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

Mustafi SM, LeMaster DM, Hernández G - Biochem. J. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: *Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201, U.S.A.

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.

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Superimposition of the FK1 domains of FKBP51 and FKBP52The 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.
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Figure 4: Superimposition of the FK1 domains of FKBP51 and FKBP52The 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.

Mentions: The 15N relaxation data for the FK1 domain of FKBP51 (Figure 3) markedly differ from that observed for the FKBP52 domain (Figure 1). Strongly elevated R2 values are observed for residues in the β3 bulge and for many of the residues throughout the long β4–β5 loop (Figure 4). These elevated R2 values were closely similar for the 0.5 mM and 1 mM samples, indicating that they did not arise from the dynamics of weak aggregation interactions. The magnetic-field-dependence of these elevated R2 values indicates substantial line-broadening arising from motion in the sub-millisecond timeframe. The three residues of the β3 bulge exhibiting the largest line-broadening effects (Ser70, Arg73 and Glu75) have R2 values that are closely similar to those observed in FKBP12 [35]. On the other hand, additional smaller conformational line-broadening effects are observed for residues within the β3a strand (Figure 3) as well as cross-strand interactions with the amides of Tyr57 and Gly59 in the β2 strand which hydrogen-bond in the X-ray structure with the side-chain Oγ of Ser70 and the carbonyl oxygen of Asp68 respectively. In analysing the magnetic-field-dependence of the 15N R2 relaxation values for the residues of the β4–β5 loop (Supplementary Figure S3 at http://www.biochemj.org/bj/461/bj4610115add.htm) as well as for those in the β3a strand and β3 bulge, the increase in conformational line-broadening is approximately proportional to the square of the magnetic field. This implies that the conformational transition rate(s) is substantially higher than the strength of the spinlock field used in the R1ρ experiments (1245 Hz at 600 MHz and 1085 Hz at 900 MHz), approaching the fast exchange limit as reported previously for FKBP12 [55–57]. The magnitude of conformational line-broadening depends upon the relative population and rate of interchange between the conformer states as well as on the differential 15N chemical shifts for these states. Near the fast exchange limit, the relative magnitude of these three contributions cannot be reliably deconvoluted.


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

Mustafi SM, LeMaster DM, Hernández G - Biochem. J. (2014)

Superimposition of the FK1 domains of FKBP51 and FKBP52The 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4060953&req=5

Figure 4: Superimposition of the FK1 domains of FKBP51 and FKBP52The 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.
Mentions: The 15N relaxation data for the FK1 domain of FKBP51 (Figure 3) markedly differ from that observed for the FKBP52 domain (Figure 1). Strongly elevated R2 values are observed for residues in the β3 bulge and for many of the residues throughout the long β4–β5 loop (Figure 4). These elevated R2 values were closely similar for the 0.5 mM and 1 mM samples, indicating that they did not arise from the dynamics of weak aggregation interactions. The magnetic-field-dependence of these elevated R2 values indicates substantial line-broadening arising from motion in the sub-millisecond timeframe. The three residues of the β3 bulge exhibiting the largest line-broadening effects (Ser70, Arg73 and Glu75) have R2 values that are closely similar to those observed in FKBP12 [35]. On the other hand, additional smaller conformational line-broadening effects are observed for residues within the β3a strand (Figure 3) as well as cross-strand interactions with the amides of Tyr57 and Gly59 in the β2 strand which hydrogen-bond in the X-ray structure with the side-chain Oγ of Ser70 and the carbonyl oxygen of Asp68 respectively. In analysing the magnetic-field-dependence of the 15N R2 relaxation values for the residues of the β4–β5 loop (Supplementary Figure S3 at http://www.biochemj.org/bj/461/bj4610115add.htm) as well as for those in the β3a strand and β3 bulge, the increase in conformational line-broadening is approximately proportional to the square of the magnetic field. This implies that the conformational transition rate(s) is substantially higher than the strength of the spinlock field used in the R1ρ experiments (1245 Hz at 600 MHz and 1085 Hz at 900 MHz), approaching the fast exchange limit as reported previously for FKBP12 [55–57]. The magnitude of conformational line-broadening depends upon the relative population and rate of interchange between the conformer states as well as on the differential 15N chemical shifts for these states. Near the fast exchange limit, the relative magnitude of these three contributions cannot be reliably deconvoluted.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: *Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201, U.S.A.

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.

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