Differential conformational dynamics in the closely homologous FK506-binding domains of FKBP51 and FKBP52

Abstract

As co-chaperones of Hsp90 (heat-shock protein 90), FKBP51 (FK506-binding protein of 51 kDa) and FKBP52 (FK506-binding protein of 52 kDa) act as antagonists in regulating the hormone affinity and nuclear transport of steroid receptor complexes. Exchange of Leu119 in FKBP51 for Pro119 in FKBP52 has been shown to largely reverse the steroid receptor activities of FKBP51 and FKBP52. To examine whether differences in conformational dynamics/plasticity might correlate with changes in the reported receptor activities, 15N-NMR relaxation measurements were carried out on the N-terminal FKBP domains of FKBP51 and FKBP52 as well as their residue-swapped variants. Both proteins exhibit a similar pattern of motion in the picosecond-nanosecond timeframe as well as a small degree of 15N line-broadening, indicative of motion in the microsecond-millisecond timeframe, in the β3a strand of the central sheet. Only the FKBP51 domain exhibits much larger line-broadening in the adjacent β3 bulge (40's loop of FKBP12) and throughout the long β4-β5 loop (80's loop of FKBP12). The L119P mutation at the tip of the β4-β5 loop completely suppressed the line-broadening in this loop while partially suppressing the line-broadening in the neighbouring β2 and β3a strands. The complementary P119L and P119L/P124S variants of FKBP52 yielded similar patterns of line-broadening for the β4-β5 loop as that for FKBP51, although only 20% and 60% as intense respectively. However, despite the close structural similarity in the packing interactions between the β4-β5 loop and the β3a strand for FKBP51 and FKBP52, the line-broadening in the β3a strand is unaffected by the P119L or P119L/P124S mutations in FKBP52.

Figure 1. ¹⁵N relaxation measurements for the backbone amide resonances in the FK1 domain of FKBP52 at 25°C

The longitudinal (R₁) and transverse (R₂) relaxation rates at 600 MHz ¹H are shown with the transverse relaxation rates at 900 MHz ¹H also indicated in red. The heteronuclear NOE and model-free [48,49] order parameters (S²) are also illustrated along with the conformational exchange line-broadening R_ex values for residues in the β_3a strand. In addition to proline residues, relaxation data are not reported for overlapped resonances and for the severely broadened resonance of Ser¹¹⁵.

Figure 2. Structural distribution for residues of the FK1 domain of FKBP52 that exhibit conformational dynamics in either the picosecond–nanosecond or microsecond–millisecond timeframe

(A) Main-chain conformational schematic diagram of the FK1 domain as viewed from the back side of the β-sheet. Discounting the termini, residues that exhibit order parameter values of S²<0.78 are indicated in red, whereas those exhibiting conformational exchange broadening above 0.5 s⁻¹ at 600 MHz ¹H and above 1.0 s⁻¹ at 900 MHz ¹H are indicated in green. (B) Sequence alignment of the FK1 domains of FKBP52 and FKBP51 with FKBP12. Residue 119 at the tip of the β₄–β₅ loop is highlighted.

Figure 3. ¹⁵N relaxation measurements for the backbone amide resonances in the FK1 domain of FKBP51 at 25°C

The longitudinal (R₁) and transverse (R₂) relaxation rates at 600 MHz ¹H are shown with the transverse relaxation rates at 900 MHz ¹H also indicated in red. The R₂ values at 900 MHz for Arg⁷³ and Leu¹¹⁹ are illustrated in pink, indicating rates that are significantly above 50 s⁻¹ for which the attenuated resonances could not be reliably quantified. The heteronuclear NOE and model-free [48,49] order parameters (S²) are also illustrated along with the conformational exchange line-broadening R_ex values for residues extending from within the β₂ strand to the end of the β₃ bulge. R_ex values for Ser⁷⁰, Arg⁷³ and Glu⁷⁵ are truncated to better illustrate the smaller line-broadening effects in the β₂ and β_3a strands. In addition to proline residues, relaxation data are not reported for overlapped resonances and for the severely broadened resonances of Tyr¹¹³ and Ser¹¹⁵.

Figure 4. Superimposition of the FK1 domains of FKBP51 and FKBP52

The FKBP51 X-ray structure from PDB code 3O5P [28] is illustrated in yellow, whereas molecule A from PDB code 4LAV [33] for FKBP52 is shown in grey. All heavy atoms are illustrated for the β₄–β₅ loop extending from Glu¹¹⁰ to Leu¹²⁸. Substantial deviations in backbone geometry are only apparent for the β₃ bulge (Ser⁷⁰–Lys⁷⁶) and the tip of the β₄–β₅ loop.

Figure 5. ¹⁵N transverse relaxation measurements for the L119P variant of FKBP51 at 25°C

The transverse (R₂) relaxation rates at 600 MHz ¹H are shown with the transverse relaxation rates at 900 MHz ¹H also indicated in red.

Figure 6. Differential ¹⁵N transverse relaxation measurements for the wild-type and L119P variant of FKBP51 at 25°C

The differential transverse relaxation rates at 600 MHz ¹H are shown in (A), whereas those for 900 MHz ¹H are shown in (B). The data for the two fields are plotted on the same vertical scale to illustrate the approximate 2.25-fold increase for the 900 MHz data indicative of conformational transitions occurring near the fast exchange time limit. As a result, the ΔR₂ values for residues 117, 119 and 122 at 900 MHz are truncated. At each field, the median R₂ values for the two datasets are scaled to correct for small variations in the global molecular correlation times. Outside the regions exhibiting significant differential line-broadening (i.e. residues 57–77 and 108–128), the RMSD for the ΔR₂ values were 0.15 and 0.18 s⁻¹ for 600 MHz and 900 MHz respectively, corresponding to 1.5% of the median R₂ values in each case. The ΔR₂ value for Leu¹¹⁹ in the wild-type protein is given relative to the median R₂ value and at 900 MHz this ΔR₂ value is too large for reliable quantification (grey). Owing to decreased statistical reliability for the more severely attenuated resonances, the residues in which the R₂ value is >18 s⁻¹ for both wild-type and the L119P variant were excluded (Ser⁷⁰, Arg⁷³ and Glu⁷⁵).

Figure 7. Structural distribution of residues in the β₂ and β_3a strands of FKBP51 that exhibit reductions in R₂ values resulting from the L119P substitution

Residues for which the ¹⁵N R₂ value decreases by more than 0.5 s⁻¹ at 900 MHz ¹H are coloured yellow. There are no other differences in R₂ greater than 0.5 s⁻¹ outside the β₄–β₅ loop. A kink in the β_3a strand occurs at Phe⁶⁷ and Asp⁶⁸ where the amide hydrogen of Asp⁶⁸ is slightly too far from the carbonyl oxygen of Gly⁵⁹ to form a canonical antiparallel β-sheet hydrogen-bonding interaction. This kink occurs at the site of direct contact with the tip of the β₄–β₅ loop as indicated by Lys¹²¹.

Figure 8. Differential ¹⁵N transverse relaxation measurements for the P119L and P119L/P124S variants of FKBP52 compared with the wild-type protein

Relative to wild-type FKBP52, the differential transverse relaxation rates at 600 MHz (black) and higher field (red) are shown for P119L (A) and P119L/P124S (B). At each field, the median R₂ values for the two datasets are scaled to correct for small variations in the global molecular correlation times. The higher field data for the two variants of FKBP52, collected at 800 MHz, were scaled to the wild-type FKBP52 data at 900 MHz under the assumption that the conformational exchange occurs in the fast limit regime. Outside the region exhibiting significant differential line-broadening (i.e. residues 110–125), the RMSD for the ΔR₂ values were 0.13 and 0.18 s⁻¹ for 600 MHz and higher field data for the P119L/P124S variant comparison respectively. The ΔR₂ values for Leu¹¹⁹ and Ser¹²⁴ are given relative to the median R₂ value.