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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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Structural distribution of residues in the β2 and β3a strands of FKBP51 that exhibit reductions in R2 values resulting from the L119P substitutionResidues 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 β3a 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.
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Figure 7: Structural distribution of residues in the β2 and β3a strands of FKBP51 that exhibit reductions in R2 values resulting from the L119P substitutionResidues 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 β3a 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.

Mentions: The 2D 1H,15N-HSQC spectrum for the FK1 domain of FKBP51 is largely unperturbed when the L119P mutation was introduced (Supplementary Figure S2). Despite this evidence for similarity in tertiary structure, the conformational exchange line-broadening in the β4–β5 loop was completely suppressed in the L119P variant (Figure 5). The pattern of line-broadening in the β3a strand and β3 bulge is quite similar to that observed for the wild-type protein, although the magnitude of that line-broadening appears to be somewhat diminished. To analyse the line-broadening in the wild-type and L119P variant FK1 domain more directly, the differences in these R2 values were considered for both the 600 MHz and 900 MHz datasets (Figure 6). In addition to the large changes in conformational exchange line-broadening in the β4–β5 loop, introduction of the L119P mutation also partially suppresses the line-broadening in the β2 and β3a strands. The largest effects in these strands occur near the highly conserved Leu61 and Phe67 whose side chains pack tightly against the similarly highly conserved Ile122 and Pro123 side chains at the tip of the β4–β5 loop. These L119P-induced changes in conformational exchange line-broadening extend along the β2 and β3a strands and possibly into the β3 bulge (Figure 7). As a potential component of the conformational transition that underlies this line-broadening behaviour, it may be noted that the canonical antiparallel hydrogen-bonding pattern between the β2 and β3a strands is disrupted by the amides of both Phe67 and Asp68 being oriented towards the carbonyl oxygen of Gly59 with the Asp68 amide being just beyond hydrogen-bonding distance (Figure 7).


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

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

Structural distribution of residues in the β2 and β3a strands of FKBP51 that exhibit reductions in R2 values resulting from the L119P substitutionResidues 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 β3a 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Structural distribution of residues in the β2 and β3a strands of FKBP51 that exhibit reductions in R2 values resulting from the L119P substitutionResidues 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 β3a 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.
Mentions: The 2D 1H,15N-HSQC spectrum for the FK1 domain of FKBP51 is largely unperturbed when the L119P mutation was introduced (Supplementary Figure S2). Despite this evidence for similarity in tertiary structure, the conformational exchange line-broadening in the β4–β5 loop was completely suppressed in the L119P variant (Figure 5). The pattern of line-broadening in the β3a strand and β3 bulge is quite similar to that observed for the wild-type protein, although the magnitude of that line-broadening appears to be somewhat diminished. To analyse the line-broadening in the wild-type and L119P variant FK1 domain more directly, the differences in these R2 values were considered for both the 600 MHz and 900 MHz datasets (Figure 6). In addition to the large changes in conformational exchange line-broadening in the β4–β5 loop, introduction of the L119P mutation also partially suppresses the line-broadening in the β2 and β3a strands. The largest effects in these strands occur near the highly conserved Leu61 and Phe67 whose side chains pack tightly against the similarly highly conserved Ile122 and Pro123 side chains at the tip of the β4–β5 loop. These L119P-induced changes in conformational exchange line-broadening extend along the β2 and β3a strands and possibly into the β3 bulge (Figure 7). As a potential component of the conformational transition that underlies this line-broadening behaviour, it may be noted that the canonical antiparallel hydrogen-bonding pattern between the β2 and β3a strands is disrupted by the amides of both Phe67 and Asp68 being oriented towards the carbonyl oxygen of Gly59 with the Asp68 amide being just beyond hydrogen-bonding distance (Figure 7).

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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