FRET-Based Trilateration of Probes Bound within Functional Ryanodine Receptors

Abstract

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.

Structural Mapping of Divergent Regions in the Type 1 Ryanodine Receptor Using Fluorescence Resonance Energy Transfer

“…Thus, minimal alterations of the native RyR structure are required for these FRET measurements. At the core of our protocol is the geometrical method termed trilateration to determine the relative location of a site by measuring distances from several well-distributed donor coordinates on FKBP, which we have defined using molecular modeling and simulated annealing (Svensson et al, 2014). These optimizations of donor positions on FKBP, combined with the well-established binding location and orientation of FKBP on RyR1 (Cornea et al, 2010; 2009; Samsó et al, 2006) provided the best available accuracy and precision for these trilaterations.…”

“…Consistency between measurements using different probes indicated that the FRET-derived distances are accurate. This implies that FRET is insignificantly affected by probe orientation, probably because donors and acceptors undergo isotropic dynamics that are fast relative to the donor fluorescence lifetime (several ns) (Svensson et al, 2014). This approach will be expanded in future studies to localize other His-tagged positions in the proximity of RyR-bound D-FKBP donor.…”

“…F-FKBP donor positions were determined computationally using a modification of a previously reported simulated annealing protocol (Svensson et al, 2014; Vrljic et al, 2010) Using Discovery Studio Visualizer (Accelrys, Inc., San Diego, CA), AF488 was attached to each of five cysteines substituted into positions 14, 32, 44, 49, and 85 of the FKBP12 atomic structure docked to RyR1. We then used a simulated annealing protocol (Brünger et al, 1990) starting with a cycle of six torsion-angle dynamics steps at 300,000 K, followed by cooling at a rate of 500 K per cycle and a 0.75 fs integration time.…”

Molecular-Scale GPS: Positioning a Biosensor Peptide on RyR

“…An alternative FRET-based method has been developed that relies on RyR modulator proteins labeled with donor and acceptor probes; binding of these molecules to RyR allows FRET measurements without affecting RyR structure (1,4,5). Two major RyR modulators have been studied, i.e., FK506 binding protein (FKBP), which promotes the closed channel conformation, and calmodulin (CaM), which exerts distinct Ca 2þ -dependent functional effects on the RyR1 and RyR2 channels.…”

“…The work by Svensson et al (1) is an in-depth analysis of binding of DPc10 to RyR2 in ventricular myocytes. The cells were detergent-permeabilized and loaded with FKBP labeled at five distinct sites with Alexa Fluor FRET donors, and DPc10 labeled with a FRET acceptor at its N-terminus.…”