The immunophilin FKBP12 is one of the most abundant and conserved proteins in biology. It is the primary receptor for the immunosuppressant actions of the drug FK506 in whose presence FKBP12 binds to and inhibits calcineurin, disrupting interleukin formation in lymphocytes. The physiologic functions of FKBP12 are less clear, although the protein has been demonstrated to physiologically interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the ryanodine receptor, and the type 1 transforming growth factor beta receptor. We now report that FKBP12 binds the IP3R at residues 1400-1401, a leucyl-prolyl dipeptide epitope that structurally resembles FK506. We further demonstrate that binding to IP3R at this site enables FKBP12 to interact with calcineurin, presumably to anchor the phosphatase to IP3R and modulate the receptor's phosphorylation status. We propose that FK506 promotes an FKBP12-calcineurin interaction by mimicking structurally similar dipeptide epitopes present within proteins that use FKBP12 to anchor calcineurin to the appropriate physiologic substrates.

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolyl isomerase that binds the macrolides FK506 and rapamycin. We have examined the role of the binding pocket residues of FKBP12 in protein-ligand interactions by making conservative substitutions of 12 of these residues by site-directed mutagenesis. For each mutant FKBP12, we measured the affinity for FK506 and rapamycin and the catalytic efficiency in the cis-frans peptidyl-prolyl isomerase reaction. The mutation of Trp59 or Phe99 generates an FKBP12 with a significantly lower affinity for FK506 than wild-type protein. Tyr26 and Tyr82 mutants are enzymatically active, demonstrating that hydrogen bonding by these residues is not required for catalysis of the cis-trans peptidyl-prolyl isomerase reaction, although these mutations alter the substrate specificity of the enzyme. We conclude that hydrophobic interactions in the active site dominate in the stabilization of FKBP12 binding to macrolide ligands and to the twisted-amide peptidyl-prolyl substrate intermediate.

We have identified a novel cDNA encoding a protein (named TX) with > 50

The 12 and 13-kDa FK506 binding proteins (FKBP12 and FKBP13) are cis-trans peptidyl-prolyl isomerases that bind the macrolides FK506 (Tacrolimus) and rapamycin (Sirolimus). The FKBP12 . FK506 complex is immunosuppressive, acting as an inhibitor of the protein phosphatase calcineurin, We have examined the role of the key surface residues of FKBP12 and FKBP13 in calcineurin interactions by generating substitutions at these residues by site-directed mutagenesis. All mutants are active catalysts of the prolyl isomerase reaction, and bind FK506 or rapamycin with high affinity, Mutations at FKBP12 residues Asp-37, Arg-42, His-87, and Ile-90 decrease calcineurin affinity of the mutant FKBP12 FK506 complex by as much as 2600-fold in the case of I90K, Replacement of three FKBP13 surface residues (Gln-50, Ala-95, and Lys-98) with the corresponding homologous FKBP12 residues (Arg-42, His-87, and Ile-90) generates an FKBP13 variant that is equivalent to FKBP12 in its affinity for FK506, rapamycin, and calcineurin, These results confirm the role of two loop regions of FKBP12 (residues 40-44 and 84-91) as part of the effector face that interacts with calcineurin.

The proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) is an early biochemical event, which occurs during apoptosis. A recent study suggested that PARP cleavage can be mediated by a novel cytosolic protease (prICE) that resembles interleukin-1 beta converting enzyme (ICE), but cannot be mediated by ICE itself (Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346-347). We have used a COS cell co-transfection assay to investigate if ICE or any known ICE-like protease is active in PARP cleavage within the cell. Here we report that coexpression of human PARP with human ICE, or the ICE homologs TX and Nedd-2, resulted in a cleavage of PARP identical to that observed in apoptotic cells. Experiments with purified recombinant human ICE indicated that PARE polypeptide can be specifically cleaved in vitro by ICE in a time- and enzyme concentration de pendent manner. PARP cleavage, however, requires a 50-100-fold higher ICE concentration than does processing of the interleukin-1 beta precursor at an equivalent substrate concentration. The abilities of ICE, TX, and Nedd-2, when expressed at high intracellular concentrations, to cleave PARP are consistent with their induction of apoptosis in transfected cells.

Interleukin-1 beta converting enzyme (ICE) processes an inactive precursor to the proinflammatory cytokine, Interleukln-1 beta, and may regulate programmed cell death in neuronal cells. The high-resolution structure of human ICE In complex with an inhibitor has been determined by X-ray diffraction. The structure confirms the relationship between human ICE and cell-death proteins in other organisms. The active site spans both the 10 and 20K subunits, which associate to form a tetramer, suggesting a mechanism for ICE autoactivation.

Parallel measurements of the thermodynamics (free-energy, enthalpy, entropy and heat-capacity changes) of ligand binding to FK506 binding protein (FKBP-12) in H2O and D2O have been performed in an effort to probe the energetic contributions of single protein-ligand hydrogen bonds formed in the binding reactions. Changing tyrosine-82 to phenylalanine in FKBP-12 abolishes protein-ligand hydrogen bond interactions in the FKBP-12 complexes with tacrolimus or rapamycin and leads to a large apparent enthalpic stabilization of binding in both H2O and D2O. High-resolution crystallographic analysis reveals that two water molecules bound to the tyrosine-82 hydroxyl group in unliganded FKBP-12 are displaced upon formation of the protein-ligand complexes. A thermodynamic analysis is presented that suggests that the removal of polar atoms from water contributes a highly unfavorable enthalpy change to the formation of C=O...HO hydrogen bonds as they occur in the processes of protein folding and ligand binding. Despite the less favorable enthalpy change, the entropic advantage of displacing two water molecules upon binding leads to a slightly more favorable free-energy change of binding in the reactions with wild-type FKBP-12.

FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond. An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor. We have determined the kinetic parameters of the human FKBP-catalyzed PPIase reaction. At 5 degrees C, the isomerization of Suc-Ala-Leu-Pro-Phe-pNA proceeds in 2.5% trifluorethanol with kcat = 600 s-1, Km = 0.5 mM and kcat/Km = 1.2 x 10(6) M-1s-1. The kcat/Km shows little pH dependence between 5 and 10. A normal secondary deuterium isotope effect is observed on both kcat and kcat/Km. To investigate dependence on enzyme nucleophiles and proton donors, we have replaced eight potential catalytic residues with alanine by site-directed mutagenesis. Each FKBP variant efficiently catalyzes the PPIase reaction. Taken together, these data support an unassisted conformational twist mechanism with rate enhancement due in part to desolvation of the peptide bond at the active site. Fluorescence quenching of the buried tryptophan 59 residue by peptide substrate suggests that isomerization occurs in a hydrophobic environment.

The mechanism of FK506 immunosuppression has been proposed to proceed by formation of a tight-binding complex with the intracellular 12-kDa FK506-binding protein (FKBP12). The FK506-FKBP12 complex then acts as a specific high-affinity inhibitor of the intracellular protein phosphatase PP2B (calcineurin), interrupting downstream dephosphorylation events required for T-cell activation. Site-directed mutagenesis of many of the surface residues of FKBP12 has no significant effect on its affinity for calcineurin. We have identified, however, three FKBP12 surface residues (Asp-37, Arg-42, and His-87) proximal to a solvent-exposed segment of bound FK506 that may be direct contacts in the calcineurin complex. Site-directed mutagenesis of two of these residues decreases the affinity of FKBP12-FK506 for calcineurin (K(i)) from 6 nm for wild-type FKBP12 to 3.7-mu-M for a R42K/H87V double mutant, without affecting the peptidylprolyl isomerase activity or FK506 affinity of the mutant protein. These FKBP12 mutations along with several substitutions on FK506 known to affect calcineurin binding form a roughly 100-angstrom2 region of the FKBP12-FK506 complex surface that is likely to be within the calcineurin binding site.