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. 2009 Mar 27;387(2):320-34.
doi: 10.1016/j.jmb.2009.01.059. Epub 2009 Feb 4.

CLIC2-RyR1 interaction and structural characterization by cryo-electron microscopy

Xing Meng  1 , Guoliang Wang, Cedric Viero, Qiongling Wang, Wei Mi, Xiao-Dong Su, Terence Wagenknecht, Alan J Williams, Zheng Liu, Chang-Cheng Yin
Affiliations

CLIC2-RyR1 interaction and structural characterization by cryo-electron microscopy

Xing Meng et al. J Mol Biol. .

Abstract

Chloride intracellular channel 2 (CLIC2), a newly discovered small protein distantly related to the glutathione transferase (GST) structural family, is highly expressed in cardiac and skeletal muscle, although its physiological function in these tissues has not been established. In the present study, [3H]ryanodine binding, Ca2+ efflux from skeletal sarcoplasmic reticulum (SR) vesicles, single channel recording, and cryo-electron microscopy were employed to investigate whether CLIC2 can interact with skeletal ryanodine receptor (RyR1) and modulate its channel activity. We found that: (1) CLIC2 facilitated [3H]ryanodine binding to skeletal SR and purified RyR1, by increasing the binding affinity of ryanodine for its receptor without significantly changing the apparent maximal binding capacity; (2) CLIC2 reduced the maximal Ca2+ efflux rate from skeletal SR vesicles; (3) CLIC2 decreased the open probability of RyR1 channel, through increasing the mean closed time of the channel; (4) CLIC2 bound to a region between domains 5 and 6 in the clamp-shaped region of RyR1; (5) and in the same clamp region, domains 9 and 10 became separated after CLIC2 binding, indicating CLIC2 induced a conformational change of RyR1. These data suggest that CLIC2 can interact with RyR1 and modulate its channel activity. We propose that CLIC2 functions as an intrinsic stabilizer of the closed state of RyR channels.

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Figures

Figure 1
Figure 1. Effects of CLIC2 on [3H]-ryanodine binding to skeletal heavy SR and purified RyR1
(A) CLIC2 increases [3H]-ryanodine binding to skeletal heavy SR. Equilibrium binding experiments were performed in the absence or presence of CLIC2. The binding buffer contained 2 nM [3H]-ryanodine and 10 μM Ca2+. CLIC2 increased [3H]-ryanodine binding to skeletal heavy SR from 1.31±0.1 pmol/ml (buffer, n=6) to 1.57±0.03 pmol/ml (15 μM CLIC2, n=6), and to 1.7±0.01 pmol/ml (30 μM CLIC2, n=6). (B) CLIC2 increases [3H]-ryanodine binding to purified RyR1. Equilibrium [3H]-ryanodine binding experiments were carried out in binding buffer containing 2 nM [3H]-ryanodine and various concentrations of Ca2+, in the absence or presence of 15 μM CLIC2. [Ca2+] was maintained, in a range between 0.1 μM and 10 mM, by a combination of EGTA and CaCl2. Free Ca2+ concentrations were calculated using the computer program of Fabiato and Fabiato. Data points shown are the mean ± S.E.M. from three separate experiments. (C) and (D) Equilibrium saturation assay of [3H]-ryanodine binding to purified RyR1. Experiments were carried out in binding buffer containing 10 μM Ca2+, and various concentrations of [3H]-ryanodine (from 1 nM to 24 nM), in the absence or presence of 15 μM CLIC2, as described in the “Materials and methods”. Panel C shows the saturation curves for [3H]-ryanodine binding to purified RyR1. Inset are the best-fit values of Bmax and Kd. Panel D shows the Scatchard analysis of panel C. Data points shown are the mean ± S.E.M., from three separate experiments.
Figure 2
Figure 2. The effect of CLIC2 on binding kinetics of [3H]-ryanodine to RyR1
(A) Time course of association of [3H]-ryanodine to RyR1. The data shown are the average of six experiments. (B) Linear transformation of A. This graph depicts the linear transformation of time-dependent association, where RLe represents the amount of [3H]-ryanodine bound at equilibrium, and RL represents the amount bound at any given time. (C) Time course of dissociation of [3H]-ryanodine from RyR1. The data shown are the average of five experiments.
Figure 3
Figure 3. Effects of CLIC2 on Ca2+ efflux from skeletal SR vesicles
(A) Representative traces of extra vesicular [Ca2+] variations measured with Antipyrylazo III as a Ca2+ indicator. These traces depict the process of Ca2+ uptake by Ca2+-ATPase/pump after 4 applications of 7.5 μM CaCl2, Ca2+ efflux through RyR1 in presence of Thapsigargin, and total Ca2+ release from skeletal heavy SR vesicles at the end evoked by A23187. Experiments were divided into two groups, buffer (black) and 30 μM CLIC2 (gray), respectively. Arrows indicate the time points at which each reagent was added. (B) Histogram of the maximal Ca2+ efflux rate from skeletal heavy SR vesicles. The asterisk denotes that the values are significantly different between the two groups, as assessed by t-test (p<0.05).
Figure 4
Figure 4. Single channel activity of purified RyR1 in presence of CLIC2
Depicted are representative single channel recordings (current flow vs. time) under EMD 41000 stimulation, with or without the addition of 7 μM CLIC2 at 3 different holding potentials: (A) +20 mV, (B) +30 mV, and (C) +40 mV. Both substances were applied to the cytosolic (cis) side of the channels. Closing and opening levels are indicated by an arrow and the letter ‘C’ or ‘O’, respectively. The traces were taken from data filtered at 1 kHz.
Figure 5
Figure 5. CLIC2 modifies gating parameters of the skeletal RyR1 channel
The analysis of three parameters is summarized in the histograms: mean open probability (Po), mean open time (To), and mean close time (Tc). Comparisons of data that showed a significant difference from the value with EMD alone (black bars) have been marked with asterisks as follows: * p≤0.05, ** p≤0.02, *** p≤0.01. White bars represent parameters following the addition of CLIC2. The data were taken from 6 to 8 single channel experiments for +20 mV (A), 5 to 6 for +30 mV (B), and 6 to 11 for +40 mV (C).
Figure 6
Figure 6. Cryo-electron microscopy of RyR1+CLIC2 complexes
Portion of cryo-EM micrograph of RyR1+CLIC2 complexes, with the protein particles embedded in a thin layer of vitreous ice. The tetrameric structure of RyR1 is well preserved as indicated by the characteristic square appearance, which represents the images of the particles lying with their 4-fold symmetry axes oriented perpendicular to the carbon support film. Several individual particles are marked with white circles. Scale bar = 500Å.
Figure 7
Figure 7. Two-dimensional averages of RyR1 and the RyR1+CLIC2 complex
(A) 2D average of the RyR1+CLIC2 complex (n = 297 particle images) in ‘top’ view; (B) Top view of the 2D average of RyR1 control (n = 317 particle images); (C) Difference map obtained by subtracting B from A. The top view represents the projection of the channel as seen from the cytoplasmic side, as shown in the cartoon in (D). The largest differences shown in (C), corresponding to the four additional masses contributed by binding of CLIC2, are seen as bright white areas, one of which is circled. The corresponding location of the major difference in (C) has been also highlighted with green dots in (A), (B), and (D). Scale bar = 100Å.
Figure 8
Figure 8. Three-dimensional surface representations of RyR1 and the RyR1+CLIC2 complex, and three-dimensional difference map
The 3D reconstruction of RyR1 is shown in yellow (panel A) and the RyR1+CLIC2 complex (panel B) is shown in orange. In panel C, the difference map (RyR1+CLIC2 minus RyR1) shown in orange is superimposed on the 3D reconstruction of RyR1 alone (yellow), another minor negative difference (RyR1 minus RyR1+CLIC2) shown in blue-violet, is also superimposed. The 3D reconstructions are shown in three views: left, top view from the cytoplasmic surface, which in situ would face the transverse tubule; middle, side view; right, bottom view showing the surface that would face the SR lumen. The numerals 1-11 on the cytoplasmic assembly indicate the distinguishable domains, according to our earlier nomenclature. Scale bar = 100Å.
Figure 9
Figure 9. Docking of the X-ray crystal structure of CLIC2 into the cryo-EM density map of the RyR1+CLIC2 complex
The atomic coordinates of the CLIC2 (PDB code: 2PER) were manually fitted into our cryo-EM density map of RyR1+CLIC2 complex, using the program O. (A) A tilted view of the RyR1+ CLIC2 complex, with one CLIC2 molecule docked in the binding site of one subunit in the RyR1 homotetramer. (B) A zoomed view of the clamp region from (A), showing that CLIC2 binds to domains 5 and 6 of RyR1. Domain rendering of CLIC2 molecule: N-terminal domain (amino acid residues 11-94), red; joint loop (residues 95-106), yellow; C-terminal domain (residues 107-152 and 179-246), green; and foot loop, (residues 153-178), blue.
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