title: N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.
abstract: We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca(2+) leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) "tags" placed within N-terminal (amino acid residues 76-619) or central (residues 2157-2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.
authors.affiliation: Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: Tanya
authors.initials: T
authors.lastname: Girgenrath
authors.forename: Mohana
authors.initials: M
authors.lastname: Mahalingam
authors.forename: Bengt
authors.initials: B
authors.lastname: Svensson
authors.forename: Florentin R
authors.initials: FR
authors.lastname: Nitu
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
authors.forename: James D
authors.initials: JD
authors.lastname: Fessenden
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D022061:Tacrolimus Binding Protein 1A
keywords: Calcium; Calcium Channels; Excitation-Contraction Coupling; FK506-binding Proteins; Fluorescence Resonance Energy Transfer (FRET); Malignant Hyperthermia; Ryanodine; Ryanodine Receptor