Structure of glutaraldehyde cross-linked ryanodine receptor

Abstract

The ryanodine receptor (RyR) family of calcium release channels plays a vital role in excitation-contraction coupling (ECC). Along with the dihydropyridine receptor (DHPR), calsequestrin, and several other smaller regulatory and adaptor proteins, RyRs form a large dynamic complex referred to as ECC machinery. Here we describe a simple cross-linking procedure that can be used to stabilize fragile components of the ECC machinery, for the purpose of structural elucidation by single particle cryo-electron microscopy (cryo-EM). As a model system, the complex of the FK506-binding protein (FKBP12) and RyR1 was used to test the cross-linking protocol. Glutaraldehyde fixation led to complete cross-linking of receptor-bound FKBP12 to RyR1, and also to extensive cross-linking of the four subunits comprising RyR to one another without compromising the RyR1 ultrastructure. FKBP12 cross-linked with RyR1 was visualized in 2D averages by single particle cryo-EM. Comparison of control RyR1 and cross-linked RyR1 3D reconstructions revealed minor conformational changes at the transmembrane assembly and at the cytoplasmic region. Intersubunit cross-linking enhanced [(3)H]ryanodine binding to RyR1. Based on our findings we propose that intersubunit cross-linking of RyR1 by glutaraldehyde induced RyR1 to adopt an open like conformation.

Fig 1

SDS-PAGE of glutaraldehyde cross-linked RyR1 and RyR1:His-FKBP12. (A) Time course of His-FKBP12 cross-linking to RyR1. Reaction solutions were prepared as described in Experimental Procedures and cross-linked with 0.4 % glutaraldehyde on ice for the indicated times. Cross-linking was terminated by addition of Laemmli buffer, and the gel was stained with a His-tag specific dye to reveal the presence of the His-FKBP12. His-tagged molecular weight protein standards indicated on gel. (B) Data were pooled from 3 independent cross-linking experiments. Error bars represent standard deviation, P-value of Students unpaired T-test calculated relative to zero time point * P < 0.05, ** P< 0.01. C, SDS-PAGE of RyR1 alone cross-linked for 20 min as in (A) and stained with Coomassie Blue. Subunits (565 kDa) of RyR1 migrate as a single band just above the 210 kDa myosin prestained molecular standard, whereas as the cross-linked RyR1 barely enters the top of the gel (as indicated by arrow).

Fig 2

Cryo-electron microscopy of RyR1 and Glutaraldehyde Cross-Linked RyR1. Typical image of glutaraldehyde cross-linked RyR1 and control RyR1. Circles placed over selected RyR1 particles. Scale bar 500 Å

Fig 3

2D Cryo-EM Analysis of Glutaraldehyde Cross-Linked RyR1 and His-FKBP12:RyR1 complex. (A and B) Two-dimensional averages of control RyR1 and glutaraldehyde cross-linked RyR1, respectively (N=369 and N=404). Fourfold symmetry was imposed on both reconstructions, averages were low- passed filtered to 29 Å resolution. (C) Difference map made by subtracting glutaraldehyde cross-linked RyR1 from control RyR1 2D average. (D) The T-test corresponding to difference map displayed at 99.99 % confidence. (E and F) Two-dimensional averages of cross-linked RyR1:His-FKBP12 complex, respectively, and cross-linked RyR1 (N=419 and N=400). Fourfold symmetry was imposed and averages were low-passed filtered to 31 Å resolution. (G) Difference map obtained by subtracting cross-linked His-FKBP12:RyR1 2D average from cross-linked RyR1 2D average. (H) T-test corresponding to difference map displayed at 99.99 % confidence. The location of His-FKBP12 revealed by 2D analysis is identical to that of FKBP12 described previously (Wagenknecht et al., 1996). Arrow points to His-FKBP12. Scale bars 100 Å.

Fig 4

3D Reconstruction of RyR1 and Glutaraldehyde Cross-Linked RyR1. (A and B) Single-particle cryo-EM 3D reconstructions of control RyR1 in red and glutaraldehyde cross-linked RyR1 in yellow, shown in top, bottom and side views. Reconstructions made by combining 4,048 and 4,567 images, respectively. Both volumes were low-pass filtered to 32 Å for comparison purposes. (C) Difference maps derived by comparing control RyR1 and glutaraldehyde cross-linked RyR1 reconstructions. The yellow differences correspond to major regions of excess mass density present in cross-linked RyR1 relative to control, and red differences correspond to excess mass present in control relative to cross-linked RyR1. The difference maps are shown superimposed on the control RyR1 volume in grey. RyR1 domains labeled according to (Radermacher et al., 1994). Density connecting domains 7 and 11 is marked by arrow and the plug in transmembrane region is marked by “p” Scale Bars 100 Å.