Structure of glutaraldehyde cross-linked ryanodine receptor

Area of Science:

• Biophysics
• Structural Biology
• Molecular Physiology

Background:

• The ryanodine receptor (RyR) is a calcium release channel crucial for muscle excitation-contraction coupling (ECC).
• The ECC machinery is a large, dynamic complex involving RyRs, dihydropyridine receptors (DHPRs), and other proteins, which is challenging to study structurally.
• Stabilizing these fragile components is essential for high-resolution structural elucidation.

Purpose of the Study:

• To develop and validate a simple cross-linking procedure to stabilize the ECC machinery for structural analysis.
• To assess the effect of cross-linking on the structure and function of the RyR1-FKBP12 complex using single particle cryo-electron microscopy (cryo-EM).

Main Methods:

• Utilized glutaraldehyde as a cross-linking agent for the FK506-binding protein (FKBP12) and RyR1 complex.
• Employed single particle cryo-electron microscopy (cryo-EM) for structural visualization and 3D reconstruction.
• Assessed functional changes by measuring [(3)H]ryanodine binding to RyR1.

Main Results:

• Glutaraldehyde effectively cross-linked FKBP12 to RyR1 and the RyR1 subunits to each other without altering RyR1 ultrastructure.
• Single particle cryo-EM visualized FKBP12 cross-linked with RyR1.
• 3D reconstructions revealed minor conformational changes in cross-linked RyR1 compared to controls.
• Intersubunit cross-linking enhanced [(3)H]ryanodine binding, indicating a functional change.

Conclusions:

• The developed cross-linking method successfully stabilizes the RyR1-FKBP12 complex for cryo-EM structural studies.
• Glutaraldehyde-induced intersubunit cross-linking of RyR1 promotes an open-like conformation.
• This technique provides a valuable approach for investigating the structure and dynamics of the ECC machinery.