Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6

Abstract

The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca(2+) channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution from P2₁ and P3₁21 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3₁21 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition `80s loop' is flipped in the P2₁ crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

Keywords: FK506-binding proteins; FKBP12.6.

Figure 1

Superposition of the region surrounding the aromatic ring of Phe59 from the two non-equivalent monomers in the 1.90 Å resolution structure of the P3₁21 crystal form of the cysteine-free variant of FKBP12.6. The C atoms of the molecule A structure are shown in yellow, while those of molecule B are indicated in green. In (a), the electron-density grid (2mF _o − DF _c at a contour level of 0.0114 e Å⁻³ = 1σ) for molecule A is also illustrated, indicating that the aromatic ring of Phe59 from this molecule is oriented perpendicular to that from molecule B. In the latter case, the plane of the ring forms the base of the active-site cleft, as seen in previously reported crystal structures of FKBP proteins. Among the residues lying beneath the ring of Phe59 (b), only Val101 C^γ2 lies within van der Waals contact with either the C^δ or C^∊ atoms of the Phe59 ring for the two orientations in the P3₁21 crystal form. The side chains of Ala63 and Leu74 C^δ are approximately 1 Å beyond van der Waals contact.

Figure 2

Backbone conformation in the active site of wild-type and E60Q FKBP12 and of the crystallographically non-equivalent monomers of FKBP12.6 in the P3₁21 crystal form. (a) shows the shift in backbone conformation and the reorientation of the Trp59 side chain that results from the altered hydrogen-bonding interactions of the side chain of residue 60 (Szep et al., 2009 ▶). A similar ∼90° rotation of the side-chain χ₂ dihedral angle for Phe59 occurs between the two non-equivalent monomers in the P3₁21 crystal form of the cysteine-free variant of FKBP12.6, but without any corresponding alteration in the conformation of the backbone (b).

Figure 3

Superposition of the region surrounding the Gly89–Val90 peptide bond from the two crystal forms of FKBP12.6 and comparison with the corresponding segment of the first FKBP domain of FKBP51. The C atoms of the P2₁ crystal form structure of cysteine-free FKBP12.6 are colored green, while those of the P3₁21 crystal form structure are shown in yellow (a). The peptide linkage between Gly89 and Val90 is flipped in the P2₁ crystal form compared with the P3₁21 crystal form as well as in comparison to either the rapamycin-inhibited FKBP12.6 structure (Deivanayagam et al., 2000 ▶) or the high-resolution apo FKBP12 structure (Szep et al., 2009 ▶). In (b), this segment of the P2₁ crystal form structure of cysteine-free FKBP12.6 is superimposed upon the homologous segment from the 1.00 Å resolution structure of the first FKBP domain of FKBP51 (PDB entry 3o5p; Bracher et al., 2011 ▶), which is illustrated in blue.

Figure 4

¹H–¹⁵N two-dimensional NMR correlation spectrum of U-²H,¹⁵N-enriched wild-type FKBP12.6. Residues exhibiting resolved resonances for the minor slow-exchange conformation are indicated in red. Resonances were not observed in this spectrum for Ala84 and for Gly89 in the major slow-exchange state presumably owing to severe line-broadening arising from both rapid amide hydrogen exchange (Hernández et al., 2009 ▶) and conformational exchange dynamics in the 80s loop (Mustafi et al., 2013 ▶) as observed in the homologous FKBP12. The Gly89 resonance for the minor exchange state is observable at an approximately twofold lower contour level. The assessment of resonance doubling for Val80 is impeded by overlap with a side-chain amide resonance. Folded side-chain resonances are indicated with ‘x’.

Figure 5

Structural distribution of residues exhibiting amide-resonance doubling owing to slow conformational exchange in FKBP12.6. Residues that yield doublings of their amide resonances separated by more than 0.15 p.p.m. (averaged as Δ¹H and 0.2Δ¹⁵N; Garrett et al., 1997 ▶) are indicated in red. Residues exhibiting smaller chemical shift differences between the two conformational states are indicated in pink. Residues that exhibit doubling in FKBP12 (Mustafi et al., 2013 ▶) but not in FKBP12.6 are indicated in blue. Prolines are marked in black.

Figure 6

Kinetics of the slow conformational exchange in the C22V+C76I variant of FKBP12.6 at 43°C. (a) zz-exchange diagonal and cross-peaks of Asp37 in the major and minor conformational states at a mixing time of 1.1 s. (b) Time course for the peak intensities for the AA and BB diagonal peaks and the AB cross-peak.