value). The reason that absolute height was not reported is mostly due to the larger variability in absolute height values under a wide number of experimental conditions. The region of interest of this measurement was defined in the center area of each molecule. In cases of limited resolution, the pore might simply not be observed in an otherwise low CorA molecule (ΔHeight <1nm). Hence, when considering straightforward changes in height, CorA formally switches between only 2 states, yet due to resolution limitations, the ΔHeight of the lower state is somewhat widened.

The major conformational difference between the closed and the multiple open states, leads to an increased height of the center or the rim by ~2nm ΔHeight in the open state(s) (e.g. one subunit ‘stands up’ in one of the bean-shaped asymmetric conformations). When all subunits are low but there is a central pore (e.g. closed state, 5-fold symmetric molecule) the observed height difference between pore and rim is ~1nm, and poorly resolved closed state molecules have a ΔHeight <1nm. In brief, failing to make significant structural state assignments, we used the best AFM criterion (i.e. height) to assign the closed state versus all other (putatively activated) states as a function of the progression of Mg2+-depletion. We thank the reviewers for pointing to these uncertainties and have changed the text accordingly as highlighted in the manuscript.

There are other possible reasons for poor fit. For instance, some of the conformational transitions in CorA is much faster than the sampling rate and this could result in aliasing. This is difficult to rule out but should be discussed.

Also the height histogram on the right side shows a very disperse distribution, only two not very distinct local maxima that were not taken as level thresholds (so why 2-state?). As this data depiction is crucial for all the analysis done in the rest of Figure 3 it needs to be clearly and convincingly communicated.

The reviewer is correct. The fluctuations of the CorA subunits are indeed faster than our sampling rate of about 2 frames s-1. In Author response image 1, we show a video section of 2 CorA molecules with a doubled scan rate of 250ms per frame and detect structural differences in single frames (see 1.75s compared to neighboring images 1.5s and 2s). Certainly, the structural fluctuations are much faster than this, and likely the reason why our classification efforts failed. Given the bandwidth limitation of image acquisition and the dynamics of the inter-state transitions in CorA, we have concluded that it would be difficult to capture individual states (other than the Mg2+-bound state and Mg2+-free states) at the current speed of image acquisition. In consequence, the only reasonable measure is whether the molecule is in its Mg2+-bound symmetric state (that can be well resolved) or an overlap of Mg2+-depleted dynamic molecules. We discuss the aspect of scan speed, subunit dynamics and the ‘merging of all activated states into one state as defined by height’ in our revised manuscript version. As detailed above and in the response to question 1a), the HS-AFM analysis essentially reduces the data to a 2-state model (inactivated or activated).

Author response image 1
HS-AFM image sequence of depicting CorA every 250ms.

STaSI is a model-independent idealization algorithm but it tends to overfit data. Have the authors tried any of the other HHM based algorithms? Given that they are assuming a two-state model, it would make more sense to use HMM.

In this point we disagree with the reviewer. We performed a thorough analysis how well STaSI performs, down to low signal to noise ratios (SNR=0.5), and found that STaSI makes the correct conservative 2-state assignment with ~95% accuracy. We compared STaSI with vbFRET (another Bayesian based approach for state assignment and state transition fitting) and StaSI performed better in our hands. We like that STaSI uses a minimum description length (MDL) and proposes the number of states in a fully unbiased way, resulting in idealized traces. How many structural states are hidden behind the assigned height-states is of course another question that would need time analysis of these height-states with large sample statistics. This is well beyond the scope of the present work.

2) The reviewers are convinced that the CorA molecule becomes highly dynamic in the absence of Mg2+ and find it very interesting that this conformation is conducting. However, we all agree that the assignment of these highly dynamic structures to discrete states is restrictive and perhaps also subjective. It is not clear how the conformational states (closed, open I, open II) taken from EM work were assigned to the conformations recorded with high speed AFM. Was it done manually? What were the objective criteria?

This is one of the key points we are trying to make. Here, we elaborate in more detail:

1) We agree with the reviewer that there is difficulty to assign snapshots of the highly dynamic molecule to the states observed by cryo-EM. In its dynamic (Mg2+-free) condition, CorA channels adopt a large variation of conformations and any attempt to automatically assign particular structures, was unsuccessful by lack of resolution, bandwidth and/or the fact that neighboring molecules touched the molecules under scrutiny (we also tried the EM-centered program Relion for particle classification).

2) We also argue that the cryo-EM structures (Open-I and Open-II) must represent conformations that sit in some sort of energy well (likely very shallow) otherwise, cryo-EM would not have been able to average any kind of structure (even at low resolution). However, the fact that cryo-EM was able to discriminate these two conformations by no means implies that there might not be many more. In fact, we are convinced this is the case and one important reason why it is so hard to make structural assignments under a regime of relatively low bandwidth.

3) Given 2), it is not unreasonable to argue that our data are somewhat equivalent to the cryo-EM open state low resolution structures.

4) How have we assigned molecules to individual classes? Manually. We used the following criteria (underlined) that correspond to features observable in the cryo-EM structures: “The channel adopts several structures that we classified into 4 categories: First, the fully-liganded channel corresponding to the 5-fold symmetric closed conformation. Second, a structure of reduced diameter with three asymmetrically distributed protrusions of increased height, assigned to the cryo-EM open-I state. Third, a round- or star-shaped conformation with an elevated center. This HS-AFM topography can be easily identified as it only displays small structural variations and resembles the cryo-EM open-II state.”

5) However, we came to the same conclusion as the reviewers that an explicit assignment to the cryo-EM open states was too restrictive. Hence, we introduced another class of “not assigned” molecules: “Finally, given that our tentative assignment to open-I and open-II states only covers a fraction of all molecular observations, a fourth category must necessarily include the remaining range of structural variations, which we named Open-+ states…”. Indeed, we see that the majority of the molecules are in this 4th class.

6) Finally, a key conclusion – see Figure 5C – is that the channel goes from a stabilized Mg2+-bound to a more-or-less stabilized Mg2+-free state over a large range of dynamic conformations. Thus, we do not think that we were restrictive trying to ‘push’ our findings towards assignment with the cryo-EM structures.

How many structures were taken? Figure 5B only gives percentages. Again, this is crucial for all the data analysis done in Figure 5. As the authors have stated, the classification of discrete state in this highly flexible molecule is a challenge.

For each Mg2+-condition between ~1,000-2,000 molecular representations were considered for classification into the 4 subgroups and then normalized to the total number of frames. In particular 943 HS-AFM images of mostly stable closed CorAs were analyzed in saturating 10mM Mg2+-condition, 1,122 frames at 3mM Mg2+, 2,394 CorA snapshots for 0mM Mg2+, and 1,104 representations were classified for the recovery experiment after re-addition of 25mM Mg2+. These numbers are now mentioned in the Materials and methods section and the corresponding figure legend.

In Figure 5A, it is not clear whether the images shown below are idealized (expected) images or averaged images for each of the states. It should be stated clearly in the legend. The actual averaged image should be shown as an inset.

The CorA molecules shown in Figure 5A bottom and Figure 5B left and top are sketches depicting the main structural classes of CorA. To avoid confusion, we now clarify this it in the figure legend.

In addition, in some instances, the state assignments are based on changes in the shape rather than height. If both heights and changes in shape are taken into consideration, it appears that the number of possible states will be much more and trying to bin these highly dynamic structures into a few discrete states becomes less meaningful.

We agree with the reviewers and believe that there are many more structures in the dynamic open state of CorA, considering the observed structural variations in the presented HS-AFM videos (see our responses to comments above).

This is why we state: “Active subunits change and the entire molecule displays high structural variability, likely adopting many more conformations than the 3 states so far identified by cryo-EM. Indeed, while we can assign the fully 5-fold symmetric state with certainty, our assignment of molecules to state open-I and open-II must be considered as putative. Perhaps more importantly, at intermediate Mg2+-concentrations ~50% of the molecules could not be assigned and were pooled in a class of undefined quaternary structures (open +).”, and, “We expect that given a larger number of particles to process (in cryo-EM) or improvements in the spatial and/or temporal resolution of HS-AFM, additional states are likely to emerge.”

Due to the current limitations in high-resolution single particle HS-AFM data in this study (~5,600 molecular representations in total) more work is required to further optimize sample preparation, which will allow more observations, and HS-AFM technology to distinguish between possible different sub-structures in the open state. However, our experiments observed a clear Mg2+- and time- depended sequence of conformational events for CorA: First, a transition into highly mobile, elongated structures occurs, followed by a transition into features resembling to those of the Mg2+-free cryo-EM structures, these molecules have a clearly increased height. As detailed above, the height changes (around Figure 3) are used to automatically detect activation of the molecules only, while the specific shapes are assigned later (see response to 2a) to describe the transition in more detail. We have added additional text in the Discussion section to point out these limitations.

3) My other main critique is that it is very difficult to understand how changes in Mg2+ concentration were achieved for each experiment, and why the experiments were designed the way that they were. It seems like two different methods were used to change Mg2+ concentrations: either pipetting in EDTA, or slow perfusion with a EDTA-containing solution over 10's of minutes (Figure 2—figure supplement 32). (Subsection “Mg2+-depletion induces large conformational changes 1 of the intracellular face” paragraph two: it's unclear whether perfusion is performed with EDTA solution or not. This could be clarified in the main text so that the reader doesn't have to go to the supplemental figure legend).

I assume that adding EDTA nearly instantaneously removes bulk Mg2+, so that any slow kinetic component is due to channel kinetics. Whereas with perfusion, changes in Mg2+ concentration and the channel's conformational journey from "phase 1" to "phase 3" both occur on over a minutes-long time scale. Because of the slow kinetic component to channel dynamics, it's really important for the logic of the paper to be clear about which method of Mg2+ depletion was used in the data in Figures 2 through 5.

Yes, the reviewer understood the two concentration change protocols and we have fully and explicitly described them now in the main text.

Do I understand correctly that many (all?) of the experiments in Figure 3 involved a ramp from high Mg2+ to low Mg2+ over about 14 minutes with another 12 minutes in Mg2+ free conditions (as shown in Figure 2—figure supplement 2)? In Figure 3, the sharp delineation of the green, blue, and red boxes seem to indicate Mg2+ concentration steps, and the legend refers to images collected at saturating, 2, and 0 mM Mg2+. This is confusing. The text goes back and forth between describing imaging at 2 mM (subsection “Mg2+-depletion induces large conformational changes 1 of the intracellular face” paragraph one and ~2 mM). According to my reading, Figure 5 then uses 3 discrete Mg++ concentrations, but the use of the color-fade arrows seems to indicate a ramp.

We thank the reviewer for pointing out the confusion of analyzed Mg2+-concentrations and the depletion methods used. We only used the fast and manual EDTA titration procedure to deplete Mg2+ in discrete steps. In all but 1 experiment (Figure 3A and 3B, bottom panel) shown in Figure 3, Mg2+ was fully depleted in a single titration step (i.e. addition of ~10mM EDTA). For further analysis (Figure 3C) only molecules monitored at 0mM Mg2+ were analyzed. For the in-depth analysis of the individual structural classes the distinct Mg2+ concentrations of 10mM, 3mM, 0mM and re-addition of Mg2+ to 25mM were achieved pipetting. We clarify the distinct Mg2+ concentrations in the text and all figure legends.

I'm not clear what advantage ramping the Mg2+ concentrations provided. Given the amount of functional data already available for this channel, including a good idea of the Kd for Mg2+, I might have gone straight to imaging at discrete Mg2+ concentrations. Could the rationale be explained?

We performed the ramping experiments using a high-precision buffer exchange system first, to test for the affinity of Mg2+ in our particular experimental set-up (reconstituted and densely packed CorA channels adsorbed on a sample surface), independent of previously determined apparent affinities. Concerning the Kd, the ramp experiments merely corroborate the findings from functional data. Anyway, ramps help us make sure that the addition of EDTA does not physically disturb and influence the measurements. We agree with the reviewer that the transition analysis itself should be performed at distinct Mg2+ concentrations, particularly when considering the long transition times. Accordingly, all CorA transition experiments used for in-depth analysis and presented in the main manuscript (Figure 2-5 and corresponding videos) were recorded and analyzed at distinct Mg2+ concentrations. We clarify this by rephrasing the main text in the revised manuscript and as discussed above (point 3).

4) Abstract: "finally equilibrates to an asymmetric structure." Is the final state a single asymmetric structure or it is at least two conformations. Based on the state identification matrix in Figure 5, it would seem that at 0 Mg, there are more than one asymmetric structures, unlike the Mg bound state.

We realize that the expression “equilibrates to an asymmetric structure” might be misleading. Our HS-AFM experiments revealed multiple, dynamically fluctuating asymmetric structures, that show an elevated Mg2+-sensor domain after spending ~10 minutes in Mg2+-free conditions. Due to the flexibility and the fast dynamics of the large and elevated substructures of CorA we are resolution limited, and thus can only make tentative assignments to the two found cryo-EM structures. Our high resolution CorA analysis at the onset of complete Mg2+-depletion (Figure 4 and 5) is highly suggestive of more than two open states of elevated heights. To this end, we changed the Abstract to “… that equilibrates to asymmetric structures of increased membrane protrusion height level.”

5) Abstract: "putative open state adopts multiple conformations through hinge-bending motions". This sentence contradicts the previous sentence and it is not clear whether the data provided in this study show hinge-bending motion. The conformational changes are compatible with hinge-bending but there is no direct evidence here.

Good catch. As mentioned in point 4, we corrected the misleading phrasing of a single asymmetric state (point 4 above) and have also clarified in the revised version of the manuscript that the observed CorA subunit movements in the putative open state are consistent with the hinge-bending movement detected by cryo-EM. As discussed in the text and Figure 5—figure supplement 1, TM1 is the only connection between the TM region and the cytoplasmic domain. Logically, the large rearrangements in the Mg2+ sensor are likely translated to the channel pore via hinge bending in TM1.

https://doi.org/10.7554/eLife.47322.sa2

Article and author information

Author details

  1. Martina Rangl

    1. Department of Anesthesiology, Weill Cornell Medical College, New York, United States
    2. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Project administration
    Competing interests
    No competing interests declared
  2. Nicolaus Schmandt

    Department of Biochemistry and Molecular Biophysics, The University of Chicago, Chicago, United States
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  3. Eduardo Perozo

    Department of Biochemistry and Molecular Biophysics, The University of Chicago, Chicago, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition
    For correspondence
    eperozo@uchicago.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7132-2793
  4. Simon Scheuring

    1. Department of Anesthesiology, Weill Cornell Medical College, New York, United States
    2. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration
    For correspondence
    sis2019@med.cornell.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3534-069X

Funding

National Institutes of Health (R01GM120561)

  • Eduardo Perozo

National Institutes of Health (DP1AT010874)

  • Simon Scheuring

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank George Heath for providing the Matlab scripts for the STaSI trace analysis, and Yi-Chih Lin for valuable comments on data analysis.

Senior Editor

  1. Richard W Aldrich, The University of Texas at Austin, United States

Reviewing Editor

  1. Baron Chanda, University of Wisconsin-Madison, United States

Reviewer

  1. Baron Chanda, University of Wisconsin-Madison, United States

Publication history

  1. Received: April 1, 2019
  2. Accepted: November 26, 2019
  3. Accepted Manuscript published: November 27, 2019 (version 1)
  4. Version of Record published: December 23, 2019 (version 2)

Copyright

© 2019, Rangl et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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