Impact of methionine oxidation on calmodulin structural dynamics.

We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron-electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous X-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM's structural heterogeneity in solution: in both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ∼4nm (closed) and another at ∼6nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each population ranging from 1 to 3nm. Both mutations (M109Q and M124Q) decrease the effect of Ca on the structure of CaM, primarily by decreasing the closed-to-open equilibrium constant in the presence of Ca. We propose that Met oxidation alters CaM's functional interaction with its target proteins by perturbing this Ca-dependent structural shift.

FRET-based trilateration of probes bound within functional ryanodine receptors.

To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca(2+) channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460-2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca(2+) leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ?35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.

N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.

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.

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.