Correspondence to: Montserrat Samsó, Wadsworth Center, Empire State Plaza, Albany, NY 12201-0509., samso{at}wadsworth.org (E-mail), (518) 474-6516 (phone), (518) 474-7992 (fax)
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Abstract |
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Cryo-electron microscopy and three-dimensional, single-particle image analysis have been used to reveal the specific binding site of imperatoxin A (IpTxa) on the architecture of the calcium release channel/ryanodine receptor from skeletal muscle (RyR1). IpTxa is a peptide toxin that binds with high affinity to RyR1 and affects its functioning. The toxin was derivatized with biotin to enhance its detection with streptavidin. IpTxa binds to the cytoplasmic moiety of RyR1 between the clamp and handle domains, 11 nm away from the transmembrane pore. The proposed mimicry by IpTxa of the dihydropyridine receptor (DHPR) II-III loop, thought to be a main physiological excitation-contraction trigger, suggests that the IpTxa binding location is a potential excitation-contraction signal transduction site.
Key Words: ryanodine receptor, imperatoxin A, cryo-electron microscopy, three-dimensional reconstruction, excitation-contraction coupling
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Introduction |
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THE ryanodine receptor isoform 1 (RyR1)1 is a large multi-subunit transmembrane protein (four identical subunits of 565-kD and four 12-kD subunits) that functions as the calcium release channel in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle (recently reviewed by
The three-dimensional (3D) structure of RyR1 as determined by cryo-EM and image processing (
A peptide toxin, imperatoxin A (IpTxa; 33 amino acid residues), isolated from the venom of scorpion Pandinus imperator, interacts specifically with the skeletal (RyR1) and cardiac (RyR2) isoforms of the RyR. It reversibly enhances binding of ryanodine to the receptors (
Image analysis of single particles in frozen solution as observed in the electron microscope is a powerful method for the structural study of large membrane-bound proteins complexes, such as membrane receptors and channels, which are not easily crystallized (
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Materials and Methods |
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Biocytin was purchased from Bachem BioScience, Inc. All other biochemicals were purchased from Sigma Chemical Co. Skeletal RyRs were isolated from terminal cisternae vesicles from rabbit skeletal muscle as described in
Affinity Chromatography and Gel Electrophoresis
Purified RyR (10 µg) was diluted and incubated with biotinylated or nonbiotinylated IpTxa in 100 µl binding buffer to yield the following final concentrations: 20 mM MOPS-NaOH (pH 7.4), 200 mM NaCl, 0.3% (vol/vol) CHAPS, 0.1 mM CaCl2, 0.24 mM DTT, 1 mM NEM, 5 µg/ml leupeptin, and 2.6 µM IpTxa. Preequilibrated streptavidin (SA)-agarose (40 µl of a 50% slurry) was added. The mixture was shaken for 15 min at room temperature. The supernatant was recovered by centrifugation for 2 min at 1,000 g. The sedimented SA-agarose was resuspended in 100 µl washing buffer of the following composition: 20 mM MOPS-NaOH (pH 7.4), 200 mM NaCl, 0.3% (vol/vol) CHAPS, 0.1 mM CaCl2, and 5 µg/ml leupeptin. The mixture was recentrifuged and the supernatant saved. This step was repeated nine times. Subsequently, the RyR was eluted batchwise in three steps with 125 µl elution buffer that contained 0.1 M sucrose, 2% SDS, 62.5 mM Tris-HCl (pH 6.8), 2 mM EDTA, 50 mM DTT, and 0.01% (wt/vol) bromophenol blue. All the supernatants recovered (100 µl each) were also mixed with 25 µl fivefold concentrated elution buffer. Aliquots (40 µl) were applied onto discontinuous SDS-polyacrylamide gels (3.5% stacking and 5% resolving gel). The resolved proteins were visualized by Coomassie staining.
Cryo-EM and Image Processing
RyR1:IpTxa-B:SA complexes were prepared in 20 mM Tris-HCl, pH 7.4, 0.15 M KCl, 0.1 mM CaCl2, and incubated between 10 and 30 min at room temperature before cryo-grid preparation. A 20-fold molar excess of IpTxa and SA over RyR1 was used. Vitrified specimens were prepared on 300-mesh carbon-coated gold grids as described in
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Results |
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IpTxa-B Activity and Stability of IpTxa-B:SA Complex
IpTxa was synthesized with a biocytin group added to the NH2 terminus to facilitate its detection by cryo-EM using SA. Biotin-derivatized IpTxa (IpTxa-B) retained the ryanodine-binding enhancement property of the native toxin, although higher concentrations were needed to obtain the same half-maximal effect, and the plateau of maximal effect was 80% of that for native IpTxa (Figure 1 a). The affinity of IpTxa-B for RyR1 is within the range suitable for cryo-EM.
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Precipitation of RyR1 by SA-agarose in the presence of IpTxa-B confirms that a stable complex forms between IpTxa-B and RyR1 (Figure 1 b). The SA-agarose resin does not precipitate RyR1 when the mixture contains IpTxa instead of IpTxa-B (Figure 1 b).
Cryo-EM and IpTxa-B:SA Difference Map
RyR1 was incubated with a molar excess of IpTxa-B and SA, and prepared for cryo-EM in parallel with a control reaction consisting of RyR1 and SA. In the presence of IpTxa-B and SA the receptors distributed homogeneously on the grid, but some dimers and a few higher oligomers formed, probably due to the tetravalent binding potential of both RyR1 for IpTxa-B and SA for biotin (Figure 2 a). The control specimen (Figure 2 b) showed mostly individual channels and some small particles on the background, presumably corresponding to free SA (in the experimental sample, less free SA was observed because a significant fraction of the SA was presumably bound by the RyR1:IpTxa-B). Although these observations are indicative of the formation of RyR1:IpTxa-B:SA complexes, it is impossible to assert by direct visual examination of the raw micrographs whether particular RyR1s contain ligand, and if so, where it is located.
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Two-dimensional (2D) Analysis
2D image processing was performed on the frequently occurring square-shaped views of RyR1 as described in
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Analysis
3D reconstructions were computed for both control and experimental samples. Surface renderings of the reconstructed volumes filtered to their limiting resolution of 29 Å (Figure 4) show the characteristic square-prism cytoplasmic moiety containing 10 well-defined domains and the smaller transmembrane assembly that have been documented previously (
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The locations of the bound IpTxa-B:SA agree with the positions of the four significant differences seen in the 2D analysis (compare Figure 4 c, violet 3D masses, left panel, and Figure 3c and Figure d, which are in the same orientation). Whereas 2D averages are performed using square projections only, most of the input data for 3D reconstruction come from other views. Thus, agreement in the differences detected by the 2D and 3D analyses is a further test of the internal consistency of the results.
The attachment of IpTxa-B:SA (Figure 4 c, violet regions) to the RyR1 appears to occur near the base of the crevice (Figure 5 a), indicating that IpTxa-B, the link between RyR1 and SA, is probably located at this region of the difference map.
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The apparent size of the surface-rendered difference between the experimental and control reconstructions appears to be smaller than SA's dimensions. This discrepancy likely results from a loss of signal at the distal regions of the SA due to its mobility, and thus dilution of the signal through averaging. Similar effects have been seen in reconstructions using antibodies as ligands (e.g.,
Although IpTxa has been shown to induce subconductance states in the RyR1 (
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Discussion |
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Allosteric Activation by IpTxa
Binding of IpTxa to the RyR1 induces the appearance of subconductance states of long lifetime. Our finding that the IpTxa binding locations are far (<11 nm) from the center of the cytoplasmic side of the transmembrane region of the channel supports an allosteric mechanism of action of IpTxa as opposed to a mechanism involving direct positioning of the toxin within the ion conducting channel (both models have been discussed by
Mimicry by the II-III Loop of the 1 Subunit of the DHPR
Recently, convincing evidence has been reported in support of the hypothesis that IpTxa mimics the effects of RyR1-activating peptides derived from the DHPR (1 subunit of the DHPR, are crucial for the activating effects that the isolated II-III loop and various derived subfragments have on the RyR1 (
In this context, the 37amino acid sequence Arg1076Asp1112 from RyR1 that interacts with the DHPR II-III loop identified by
To correlate further our results with the work implicating IpTxa as a DHPR mimic, we examined how the density attributed to IpTxa-B:SA in our reconstruction (Figure 4 c, violet) fits into the known quaternary arrangement of DHPRs at the triad junction (
Regardless of the potential use of IpTxa as a tool to help elucidate E-C coupling, it is important to emphasize that IpTxa produces discrete functional effects on RyR1 that ultimately lead to calcium release in isolated SR vesicles (
In conclusion, we have found by cryo-EM and 3D reconstruction that IpTxa binds to RyR1 along the edges of the cytoplasmic assembly, in a crevice between the clamp and handle domains. We suggest that a subtle conformational change mediates pore gating and toxin binding, and we discuss the possibility that the toxin binding location represents one of the physiological activating sites of RyR1 during E-C coupling.
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Acknowledgements |
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The authors gratefully acknowledge the use of the Wadsworth Center Electron Microscopy and Research Computing core facilities. We thank the Peptide Synthesis, Mass Spectroscopy and Biochemistry core facilities for synthesis and analysis of IpTxa and IpTxa-B; Jon Berkowitz for the purification of RyR1; and Brenda Benacquista for help in preparation of Figure 2.
R. Trujillo was supported in part by the Ministry of Education and Culture of Spain. This work was supported by grants from the National Institutes of Health AR40615 (to T. Wagenknecht) and HL55438 (to H.H. Valdivia).
Submitted: May 14, 1999; Accepted: June 22, 1999.
1.used in this paper: 2D, two-dimensional; 3D, three-dimensional; DHPR, dihydropyridine receptor; E-C, excitation-contraction; IpTxa, imperatoxin A; IpTxa-B, IpTxa-biotin conjugate; RyR, ryanodine receptor; RyR1, skeletal muscle isoform of the ryanodine receptor; SA, streptavidin; SR, sarcoplasmic reticulum; T tubule, transverse tubule
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