Human junctophilin-2 undergoes a structural rearrangement upon binding PtdIns(3,4,5) P ₃ and the S101R mutation identified in hypertrophic cardiomyopathy obviates this response

Abstract

JP2 (junctophilin-2) is believed to hold the transverse tubular and jSR (junctional sarcoplasmic reticulum) membranes in a precise geometry that facilitates excitation–contraction coupling in cardiomyocytes. We have expressed and purified human JP2 and shown using electron microscopy that the protein forms elongated structures ~15 nm long and 2 nm wide. Employing lipid-binding assays and quartz crystal microbalance with dissipation we have determined that JP2 is selective for PS (phosphatidylserine), with a K _d value of ~0.5 μM, with the N-terminal domain mediating this interaction. JP2 also binds PtdIns(3,4,5) P ₃ at a different site than PS, resulting in the protein adopting a more flexible conformation; this interaction is modulated by both Ca ²⁺ and Mg ²⁺ ions. We show that the S101R mutation identified in patients with hypertrophic cardiomyopathy leads to modification of the protein secondary structure, forming a more flexible molecule with an increased affinity for PS, but does not undergo a structural transition in response to binding PtdIns(3,4,5) P ₃. In conclusion, the present study provides new insights into the structural and lipid-binding properties of JP2 and how the S101R mutation may have an effect upon the stability of the dyad organization with the potential to alter JP2–protein interactions regulating Ca ²⁺ cycling.

Abstract

We have purified human JP2 (junctophilin-2) and the S101R hypertrophic cardiomyopathy mutant. JP2 interacts with phosphatidylserine, K _d ~0.5 μM, and PtdIns(3,4,5) P ₃ at different sites; divalent cations perturb the association. S101R has a modified structure and phospholipid-binding properties.