Three-dimensional visualization of FKBP12.6 binding to an open conformation of cardiac ryanodine receptor.
The cardiac isoform of the ryanodine receptor (RyR2) from dog binds predominantly a 12.6-kDa isoform of the FK506-binding protein (FKBP12.6), whereas RyR2 from other species binds both FKBP12.6 and the closely related isoform FKBP12. The role played by FKBP12.6 in modulating calcium release by RyR2 is unclear at present. We have used cryoelectron microscopy and three-dimensional (3D) reconstruction techniques to determine the binding position of FKBP12.6 on the surface of canine RyR2. Buffer conditions that should favor the "open" state of RyR2 were used. Quantitative comparison of 3D reconstructions of RyR2 in the presence and absence of FKBP12.6 reveals that FKBP12.6 binds along the sides of the square-shaped cytoplasmic region of the receptor, adjacent to domain 9, which forms part of the four clamp (corner-forming) structures. The location of the FKBP12.6 binding site on "open" RyR2 appears similar, but slightly displaced (by 1-2 nm) from that found previously for FKBP12 binding to the skeletal muscle ryanodine receptor that was in the buffer that favors the "closed" state. The conformation of RyR2 containing bound FKBP12.6 differs considerably from that depleted of FKBP12.6, particularly in the transmembrane region and in the clamp structures. The x-ray structure of FKBP12.6 was docked into the region of the 3D reconstruction that is attributable to bound FKBP12.6, to show the relative orientations of amino acid residues (Gln-31, Asn-32, Phe-59) that have been implicated as being critical in interactions with RyR2. A thorough understanding of the structural basis of RyR2-FKBP12.6 interaction should aid in understanding the roles that have been proposed for FKBP12.6 in heart failure and in certain forms of sudden cardiac death.
Sharma
Manjuli Rani
MR
Wadsworth Center, New York State Department of Health, School of Public Health, State University of New York at Albany, Albany, New York 12201-0509, USA.
Jeyakumar
Loice H
LH
Fleischer
Sidney
S
Wagenknecht
Terence
T
D000818:Animals; D001665:Binding Sites; D001703:Biophysics / Q000379:methods*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D020285:Cryoelectron Microscopy; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012433:Ryanodine / Q000737:chemistry; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D014965:X-Rays
D020033:Protein Isoforms; D011506:Proteins; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B; D002118:Calcium