The HCM-associated R347C mutation in JPH2 abolishes its membrane localization by disrupting a positively charged linker required for membrane binding.
The study reveals the structural basis of JPH2 membrane binding and demonstrates that the HCM-associated R347C mutation disrupts this interaction, contributing to HCM pathogenesis.
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Objective Junctophilin-2 (JPH2) is an essential structural protein that maintains junctional membrane complexes (JMCs) in cardiomyocytes by tethering the plasma membrane to the sarcoplasmic reticulum, thereby facilitating excitationcontraction (E-C) coupling. Mutations in JPH2 have been associated with hypertrophic cardiomyopathy (HCM), but the molecular mechanisms governing its membrane-binding properties and the functional relevance of its membrane occupation and recognition nexus (MORN) repeat motifs remain incompletely understood. This study aimed to elucidate the structural basis of JPH2 membrane association and its implications for HCM pathogenesis. Methods A recombinant N-terminal fragment of mouse JPH2 (residues1-440), encompassing the MORN repeats and an adjacent helical region, was purified under near-physiological buffer conditions. X-ray crystallography was employed to determine the structure of the JPH2 MORN-Helix domain. Sequence conservation analysis across species and junctophilin isoforms was performed to assess the evolutionary conservation of key structural features. Functional membrane-binding assays were conducted using liposome co-sedimentation and cell-based localization studies in COS7 and He La cells. In addition, site-directed mutagenesis targeting positively charged residues and known HCM-associated mutations, including R347C,was used to evaluate their effects on membrane interaction and subcellular localization. Results The crystal structure of the mouse JPH2 MORN-Helix domain was resolved at 2.6 ?, revealing a compact, elongated architecture consisting of multiple tandem MORN motifs arranged in a curved configuration, forming a continuous hydrophobic core stabilized by alternating aromatic residues. A C-terminal α-helix further reinforced structural integrity. Conservation analysis identified the inner groove of the MORN array as a highly conserved surface, suggesting its role as a protein-binding interface. A flexible linker segment enriched in positively charged residues, located adjacent to the MORN motifs, was found to mediate direct electrostatic interactions with negatively charged phospholipid membranes. Functional assays demonstrated that mutation of these basic residues impaired membrane association, while the HCM-linked R347C mutation completely abolished membrane localization in cellular assays, despite preserving the overall MORN-Helix fold in structural modeling. Conclusion This study provides structural insight into the membrane-binding mechanism of the cardiomyocyte-specific protein JPH2, highlighting the dual roles of its MORN-Helix domain in membrane anchoring and protein interactions. The findings clarify the structural basis for membrane targeting via a positively charged linker and demonstrate that disruption of this interaction—such as that caused by the R347C mutation—likely contributes to HCM pathogenesis. These results not only enhance current understanding of JPH2 function in cardiac E-C coupling but also offer a structural framework for future investigations into the assembly and regulation of JMCs in both physiological and disease contexts.
Wang et al. (Wed,) reported a other. The HCM-associated R347C mutation in JPH2 abolishes its membrane localization by disrupting a positively charged linker required for membrane binding.