Structure comparison of native and mutant human recombinant FKBP12 complexes with the immunosuppressant drug FK506
Abstract:
The consequences of site-directed mutagenesis experiments are often anticipated by empirical rules regarding the expected effects of a given amino acid substitution. Here, we examine the effects of "conservative" and "nonconservative" substitutions on the X-ray crystal structures of human recombinant FKBP12 mutants in complex with the immunosuppressant drug FK506 (tacrolimus). R42K and R42I mutant complexes show 110-fold and 180-fold decreased calcineurin (CN) inhibition, respectively, versus the native complex, yet retain full peptidyl prolyl isomerase (PPIase) activity, FK506 binding, and FK506-mediated PPIase inhibition. Interestingly, the structure of the R42I mutant complex is better conserved than that of the R42K mutant complex when compared to the native complex structure, within both the FKBP12 protein and FK506 ligand regions of the complexes, and with respect to temperature factors and RMS coordinate differences. This is due to compensatory interactions mediated by two newly ordered water molecules in the R42I complex structure, molecules that act as surrogates for the missing arginine guanidino nitrogens of R42. The absence of such surrogate solvent interactions in the R42K complex leads to some disorder in the so-called "40s loop" that encompasses the substituent. One rationalization proposed for the observed loss in CN inhibition in these R42 mutant complexes invokes indirect effects leading to a misorientation of FKBP12 and FK506 structural elements that normally interact with calcineurin. Our results with the structure of the R42I complex in particular suggest that the observed loss of CN inhibition might also be explained by the loss of a specific R42-mediated interaction with CN that cannot be mimicked effectively by the solvent molecules that otherwise stabilize the conformation of the 40s loop in that structure.
Insights
Site-directed mutagenesis of FKBP12 revealed that specific arginine substitutions (R42K, R42I) significantly reduce calcineurin inhibition while maintaining FK506 binding and PPIase activity. Structural analysis highlights compensatory water molecules in R42I mutants, explaining preserved structure despite reduced drug efficacy.
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- Site-directed mutagenesis is crucial for understanding protein function, often guided by empirical rules for amino acid substitutions.
- FKBP12 is a key protein interacting with immunosuppressants like FK506 (tacrolimus) to inhibit calcineurin (CN).
Purpose of the Study:
- To investigate the structural and functional consequences of conservative and nonconservative amino acid substitutions in FKBP12 mutants.
- To elucidate the molecular mechanisms behind altered calcineurin inhibition in FKBP12 mutants complexed with FK506.
Main Methods:
- X-ray crystallography was used to determine the structures of human recombinant FKBP12 mutants (R42K, R42I) complexed with FK506.
- Enzyme activity assays were performed to assess peptidyl prolyl isomerase (PPIase) activity and FK506 binding.
- Calcineurin (CN) inhibition assays were conducted to quantify the functional impact of the mutations.
Main Results:
- R42K and R42I FKBP12 mutants exhibited significantly reduced calcineurin inhibition (110-fold and 180-fold, respectively) compared to the native complex.
- These mutants retained full FK506 binding and FK506-mediated PPIase inhibition.
- The R42I mutant complex structure showed better conservation than R42K, attributed to ordered water molecules compensating for the loss of arginine interactions.
Conclusions:
- Amino acid substitutions in FKBP12 can drastically alter calcineurin inhibition while preserving drug binding and PPIase activity.
- Structural plasticity, including the role of ordered water molecules, plays a critical role in maintaining complex stability after mutation.
- The loss of a specific R42-mediated interaction with calcineurin, rather than just indirect effects, likely explains the reduced CN inhibition in R42 mutants.
