Three-dimensional visualization of FKBP12.6 binding to an open conformation of cardiac ryanodine receptor

Abstract

The cardiac isoform of the ryanodine receptor (RyR2) from dog binds predominantly a 12.6-kDa isoform of the FK506-binding protein (FKBP12.6), whereas RyR2 from other species binds both FKBP12.6 and the closely related isoform FKBP12. The role played by FKBP12.6 in modulating calcium release by RyR2 is unclear at present. We have used cryoelectron microscopy and three-dimensional (3D) reconstruction techniques to determine the binding position of FKBP12.6 on the surface of canine RyR2. Buffer conditions that should favor the "open" state of RyR2 were used. Quantitative comparison of 3D reconstructions of RyR2 in the presence and absence of FKBP12.6 reveals that FKBP12.6 binds along the sides of the square-shaped cytoplasmic region of the receptor, adjacent to domain 9, which forms part of the four clamp (corner-forming) structures. The location of the FKBP12.6 binding site on "open" RyR2 appears similar, but slightly displaced (by 1-2 nm) from that found previously for FKBP12 binding to the skeletal muscle ryanodine receptor that was in the buffer that favors the "closed" state. The conformation of RyR2 containing bound FKBP12.6 differs considerably from that depleted of FKBP12.6, particularly in the transmembrane region and in the clamp structures. The x-ray structure of FKBP12.6 was docked into the region of the 3D reconstruction that is attributable to bound FKBP12.6, to show the relative orientations of amino acid residues (Gln-31, Asn-32, Phe-59) that have been implicated as being critical in interactions with RyR2. A thorough understanding of the structural basis of RyR2-FKBP12.6 interaction should aid in understanding the roles that have been proposed for FKBP12.6 in heart failure and in certain forms of sudden cardiac death.

FIGURE 1

Surface representations of 3D cryo-EM reconstructions of RyR2 with and without bound FKBP12.6. (A) The 3D map of RyR2 (+FKBP), obtained by assembly in vitro of purified RyR2 incubated with FKBP12.6 alone, and (B) the 3D map of RyR2 (−FKBP), obtained by incubating RyR2 with FKBP12.6 and excess FK506, with the superimposed map of difference density (shown in blue), as obtained by subtracting the 3D map of RyR2 (−FKBP) from that of RyR2 (+FKBP). In panel A, arrows point to the extra density in RyR2 (+FKBP12.6) that accounts for the FKBP12.6 mass that is enhanced in the difference map shown in panel B. The 3D volumes for the two conditions are each shown in three views: left, cytoplasmic view; middle, SR junctional face; and right, side view. Abbreviation: TA, transmembrane assembly. Numerals refer to established nomenclature of domains. Scale bar ∼100 Å.

FIGURE 2

3D maps of RyR2 (+FKBP) and RyR2 (−FKBP) shown at two different contour levels with a previously determined structure of RyR3 (59). (A) Two superimposed volumes of RyR2 (+FKBP12.6) are shown, one in semitransparent pink and the other as solid pink. For comparison, the “open” RyR3 structure is shown as a thumbnail to the lower left. (B) Similarly, as in panel A, two volumes of RyR2 (−FKBP12.6) are superimposed, one shown in solid red and the other as semitransparent red. The comparative structure of RyR3 in the “closed” state is shown as a thumbnail to the lower right. For both panels A and B, the semitransparent structures are displayed at the same threshold as in Fig. 1, whereas the solid densities of both complexes are shown at a higher threshold such that their total volume is ∼1/4 of the semitransparent map. Note the similar conformations of the domains “6” in (RyR2 -FKBP12.6) complexes and changes in transmembrane assembly that correlate to RyR3 volumes, respectively. Scale bar ∼100 Å.

FIGURE 3

Manual docking of FKBP12.6 atomic structure (68) into the 3D density difference map. To the left, a reconstruction of the RyR2 (−FKBP) complex is shown with the difference density map (blue), attributed to FKBP12.6, superimposed (blue; also see Fig. 1). To the right is an enlargement of one of the four FKBP12.6 difference masses with a ribbon representation of the FKBP12.6 atomic structure (68) docked inside it. To obtain an optimal fitting, the difference map density is displayed at a slightly lower threshold as that shown in Fig. 1 B, keeping the maximum limit of the difference density close to the known molecular mass of FKBP12.6. Side chains of residues of interest (Ala-63, Phe-59, Ile-90, Gln-31, and Asn-32) are shown in dark blue (see text).