Mapping the Ryanodine Receptor FK506-binding Protein Subunit Using Fluorescence Resonance Energy Transfer

Abstract

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. The 2.3-MDa ryanodine receptor (RyR)2 /Ca 2ϩ release channel isoforms expressed in skeletal muscle (RyR1) and cardiac muscle (RyR2) function in complex with smaller regulatory proteins, which include FK506-binding proteins (FKBP12 and FKBP12.6) and calmodulin (CaM) (1, 2). The FKBPs tightly bind to RyR channels and may function to stabilize channels in a fully closed conformational state while minimizing the occurrence of subconductance states (3, 4).Disruption of FKBP binding to RyRs has been proposed to underlie increased channel openings in response to ␤-adrenergic stimulation (5), oxidation/nitrosylation (6 -8), or diseasecausing channel mutations (9 -11), and the FKBP binding interface is now under investigation as a therapeutic target for disordered Ca 2ϩ regulation in cardiac and skeletal muscle. However, the fundamental cellular and molecular mechanisms that govern FKBP binding remain poorly defined, and the significance of reduced FKBP binding in RyR channelopathies is controversial (12-16). Because no atomic structure of an FKBP⅐RyR complex is currently available, uncertainty regarding the specific structural domains that comprise the binding interface remains a significant gap in understanding.Molecular determinants of FKBP binding have been investigated previously through mutagenesis of FKBP12 and FKBP12.6 (17,18). However, the key determinants thus far identified are broadly distributed throughout the FKBP threedimensional structure and do not provide a clear indication of a major RyR binding interface. Cryoelectron microscopy (cryo-EM) and single-particle three-dimensional reconstruction of RyRs in the absence and presence of FKBP12/12.6 have demonstrated that the FKBPs bind within a pocket formed by the intersection of domains 3 and 9 of the RyR1 and RyR2 cytoplasmic assemblies (19,20). More recently, Samsó et al. (21) advanced this approach to higher resolution and were able to dock the atomic structure of FKBP12 into the three-dimensional difference map of FKBP12 in a unique orientation. These results suggested a distinct mode of binding, in which the surface formed by the ␤-sheet and adjacent loops of FKBP12 forms a major binding interface with domain 9 of the RyR1 channel, and the hydrophobic drug-binding pocket of FKBP12 faces RyR1 domain 3.Here, we extend an approach (22) based on site-directed labeling of channel regulatory proteins and fluorescence resonance energy transfer (FRET) to further investigate the structural basis of FKBP binding to RyR channels. We identify structural determinants of both high affinity binding and RyR inhibition. We define the orientation of FKBP12.6 when bound to both the RyR1 and the RyR2 channel isoforms. Ou...

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…Difference mapping of threedimensional reconstructions of RyR with and without FKBP12 or 12.6 places the FKBPs binding site between subdomains 3, 5, and 9 (Wagenknecht et al 1996;Wagenknecht et al 1997;Samsó et al 2006;Sharma et al 2006). In agreement with this localization, FRET studies have localized the FKBP12 (Cornea et al 2009) and the FKBP12.6 (Cornea et al 2010) binding site to the same area as the model from Samsó et al 2006. Furthermore, both FKBP12 and 12.6 bind RyR1 and RyR2 in the same orientation (Cornea et al 2010).…”

“…In agreement with this localization, FRET studies have localized the FKBP12 (Cornea et al 2009) and the FKBP12.6 (Cornea et al 2010) binding site to the same area as the model from Samsó et al 2006. Furthermore, both FKBP12 and 12.6 bind RyR1 and RyR2 in the same orientation (Cornea et al 2010). Comparison of this location with the docking of the IP3 homology model, which includes the suppressor domain and the IP3-binding core region both with high sequence similarity to RyR1 aminoterminus, suggests a binding pocket for FKBP12 formed by Glu161, Arg164, Arg402, and Ile404 (Serysheva et al 2008).…”

N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins

“…Within the RyR1 cryo-EM map, FKBP12.6 binds to a well defined site between the clamp and handle structural subdomains (Fig. 1B) in the same location and orientation as FKBP12, the naturally occurring FKBP isoform in skeletal muscle (15). However, because FKBP12.6 dissociates from RyR1 extremely slowly, and has an almost identical backbone structure as FKBP12, it has been used for FRET-based analysis of RyR structure in previous studies (12,15,27,28) as well as in the current study.…”

“…Synthesis and Purification of FRET Donors-Fluorescent derivatives of FKBP12.6 were prepared as previously described (15). A maleimide derivative of Alexa Fluor 488 (AF488; Invitrogen) was used to label single cysteines substituted at positions 14,32,44,49, and 85 into a null-cysteine variant of human FKBP12.6.…”

“…Using cryo-electron microscopic (EM) single particle analysis, bound FKBP12 has been localized to a position between the "clamp" and "handle" domains of the large cytoplasmic "foot" structure of RyR1 (13,14). In addition, FRET measurements indicate that the orientations of bound FKBP12 and FKBP12.6 on either RyR1 or RyR2 are similar (15), thus suggesting that the FKBP binding sites on each RyR isoform are in similar locations. However, despite this detailed understanding of the structural nature of the RyR1 FKBP binding site, the location of this site within the RyR1 protein sequence is controversial.…”