title: Human junctophilin-2 undergoes a structural rearrangement upon binding PtdIns(3,4,5)P3 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 Kd value of ~0.5 μM, with the N-terminal domain mediating this interaction. JP2 also binds PtdIns(3,4,5)P3 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)P3. 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.
authors.affiliation: *Institute of Cardiovascular Sciences, Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9NT, U.K.
authors.forename: Hayley J
authors.initials: HJ
authors.lastname: Bennett
authors.forename: John Bernard
authors.initials: JB
authors.lastname: Davenport
authors.forename: Richard F
authors.initials: RF
authors.lastname: Collins
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Trafford
authors.forename: Christian
authors.initials: C
authors.lastname: Pinali
authors.forename: Ashraf
authors.initials: A
authors.lastname: Kitmitto
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000235:genetics; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058525:Quartz Crystal Microbalance Techniques; D013816:Thermodynamics
chemical_list: D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; C412507:junctophilin; C060974:phosphatidylinositol 3,4,5-triphosphate; D008274:Magnesium; D002118:Calcium