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Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study
12:43

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Published on: July 27, 2016

Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor

H B Xin1, K Rogers, Y Qi

  • 1Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

The Journal of Biological Chemistry
|May 21, 1999
PubMed

Insights

FKBP12.6 selectively binds to cardiac RyR2 calcium channels, unlike FKBP12. Three specific amino acid differences in FKBP12.6 are crucial for this selective binding to RyR2, impacting cardiac function.

Area of Science:

  • Molecular Biology
  • Cardiovascular Physiology
  • Protein-Protein Interactions

Background:

  • FKBP12 binds to skeletal muscle RyR1, while FKBP12.6 binds to cardiac RyR2.
  • Both RyR1 and RyR2 channels have a stoichiometry of 4 FKBP proteins per receptor.
  • FKBP12.6 and FKBP12 differ by only 18 amino acids, yet exhibit distinct receptor selectivity.

Purpose of the Study:

  • To identify the specific amino acid residues in FKBP12.6 responsible for its selective binding to RyR2.
  • To understand the molecular basis of FKBP isoform selectivity for different ryanodine receptor subtypes.

Main Methods:

  • Site-directed mutagenesis was used to create FKBP12.6 mutants with residues changed to match FKBP12.
  • Cardiac RyR2, prelabeled with [35S]FKBP12.6, served as the assay system for binding and exchange studies.
  • FKBP12 mutants were created by introducing the three critical FKBP12.6 amino acids.

Main Results:

  • A triple mutant of FKBP12.6 (Q31E/N32D/F59W) lost selective binding to RyR2, behaving like FKBP12.
  • Mutating FKBP12 to incorporate the three critical FKBP12.6 residues conferred selective RyR2 binding.
  • All FKBP mutants retained binding to skeletal muscle RyR1.

Conclusions:

  • Gln31, Asn32, and Phe59 in human FKBP12.6 are the key residues driving selective binding to cardiac RyR2.
  • These findings elucidate the molecular determinants of FKBP-RyR subtype specificity.
  • Understanding this interaction is vital for exploring cardiac channelopathies and therapeutic interventions.

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