Three Amino Acid Residues Determine Selective Binding of FK506-binding Protein 12.6 to the Cardiac Ryanodine Receptor

Abstract

FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2. FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2. FK506, a fungal macrolide antibiotic, possesses potent immunosuppressive activity. The drug has been used both experimentally and clinically to prevent organ graft rejection and to treat autoimmune diseases (1Ochiai T. Nakajima K. Nagata M. Transplantation. 1987; 44: 734-738Crossref PubMed Scopus (169) Google Scholar, 2Ochiai T. Nagata M. Nakajima K. Suzuki T. Sakamoto K. Enomoto K. Gunji Y. Uematsu T. Goto T. Hori S. et al.Transplantation. 1987; 44: 729-733Crossref PubMed Scopus (128) Google Scholar, 3Todo S. Demetris A. Ueda Y. Imventarza O. Cadoff E. Zeevi A. Starzl T.E. Surgery. 1989; 106: 444-450PubMed Google Scholar, 4Sterzl T.E. Donner A. Eliasziw M. Meier P. Fung J.J. McMichael J.P. Todo S. Lancet. 1995; 346: 1346-1350Crossref PubMed Scopus (52) Google Scholar, 5Winkler M. Christians U. Drug Saf. 1995; 12: 348-357Crossref PubMed Scopus (55) Google Scholar). FK506 suppresses activation of human T-lymphocytes by blocking a key step required in the synthesis or activation of transcription factors (NF-AT, AP-3, and NF-κB), which promote expression of lymphokine genes such as interleukin-2 (IL-2) (6Crabtree B.R. Science. 1989; 243: 355-361Crossref PubMed Scopus (906) Google Scholar). The immunosuppressive action of the drug is related to its inhibition of calcineurin, a calcium-dependent protein phosphatase involved in intracellular signaling transduction, by way of binding to FK506-binding proteins (FKBPs), 1The abbreviations used are: FKBP, FK506-binding protein; CSR, cardiac SR; FKBP12, a 12-kDa FKBP; FKBP12.6, an isoform of FKBP with slightly slower mobility than FKBP12; RyR1 and RyR2, ryanodine receptor isoform 1 from skeletal muscle and isoform 2 from heart, respectively; SR, sarcoplasmic reticulum; TC, terminal cisternae of SR; HPLC, high performance liquid chromatography1The abbreviations used are: FKBP, FK506-binding protein; CSR, cardiac SR; FKBP12, a 12-kDa FKBP; FKBP12.6, an isoform of FKBP with slightly slower mobility than FKBP12; RyR1 and RyR2, ryanodine receptor isoform 1 from skeletal muscle and isoform 2 from heart, respectively; SR, sarcoplasmic reticulum; TC, terminal cisternae of SR; HPLC, high performance liquid chromatography a family of related intracellular receptors including FKBP12 and FKBP12.6 (7Liu J. Farmer J. Lane W. Friedman J. Weissman I. Schreiber S. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3546) Google Scholar, 8Sewell T. Lam E. Martin M. Leszyk J. Weinder J. Calaycay J. Griffin P. Williams H. Hung S. Cryan J. Sigal N. Wiederrecht G.J. J. Biol. Chem. 1994; 269: 21094-21102Abstract Full Text PDF PubMed Google Scholar, 9Lam E. Martin M.M. Timerman A.P. Sabers C. Fleischer S. Wiederrecht G.J. J. Biol. Chem. 1995; 270: 26511-26522Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In skeletal muscle and heart, the ryanodine receptor (RyR)/calcium release channel serves to release Ca2+ from sarcoplasmic reticulum (SR), thereby triggering muscle contraction (10Fleischer S. Inui M. Annu. Rev. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (441) Google Scholar, 11McPherson P.S. Campbell K.P. J. Biol. Chem. 1993; 268: 13765-13768Abstract Full Text PDF PubMed Google Scholar, 12Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (834) Google Scholar). FKBP12 has been found to be tightly associated with skeletal muscle RyR (RyR1), and more recently its novel isoform, FKBP12.6, was found to be selectively associated with cardiac RyR (RyR2). Thus, the structural formulae for RyR1 and RyR2 are (RyR1 protomer)4(FKBP12)4 and (RyR2 protomer)4(FKBP12.6)4, respectively (13Jayaraman T. Brillantes A.M. Timerman A.P. Fleischer S. Erdjument-Bromage H. Tempst P. Marks A.R. J. Biol. Chem. 1992; 267: 9474-9477Abstract Full Text PDF PubMed Google Scholar, 14Timerman A.P. Jayaraman T. Wiederrecht G. Onoue H. Marks A.R. Fleischer S. Biochem. Biophys. Res. Communs. 1994; 198: 701-706Crossref PubMed Scopus (113) Google Scholar, 15Timerman A.P. Ogunbumni E. Freund E. Wiederrecht G. Marks A.R. Fleischer S. J. Biol. Chem. 1993; 268: 22992-22999Abstract Full Text PDF PubMed Google Scholar, 16Timerman A.P. Onoue H. Xin H.-B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The ultrastructural localization of FKBP12 has been pinpointed within the three dimensional structure of RyR1 by cryoelectron microscopy and image enhancement analysis. There are four FKBPs/receptor with 4-fold symmetry, and FKBP is located in the vicinity of RyR1 that is close to the transverse tubule, consistent with its serving a role in E-C coupling (17Wagenknecht T. Grassuccci R. Berkowitz J. Wiederrecht G.J. Xin H.-B. Fleischer S. Biophys. J. 1996; 70: 1709-1715Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 18Wagenknecht T. Radermacher M. Grassuccci R. Berkowitz J. Xin H.-B. Fleischer S. J. Biol. Chem. 1997; 272: 32463-32471Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Both FKBP12 and FKBP12.6 share a number of structural and functional similarities, including: 1) 85% amino acid sequence homology (only 18 of 108 amino acid residues differ); 2) PPIase activity that is inhibited by FK506 or rapamycin; and 3) inhibition of calcineurin when the ternary complexes are formed with FKBP and FK506 (6Crabtree B.R. Science. 1989; 243: 355-361Crossref PubMed Scopus (906) Google Scholar, 7Liu J. Farmer J. Lane W. Friedman J. Weissman I. Schreiber S. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3546) Google Scholar, 8Sewell T. Lam E. Martin M. Leszyk J. Weinder J. Calaycay J. Griffin P. Williams H. Hung S. Cryan J. Sigal N. Wiederrecht G.J. J. Biol. Chem. 1994; 269: 21094-21102Abstract Full Text PDF PubMed Google Scholar). FKBP12 appears to have a role in excitation-contraction coupling in skeletal muscle by modulating the release of Ca2+ from SR (15Timerman A.P. Ogunbumni E. Freund E. Wiederrecht G. Marks A.R. Fleischer S. J. Biol. Chem. 1993; 268: 22992-22999Abstract Full Text PDF PubMed Google Scholar, 19Timerman A.P. Wiederrecht G.J. Marcy A. Fleischer S. J. Biol. Chem. 1995; 270: 2451-2459Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 20Brillantes A.M. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Lander M. Ehlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (697) Google Scholar). However, the action of FKBP12.6 on cardiac excitation-contraction coupling is less clear (16Timerman A.P. Onoue H. Xin H.-B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Recently, it has been suggested that FKBP12.6 may be involved in the regulation of insulin biosynthesis and secretion by modulating intracellular Ca2+ levels via RyR2 in rat pancreatic islets. It has been proposed that FKBP12.6 bound on RyR2 can be released by cyclic ADP-ribose, a metabolite of NAD+ during insulin secretion by glucose stimulation (21Noguchi N. Takasawa S. Nata K. Tohgo A. Kato I. Ikehata F. Yonekura H. Okamoto H. J. Biol. Chem. 1997; 272: 3133-3136Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The precise role of FKBP12.6 in cardiac excitation-contraction coupling and insulin secretion remains to be elucidated. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acid residues 2We are using the numbering system in the literature (35Standaert R.F. Galat A. Verdine G.L. Schreiber S.L. Nature. 1990; 346: 671-674Crossref PubMed Scopus (270) Google Scholar) for consistency (see Figs. 1 and 4), that is, the N-terminal methionine of FKBP12 is designated as the 0 amino acid residue so that the C-terminal glycine is numbered 107.2We are using the numbering system in the literature (35Standaert R.F. Galat A. Verdine G.L. Schreiber S.L. Nature. 1990; 346: 671-674Crossref PubMed Scopus (270) Google Scholar) for consistency (see Figs. 1 and 4), that is, the N-terminal methionine of FKBP12 is designated as the 0 amino acid residue so that the C-terminal glycine is numbered 107. (9Lam E. Martin M.M. Timerman A.P. Sabers C. Fleischer S. Wiederrecht G.J. J. Biol. Chem. 1995; 270: 26511-26522Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 35Standaert R.F. Galat A. Verdine G.L. Schreiber S.L. Nature. 1990; 346: 671-674Crossref PubMed Scopus (270) Google Scholar), FKBP12.6 selectively binds to cardiac RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to skeletal muscle RyR1 and exchange with bound FKBP12 of RyR1 (16Timerman A.P. Onoue H. Xin H.-B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 22Barg S. Copello J.A. Wiederrecht G. Fleischer S. Am. J. Physiol. 1997; 272: C1726-C1733Crossref PubMed Google Scholar). In the present study, site-directed mutagenesis was used to determine the amino acid residues of FKBP12.6 that confer its selective association with the cardiac RyR2. The preparation of human FKBP12.6 mutants was carried out using site-directed mutagenesis system (QuickChangeTMsite-directed mutagenesis kit, Stratagene, La Jolla, CA). Briefly, the complementary DNA coding region of human FKBP12.6 was subcloned into the protein expression vector pET-3d at NcoI andBamHI sites as described previously (9Lam E. Martin M.M. Timerman A.P. Sabers C. Fleischer S. Wiederrecht G.J. J. Biol. Chem. 1995; 270: 26511-26522Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Using this plasmid as a DNA template, the mutants of FKBP12.6 were synthesized by polymerase chain reaction in the presence of a pair of complementary synthetic oligonucleotide primers containing the desired mutation usingPfu DNA polymerase, which replicates both plasmid strands with high fidelity and without displacing the mutant oligonucleotide primers. Following temperature cycling of polymerase chain reaction, the parental DNA template was digested by DpnI endonuclease, which is specific for cleaving methylated and hemimethylated DNA, and the nicked vector DNA incorporating the desired mutations was then transformed into XL1-Blue supercompetent cells. The mutants were identified by sequencing using an Applied Biosystems Prism 377 automatic sequencer (Applied Biosystems) with a Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer). The mutants with double or multiple amino acids mutations were subsequently made by using the single or multiple mutants as a template. The oligonucleotides primers used for mutagenesis were synthesized by changing the DNA sequences of FKBP12.6 to FKBP12. The same procedure described above was used to make FKBP12 mutants except using human recombinant FKBP12, which was also subcloned in the pET-3d expression vector, as a template and changing the DNA sequences from FKBP12 to FKBP12.6. The pET-3d plasmid containing human FKBP12.6, FKBP12, and their mutants were transformed into BL21 (DE3) competent cells, and the recombinant and mutated proteins were induced by isopropy-β-d-thiogalactopyranoside. FKBPs and their mutants were purified by high pressure liquid chromatography (HPLC) using a TSK G3000SW column as described previously (23Xin H.B. Timerman A.P. Onoue H. Wiederrecht G.J. Fleischer S. Biochem. Biophys. Res. Commun. 1995; 214: 263-270Crossref PubMed Scopus (27) Google Scholar). The biosynthesis of35S-labeled FKBP12.6 or 35S-Labeled FKBP12 were carried out essentially as described previously (9Lam E. Martin M.M. Timerman A.P. Sabers C. Fleischer S. Wiederrecht G.J. J. Biol. Chem. 1995; 270: 26511-26522Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) with some modification. Briefly, the pET-3d plasmid DNA containing human FKBP12.6 or FKBP12 was transformed into BL21 (DE3) host cell. Two ml of an overnight culture from a single colony were inoculated into 200 ml of M9 medium containing 50 μg/ml ampicillin and incubated at 37 °C with shaking (250 rpm). The bacteria were collected by centrifugation at 3000 rpm for 5 min (Beckman JA-14 rotor) when the OD600value of the bacteria reached to 0.5. The pelleted bacteria were resuspended in 200 ml of RPMI 1640 medium (Life Technologies, Inc.) containing 50 mg/ml ampicillin, 1/40th of methionine, cystine, and glucose concentration compared with the regular RPMI 1640 medium and then isopropy-β-d-thiogalactopyranoside (1 mmfinal concentration) was added. A 2.5 mCi of [35S]methionine and [35S]cystine mixture (NEN Life Science Products) was then added to the medium after incubation at 37 °C for 15 min with shaking, and the bacteria were cultured for 5–6 h at the same conditions. The bacteria were collected by centrifugation, and the pellets were resuspended in 10 ml of phosphate-buffered saline and then lysed by sonication (50% pules, 30-s intervals for 3 min, Branson Sonic Power Co.). The supernatant was collected by centrifugation at 15,000 × g for 30 min at 4 °C, and the DNA was precipitated by adding protamine sulfate (final concentration: 0.04%). Finally, the 35S-labeled FKBP12.6 or 35S-labeled FKBP12 was purified by HPLC using a TSK G3000SW column. The specific radioactivity of [35S]FKBP12.6 or [35S]FKBP12 in the time span of these experiments were changed from 6500 to 4500 cpm/pmol and from 2100 to 1500 cpm/pmol, respectively. Cardiac SR (CSR) was isolated from canine heart, and TC vesicles of SR were isolated from fast twitch rabbit skeletal muscle as described previously (24Chamberlain B. Levitsky D. Fleischer S. J. Biol. Chem. 1983; 258: 6602-6609Abstract Full Text PDF PubMed Google Scholar, 25Saito A. Seiler S. Chu A. Fleischer S. J. Cell Biol. 1984; 99: 875-885Crossref PubMed Scopus (416) Google Scholar). The R3 fraction from the TC preparation of skeletal muscle SR was used in the35S-labeled FKBP12 exchange experiments, because in comparison with the highly enriched R4 TC fraction, which has higher concentration of high affinity ryanodine binding (of about 25 pmol/mg protein), the concentration of high affinity ryanodine binding site in the R3 fraction (about 8 pmol/mg of protein) was in a similar range to that of the CSR (5 pmol/mg of protein). Cardiac SR or skeletal muscle TC of SR (2.5 mg/ml) were prelabeled with [35S]FKBP12.6 or [35S]FKBP12 (3 μm) by exchange as described previously by incubating 30 min at 37 °C (15Timerman A.P. Ogunbumni E. Freund E. Wiederrecht G. Marks A.R. Fleischer S. J. Biol. Chem. 1993; 268: 22992-22999Abstract Full Text PDF PubMed Google Scholar, 16Timerman A.P. Onoue H. Xin H.-B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The unbound [35S]FKBPs were separated from the [35S]FKBPs bound on CSR or skeletal muscle TC vesicles by sedimentation of 35,000 rpm (Beckman TL100.2 rotor) for 15 min at 4 °C. The cardiac SR or skeletal muscle TC vesicles were resuspended in imidazole homogenization medium (5 mm imidazole-HCl, pH 7.4, and 0.3 m sucrose) to a final concentration of 2.5 mg/ml. Skeletal muscle terminal cisternae of SR or cardiac SR were prelabeled by exchange with [35S]FKBP12.6 or [35S]FKBP12, respectively (see above), as described previously (15Timerman A.P. Ogunbumni E. Freund E. Wiederrecht G. Marks A.R. Fleischer S. J. Biol. Chem. 1993; 268: 22992-22999Abstract Full Text PDF PubMed Google Scholar, 16Timerman A.P. Onoue H. Xin H.-B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The EC50 for the release of [35S]FKBPs from such prelabeled SR by FKBPs and their mutants was used an index of FKBP binding to the ryanodine receptors. Briefly, [35S]FKBP12.6 and [35S]FKBP12 were used to label cardiac SR and skeletal muscle TC, respectively. The SR vesicles in a final concentration of 2 mg/ml were incubated with increasing concentrations of FKBPs or mutants at 37 °C for 30 min. The free [35S]FKBPs was separated from bound [35S]FKBPs by diluting a 50 μl of sample (120 μg of protein) into 200 μl of ice-cold imidazole homogenization medium buffer and immediately sedimenting the vesicles by centrifugation (95,000 rpm for 15 min at 4 °C). The pellet was quickly rinsed, then resuspended in 200 μl of distilled water and counted in 5 ml of Cytoscint liquid scintillation mixture. The protein concentration of SR membrane fractions was determined by the Lowry procedure (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and FKBPs and mutants by scanning densitometry of Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis gels using a gel analysis and image processing system (Technology Resources Inc., Nashville, TN) (15Timerman A.P. Ogunbumni E. Freund E. Wiederrecht G. Marks A.R. Fleischer S. J. Biol. Chem. 1993; 268: 22992-22999Abstract Full Text PDF PubMed Google Scholar). Bovine serum albumin was used as the protein standard. SDS-polyacrylamide gel electrophoresis assay was performed with a mini-slab gel apparatus (Hoeffer Scientific) using the buffer system described by Laemmli (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Human FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids (9Lam E. Martin M.M. Timerman A.P. Sabers C. Fleischer S. Wiederrecht G.J. J. Biol. Chem. 1995; 270: 26511-26522Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), yet FKBP12.6 selectively binds to and exchanges with bound FKBP12.6 on cardiac RyR2, whereas both FKBP isoforms bind to and exchange with bound FKBP12 of skeletal muscle RyR1 (16Timerman A.P. Onoue H. Xin H.-B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 22Barg S. Copello J.A. Wiederrecht G. Fleischer S. Am. J. Physiol. 1997; 272: C1726-C1733Crossref PubMed Google Scholar). The selective binding of FKBP12.6 to RyR2 must be referable to some of the 18 differing amino acid residues between the two FKBP isoforms In to which amino acid residues determine selective binding and mutated some of the 18 differing amino acids of FKBP12.6 and the in selective exchange to RyR2. binding and exchange have been found to the EC50 for the concentration of the FKBP mutants to release of [35S]FKBP12.6 as an index of FKBP12.6 binding to RyR2. A of single or multiple recombinant mutants of FKBP12.6 were and the concentration for release of [35S]FKBP12.6 from prelabeled SR was A and We made single and double mutants by the amino acids of FKBP12.6 with of FKBP12. In the of the the EC50 for exchange of FKBP12.6 to cardiac RyR2 was found to be than for FKBP12.0. is, the EC50 of FKBP12.6 for release of [35S]FKBP12.6 from cardiac SR by exchange was compared with for FKBP12. Two mutants of FKBP12.6, and with the EC50 of and have and affinity for cardiac RyR2, compared with FKBP12.6. single mutants the affinity to cardiac RyR2. is, the EC50 are in a similar range μm) and 2 as FKBP12.6 on these double mutant and three triple mutants were The triple mutant (Q31E/N32D/F59W) was of selective binding to cardiac SR an EC50 of similar to FKBP12 Two double mutants and were made to assess two amino acids from this triple mutant were for the selective binding to cardiac RyR2. The that both double mutants and retained binding activity to cardiac RyR2 with of and respectively of FKBPs mutants with FKBP12 prelabeled CSR CSR vesicles (2.5 B. Levitsky D. Fleischer S. J. Biol. Chem. 1983; 258: 6602-6609Abstract Full Text PDF PubMed Google Scholar) were prelabeled by exchange with [35S]FKBP12.6 (3 μm) by incubation for 30 min at 37 °C. The of [35S]FKBP12.6 from prelabeled CSR was carried out at 37 °C for 30 min in the presence of concentrations of FKBP12, FKBP12.6, and FKBP12.6 mutants or FKBP12 mutants The [35S]FKBP12.6 in the CSR vesicles was determined as described Each was performed with two or three with in The EC50 are in image of the EC50 and the exchange of FKBP12, FKBP12.6, and their mutants on EC50 in were from exchange as in A and B. in a The EC50 in were from exchange as in A and B. mutation of these three amino acid residues in FKBP12.6 is to out selective binding to RyR2, then mutation of these residues in FKBP12 to that of FKBP12.6 confer selective mutants of FKBP12 were in which the three key amino acids of FKBP12 were by the respective residues of FKBP12.6. The that the triple mutant binds to cardiac RyR2 with a similar EC50 μm) as FKBP12.6 The double mutants and bind to cardiac RyR2, with EC50 of and respectively 2 and A key for the role of the three amino acid residues to confer selective binding to RyR2 is that the mutants their binding to RyR1. For this skeletal muscle SR prelabeled by exchange with [35S]FKBP12 as the assay We found that of the FKBP12.6 and FKBP12 mutants retained their to exchange with FKBP bound to skeletal muscle RyR1. is, the mutants comparable binding affinity as FKBP12 or FKBP12.6, in the range of 3 and of the EC50 and the exchange of FKBP12, FKBP12.6, and their mutants on TC of skeletal muscle EC50 in were from exchange as in in a The EC50 in were from exchange as in We conclude that three amino acids of FKBP12.6 (Gln31, Asn32, and Phe59) determine selective binding to RyR2. In this study, is into the of the of FKBP with the of and skeletal muscle FKBP12 and FKBP12.6 both bind to whereas FKBP12.6 selectively binds to RyR2. In to assess which amino acids of FKBP12.6 confer selective binding to RyR2, carried out site-directed mutagenesis in amino acid residues that differ between the two FKBP The assay for selective binding/exchange of FKBP12.6 to RyR2 was then used to assess which mutations the of We that only three amino acid residues of FKBP12.6 determine the of binding to RyR2. The triple amino acid mutation of FKBP12.6 the amino acids of FKBP12.6 with the respective three amino acid residues in FKBP12, the selective binding for RyR2. In complementary experiments, FKBP12 can be made to selectively bind to RyR2 by these same three amino acid residues to in FKBP12.6. The that mutants binding to skeletal muscle RyR1 with a similar affinity as that the of both FKBPs are and We conclude that three amino acid residues (Gln31, Asn32, and Phe59) determine the selective binding of FKBP12.6 to the cardiac RyR2. The of FKBP12 and complexes have been determined to high by and J.A. Nature. 1991; PubMed Scopus Google Scholar, M. Schreiber S.L. Science. 1991; PubMed Scopus Google Scholar, R.F. Schreiber S.L. J. Science. 1991; PubMed Scopus Google Scholar, et 1995; PubMed Scopus Google Scholar, J.P. J.A. K. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). is close sequence homology between FKBP12 and FKBP12.6 (9Lam E. Martin M.M. Timerman A.P. Sabers C. Fleischer S. Wiederrecht G.J. J. Biol. Chem. 1995; 270: 26511-26522Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), carried out structure of FKBP12.6 and its mutants using the structure of FKBP12 as the The 18 amino acid residues of FKBP12.6 were and was used to assess the structure structure of FKBP12.6 were using the system on the structure of FKBP12 The of the structure of FKBP12.6 is similar to that of FKBP12. were between FKBP12.6 and its double and triple mutants and Thus, structural are between the two FKBP isoforms or when the three amino acid residues that are critical for selective binding to RyR2. Schreiber and that and of FKBP12 to a FK506 and The of is at the of the and serves as a for the of FK506 M. Schreiber S.L. Science. 1991; PubMed Scopus Google Scholar, R.F. Schreiber S.L. J. Science. 1991; PubMed Scopus Google Scholar). It is that is a similar in FKBP12.6 for binding to FK506 as for FKBP12. The amino acids that the are in both FKBPs with the of in FKBP12.6, which in FKBP12. The of the in FKBP12.6 be to be changed when is mutated to both are amino However, these two amino acid residues differ in so that may in to the of The is in FKBP12.6, it the residue of the residue in FKBP12. Schreiber and Schreiber S.L. J. Chem. 1990; Scopus (128) Google Scholar) the of activity of FKBP12 and suggested that FK506 as a of and Fung J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) suggested that FKBP12 binds RyR1 at a sequence There is a similar in cardiac RyR2 containing an of a The in such a binding of RyR2 may be by the of FKBP12.6, which the of Thus, factors may in determine the selective binding of FKBP12.6 to cardiac RyR2, whereas both FKBPs bind to skeletal muscle RyR1. The affinity for binding to cardiac RyR2 by and of FKBP12.6 to and of FKBP12 an in two that may be a in the lack of binding of FKBP12 to RyR2. the amino acid residues of FKBP12.6 that determine the selective with RyR2. the structure of FKBP12.6 and FKBP12 to be that a in the structure of RyR1 and RyR2 also to the of the We of for human FKBP12 and FKBP12.6 We also of for with the of FKBP12.6