Mapping the ryanodine receptor FK506-binding protein subunit using fluorescence resonance energy transfer.
The 12-kDa FK506-binding proteins (FKBP12 and FKBP12.6) are regulatory subunits of ryanodine receptor (RyR) Ca(2+) release channels. To investigate the structural basis of FKBP interactions with the RyR1 and RyR2 isoforms, we used site-directed fluorescent labeling of FKBP12.6, ligand binding measurements, and fluorescence resonance energy transfer (FRET). Single-cysteine substitutions were introduced at five positions distributed over the surface of FKBP12.6. Fluorescent labeling at position 14, 32, 49, or 85 did not affect high affinity binding to the RyR1. By comparison, fluorescent labeling at position 41 reduced the affinity of FKBP12.6 binding by 10-fold. Each of the five fluorescent FKBPs retained the ability to inhibit [(3)H]ryanodine binding to the RyR1, although the maximal extent of inhibition was reduced by half when the label was attached at position 32. The orientation of FKBP12.6 bound to the RyR1 and RyR2 was examined by measuring FRET from the different labeling positions on FKBP12.6 to an acceptor attached within the RyR calmodulin subunit. FRET was dependent on the position of fluorophore attachment on FKBP12.6; however, for any given position, the distance separating donors and acceptors bound to RyR1 versus RyR2 did not differ significantly. Our results show that FKBP12.6 binds to RyR1 and RyR2 in the same orientation and suggest new insights into the discrete structural domains responsible for channel binding and inhibition. FRET mapping of RyR-bound FKBP12.6 is consistent with the predictions of a previous cryoelectron microscopy study and strongly supports the proposed structural model.
Cornea
Razvan L
RL
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Nitu
Florentin R
FR
Samsó
Montserrat
M
Thomas
David D
DD
Fruen
Bradley R
BR
D000818:Animals; D002147:Calmodulin / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D020104:Immunophilins / Q000737:chemistry; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000473:pathology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012433:Ryanodine / Q000737:chemistry; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013552:Swine; D022021:Tacrolimus Binding Proteins / Q000737:chemistry*
D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins
Circ Res. 2007 Nov 9;101(10):1020-9
17872463
Proc Natl Acad Sci U S A. 2009 Jul 7;106(27):11040-4
19541610
Biochem J. 2009 Apr 15;419(2):273-8
19226252
Biophys J. 2006 Jan 1;90(1):164-72
16214874
J Comput Chem. 2004 Oct;25(13):1605-12
15264254
J Biol Chem. 2002 Jan 11;277(2):1349-53
11694536
J Biol Chem. 2007 Nov 30;282(48):34828-38
17921453
Cell. 1994 May 20;77(4):513-23
7514503
Biochemistry. 2005 Jan 11;44(1):278-84
15628869
Front Biosci. 2002 Jun 01;7:d1464-74
12045016
J Biol Chem. 2004 Jun 18;279(25):26481-8
15033987
Antioxid Redox Signal. 2005 Jul-Aug;7(7-8):870-81
15998242
Biochem J. 2006 May 15;396(1):e1-3
16626281
Acta Crystallogr D Biol Crystallogr. 2000 Mar;56(Pt 3):266-71
10713512
Cell. 2003 Jun 27;113(7):829-40
12837242
Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6128-33
19332786
J Biol Chem. 1992 Feb 15;267(5):3316-24
1371117
Biochemistry. 2003 Mar 11;42(9):2740-7
12614169
J Mol Cell Cardiol. 2004 Aug;37(2):417-29
15276012
Circulation. 2004 Jun 29;109(25):3208-14
15197150
Nat Med. 2009 Mar;15(3):325-30
19198614
Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3456-61
16481613
J Biol Chem. 1991 Feb 5;266(4):2474-9
1989998
Am J Physiol. 1997 May;272(5 Pt 1):C1726-33
9176165
J Biol Chem. 1997 Dec 19;272(51):32463-71
9405457
J Biol Chem. 2007 Mar 9;282(10):6976-83
17200109
Circ Res. 2006 Aug 4;99(3):292-8
16825580
Structure. 2009 Nov 11;17(11):1505-14
19913485
Biochem Biophys Res Commun. 1991 Oct 31;180(2):846-52
1719972
J Mol Biol. 2006 Mar 3;356(4):917-27
16405911
Cell Calcium. 1994 Feb;15(2):99-108
7511990
J Biol Chem. 1998 Dec 25;273(52):34813-9
9857007
Proc Natl Acad Sci U S A. 2008 Jul 15;105(28):9610-5
18621707
Cell. 2000 May 12;101(4):365-76
10830164
Circ Res. 2010 Jun 11;106(11):1743-52
20431056
J Biol Chem. 2007 Aug 31;282(35):25929-39
17606610
J Biol Chem. 2009 Feb 13;284(7):4047-51
18927076
J Biol Chem. 1999 May 28;274(22):15315-9
10336416