©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
FK506 Binding Protein Mutational Analysis
DEFINING THE SURFACE RESIDUE CONTRIBUTIONS TO STABILITY OF THE CALCINEURIN CO-COMPLEX (*)

(Received for publication, November 15, 1994; and in revised form, May 16, 1995)

Olga Futer Maureen T. DeCenzo Robert A. Aldape David J. Livingston (§)

From theFrom Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4211

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 FKBP12bulletFK506 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 FKBP12bulletFK506 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.


INTRODUCTION

The immunophilins are the intracellular high affinity receptors for the potent immunosuppressive agents cyclosporin, FK506, and rapamycin. The structure and function of the immunophilins have been investigated intensely since these proteins were first identified in thymus and T-cell extracts(1, 2, 3) . The immunophilins FKBP12 (^1)and FKBP13 are members of the multigene FK506-binding protein family, encoded by distinct genes (4, 5, 6) and performing distinct cellular functions. FKBP12 and FKBP13 are homologous proteins of molecular mass 12 and 13 kDa respectively, with 43% amino acid sequence identity and 51% nucleotide sequence identity (Fig.1)(7) . Upon binding of FK506 to FKBP12 in the T-cell, cytosolic signaling events are interrupted, resulting in inhibition of interleukin-2 expression during T-cell activation(8) . Inhibition of this signal transduction pathway is mediated through binding of the FKBP12bulletFK506 complex to the protein calcineurin (K = 6 nM)(9) , a Ca-activated, calmodulin-dependent serine/threonine phosphatase(10) . Human FKBP13 is a membrane-associated protein that contains a 21-residue N-terminal signal peptide(7) . Gene expression and immunolocalization studies of FKBP13 indicate that this protein may act as an endoplasmic reticulum chaperone and that its expression is induced by cellular stress such as heat shock(11, 12) . Its FK506 complex does not inhibit calcineurin, as demonstrated herein.


Figure 1: Primary sequences of FKBP12 and FKBP13. Crystallographic sequence alignment (modified from (9) and (13) ). Mutations introduced into FKBP12 are listed above the human sequence (hFKBP12). Numbering for human FKBP13 residues (hFKBP13) refers to the mature protein N terminus. Other sequences shown are bovine (bFKBP12), murine (mFKBP12), Saccharomyces cerevisiae (ScFKBP12), Neurospora crassa (NcFKBP12). The hydrophobic pocket forming residues of FKBP12 are Tyr-26, Phe-46, Val-55, Ile-56, Trp-59, His-87, Ile-91, and Phe-99(15, 16, 34) . Trp-59 indole serves as platform for the FK506 pipecolinyl ring, and His-87 forms a surface complementary to the FK506 C-10-C-14 pyranose methyl region. Asp-37 forms a hydrogen bond with the FK506 C-10 hemiketal hydroxyl.



The FKBPs share the common activity of catalyzing the cis-trans isomerization of proline amide bonds of polypeptide substrates (the peptidyl-prolyl isomerase reaction), but beyond this, their biochemical properties diverge. Different substrate specificities are observed for catalysis of the peptidyl-prolyl isomerase reaction as well as in binding affinity for FK506 and rapamycin(13) . High resolution three-dimensional structures have been determined of FKBP12(14, 15) , FKBP12 in complex with FK506 and rapamycin(15, 16) , and the FKBP13bulletFK506 complex(17) . A network of hydrophobic interactions and hydrogen bonds from macrolide ligand to protein side chain and backbone heteroatoms are known to stabilize these complexes.

A ternary structure at atomic resolution of the FKBP12bullet FK506bulletcalcineurin complex is not yet available. However, by studying the effects of mutations on the affinity of FKBP12 for calcineurin, we reported the first direct evidence that the calcineurin-inhibitory binding surface of the FKBP12bulletFK506 complex must include the Arg-42, His-87(9) , and Ile-90 (^2)surface residues of the protein itself, a finding later confirmed by other groups(18, 19) . Yang et al.(18) raised questions about the role of Arg-42 in calcineurin binding versus its role in stabilization of local structure. In this investigation, we have further explored the effects of FKBP12 surface mutations and we have examined the role of the homologous surface residues of FKBP13 in calcineurin inhibition. Interpretation of these data was aided by the availability of the high resolution structures of several of these mutant FKBPs solved by high field NMR (20) and x-ray crystallography (^3)(21) . We report here the separable effects of these FKBP surface mutations on binding interactions with peptidyl-prolyl isomerase substrates, macrolides, and calcineurin.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes and polynucleotide kinase were from New England Biolabs. Sequenase® and other sequencing reagents were from U. S. Biochemical Corp. Phagemid, helper phage, the Muta-Gene kit, and protein assay reagents were from Bio-Rad. Alkaline phosphatase was from Boehringer Mannheim; Gene-Clean kits were from Bio-101; and Sep-Pak cartridges were from Millipore Corp. Peptide substrates for peptidyl-prolyl isomerase assay were from Bachem Biosciences (Philadelphia, PA), and the calcineurin peptide substrate was from Penninsula Labs. Glutathione-Sepharose 4B was purchased from Pharmacia Biotech Inc. FK506 and rapamycin were kindly provided by Chugai Pharmaceuticals.

Bacteria and Plasmids

For site-directed mutagenesis, the dutungEscherichia coli strain CJ236 was used for uracil enrichment of single strand DNA, and the Nova Blue strain (NovaGene) was used for selection of heteroduplex DNA after extension-ligation reactions. The CAG626 host strain, obtained from C. Gross (University of Wisconsin, Madison, WI) was used for expression of FKBP12 mutants. The XA90 strain was used as a host for expression of recombinant glutathione S-transferase (GST) FKBP13 fusion proteins.

A pKEN2 phagemid (G. Verdine, Harvard University, Cambridge, MA) was used for site-directed mutagenesis. FKBP12 proteins were expressed in this vector as described previously(22) . The pGEX-2T phagemid (Pharmacia) was used for expression of fusion GST-FKBP13 proteins. This vector is a derivative of pGEX-1 vector, which includes an isopropyl-1-thio-beta-D-galactopyranoside-inducible tac promoter, complete coding sequence of glutathione S-transferase followed by oligonucleotides encoding the cleavage-recognition sequence of thrombin, and a polylinker containing unique recognition sites for BamHI, SmaI, and EcoRI. The phagemid also contains an ampgene and a lacI allele of the lac repressor(23) .

Site-directed Mutagenesis

A 362-base pair BamHI fragment derived from a cDNA encoding mature human FKBP13 and a 431-base pair EcoRI fragment encoding human FKBP12 cDNA were ligated into phagemid pKEN2. All oligonucleotide-directed mutagenesis was performed on the pKEN2bulletFKBP12 and pKEN2bulletFKBP13 constructs using uracil-enrichment of single strand DNA by the modification of Kunkel (24, 25) of the method originally described for M13 mutagenesis(26, 27) . Two-primer mutagenesis was performed to construct the double mutants. All mutants were sequenced by dideoxy method (28) from the promoter through the region surrounding the mutagenesis site, and most were sequenced in their entirety in the coding region.

Expression of Wild-type and Mutant Proteins

E. coli strain CAG626 (a lon strain on a SC122 background) was used as host for expression of FKBP12 mutants in the pKEN2 expression vector(22) . For expression of FKBP13, a cDNA encoding the 13-kDa mature form was used for mutagenesis and expression. cDNA inserts carrying a confirmed mutation were cut out of the pKEN2 vector by BamHI restriction digest, purified from the agarose gel using Gene-Clean kit, and subcloned into BamHI site of the pGEX-2T vector. The 5` ends of the BamHI-cut vector were dephosphorylated with calf intestine alkaline phosphatase. Orientation of the insert was determined by restriction digest with PstI. Positive clones were sequenced in their entirety. The GST-fusion expression system allowed us to obtain expression levels of 3-12 mg of the GST-FKBP13 chimera/g of cell paste.

Mutant duplex DNA was transformed into XA90 cells for expression and plated on LB plates containing 100 µg/ml ampicillin. Five-ml overnight cultures were started from multiple colonies and grown at 37 °C on LB-ampicillin medium. For expression, cultures were grown at 37 °C in 2-liter shake flasks or in a 10-liter fermentor (Biostat ED, B. Braun). Culture medium (LB-ampicillin) was inoculated at a density of 1:500 and grown to A of 0.5-1.0. Protein expression was then induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside at 1 mM final concentration. Cells were then grown for 15-19 h, and harvested by centrifugation at 4230 g for 15 min at 4 °C. Cell pastes were washed in 0.1 M Tris, pH 7.4, and frozen at -70 °C.

Purification of Wild-type and Mutant Proteins-The purification scheme used for the wild-type and mutant FKBP12 has been described previously(22) . Wild-type and mutant FKBP13 were purified by a procedure modified from Smith and Johnson(23) . Cell paste (9-12 g) was resuspended in 10-15 ml of phosphate-buffered saline buffer containing 1% Triton X-100, incubated with lysozyme (0.4 mg/g of cell paste, 10 mg/ml stock solution) for 15-20 min at room temperature, sonicated 3 30 s at 20% with microtip sonicator, and spun for 15 min at 31,000 g. Supernatant fractions were transferred to a clean centrifuge tube and spun again to remove residual particulates. The cell pellet was again resuspended in a smaller volume of buffer, sonicated once for 30 s, and centrifuged. The resulting supernatant fractions were pooled. The purification was monitored by peptidyl-prolyl isomerase enzyme activity, using FK506-inhibitable activity to distinguish FKBP13 activity from endogenous cyclophilin activity.

All subsequent purification steps were performed at 4 °C unless otherwise indicated. The clarified cell lysate was loaded on glutathione-Sepharose column (20 ml bed volume) preequilibrated with phosphate-buffered saline/Triton buffer. The column was washed with at least 10 bed volumes of phosphate-buffered saline and/or until A returned to base line. GST fusion protein was eluted with 5 mM glutathione in 50 mM Tris buffer, pH 8.0, concentrated, and dialyzed overnight versus 50 mM Tris, pH 8.0. The >50% pure protein was comprised of two major bands on SDS-polyacrylamide gel electrophoresis (Fig.2) corresponding to fusion protein (43 kDa) and free GST (29 kDa). The protein was then subjected to cleavage by thrombin (1.2 NIH units/mg of fusion protein) by incubating at room temperature for 1 h in 50 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCl(2) as described in (23) . The reaction was stopped by addition of phenylmethylsulfonyl fluoride and EGTA to 0.025 and 2.0 mM final concentration, respectively.


Figure 2: SDS-polyacrylamide gel electrophoresis of purification steps of the GST-FKBP13 fusion proteins. The samples were denatured in the sample buffer and loaded on 4%-20% gradient polyacrylamide, 0.1% SDS gel (Novex). Proteins were detected by staining with Coomassie Blue R-250. Lanes are as follows: 1, molecular weight markers (from top, 97.4, 66.2, 45, 31, 21.5, and 14.4 kDa); 2, crude lysate; 3, eluate from the first glutathione-Sepharose column (the two major bands are the GST-FKBP13 fusion, 43 kDa and free GST, 29 kDa); 4, the same material as in lane3 after it was subjected to thrombin cleavage (the two bands are free GST and free FKBP13, which runs at about 17 kDa); 5, eluate from the second glutathione-Sepharose column; 6, hydrophobic interaction chromatography-purified FKBP13.



Thrombin-cleaved protein was loaded on a glutathione-Sepharose column (20 ml bed volume) equilibrated with 50 mM Tris, pH 8.0; the column was washed with the same buffer, and free FKBP was collected in the flow-through. The remaining GST was retained on the column, while FKBP13 was collected in the flow-through fractions. To remove remaining contaminants, the sample was purified further by hydrophobic interaction chromatography. Active fractions were pooled and concentrated to 10 ml and brought to 2 M potassium phosphate, pH 7.0. The sample was then loaded onto a 10 100 mm hydrophobic interaction chromatography column (Hydropore-HIC, Rainin) mounted on a Waters 650E HPLC. The conditions used were as described previously(21) , except we used a 2-h gradient. All purified proteins were stored in 50 mM Tris, pH 8.0, at 4 °C. 1% polyethylene glycol(8000) was added for longer storage of proteins at -70 °C.

Kinetic Methods

Measurement of catalytic efficiency (k/K) for the peptidyl-prolyl isomerase reaction was performed essentially according to Harrison and Stein (29) with the modifications described previously (22) . The substrate used for determinations of k/K and inhibition constants (K) was Suc-Ala-Leu-Pro-Phe-pNA. k/K and K were measured at enzyme concentrations such that k was at least 5-fold higher than k. Stock solutions of FK506 and rapamycin were prepared in Me(2)SO. Final Me(2)SO concentration in the peptidylprolyl isomerase assay was 0.5% (v/v). Reactions were initiated with chymotrypsin (Sigma) at a final concentration of 50 µg/ml. Absorbance at 400 nm was monitored at 1-s intervals on a Hewlett-Packard 8452A spectrophotometer with a thermostatted cuvette holder, interfaced to a model 300 Hewlett-Packard computer.

The calcineurin assay was performed essentially as described by Klee and Cohen(30) . A commercial preparation of bovine brain calcineurin was used (Sigma, catalog no. C-1907, specific activity = 16 nmol/min/mg under the conditions of the assay). Radiolabeled phosphorylated peptide substrate, derived from the serine phosphorylation site sequence of the RII subunit of cAMP-dependent protein kinase, was prepared as described previously(9) . The serine phosphatase assay was performed in 60 µl of buffer containing 20 mM Tris, pH 8.0, 0.1 M NaCl, 6 mM MgCl(2), 0.1 mM CaCl(2), 0.5 mM dithiothreitol, and 0.1 mg/ml bovine serum albumin(30) . The following ordered additions were made for the assays: 5 nM to 15 µM FKBP, 5 nM to 15 µM FK506, 160 nM bovine calmodulin (Sigma, catalog no. P-2277), and 40 nM bovine brain calcineurin. P-Labeled phosphorylated peptide was added to 1-2 µM final concentration, followed by a 15-min incubation at 30 °C. Reactions were quenched with 540 µl of 0.1 M potassium phosphate, 5% trichloracetic acid (w/v). Cation-exchange columns (Dowex AG1-X8, 0.6 ml) were used for separation of free P(i)(31) . The quenched reaction mixtures (0.6 ml) were applied to the columns, followed by a 0.6-ml H(2)0 wash, and the effluents were collected in scintillation vials and counted with 5 ml of scintillation mixture (Beckmann Liquiscint). All assays were performed in duplicate.

Affinity of the mutant FKBPbulletFK506 complexes for calcineurin was determined by varying the concentrations of mutant FKBP and FK506 at 30 °C, using a drug/FKBP ratio of 1.35:1. FK506/FKBP ratios were increased appropriately for the lower affinity mutants to ensure saturation of the mutant FKBP with drug. Control reactions at 30 °C were run in the presence of the same concentration of Me(2)SO (0.1% (v/v)) as reactions containing FK506.

Data Analysis

For the peptidyl-prolyl isomerase reaction data, absorbance data points were fit to a first-order function using Hewlett-Packard data analysis software. K values were calculated using nonlinear fitting to a competitive tight-binding equation (32) using KineTic software version 3.0 (BioKin, Ltd.) running on a Macintosh IIcx. An example of a fit of K data is shown in Fig.3A.


Figure 3: Determination of K for peptidyl-prolyl isomerase activity (A) and calcineurin inhibition (B) of FKBP12 R42Q mutant. Peptidyl-prolyl isomerase inhibition data were analyzed by nonlinear fitting to a tight binding competitive inhibition equation(31) . Fractional inhibition of calcineurin was determined over a range of FKBP12bulletFK506 concentrations. The concentrations of free FKBP12 [R] and FK506 [L] were calculated from the equilibrium equations derived by Liu et al.(32) , and these concentrations were replotted versus fractional inhibition using the equation shown in the text. K for calcineurin is calculated by taking the inverse of the product of the slope of the replot and K for FK506.



In control experiments with the homogeneous major isoform (Aalpha) of bovine calcineurin (provided by M. Fitzgibbon and J. Thomson, Vertex), we developed an active site titration method for determining active calcineurin concentration. We observed identical inhibition constants (K = 5.5-6 nM) for the commercial bovine and homogenous Aalpha isoform preparations. The inhibition constant for calcineurin by the mutant FKBPbulletFK506 complexes (K) was calculated by computer-fitting the fractional inhibition data as a function of concentration of free FKBP mutant and FK506 to an the equilibrium equation derived by Liu et al.(33) . Quadratic equations were first used to calculate the free concentrations of these reaction components from the concentrations of calcineurin, FKBP mutant, and FK506 in the experiment, as well as the K of the FKBP mutant for FK506. A rearranged form of Equation 7 from (33) was then used to calculate the calcineurin affinity of the FKBP mutantbulletFK506 complex: I/(1 - I) = [FK506][FKBP12]/(K(i)K), where I is the fractional inhibition of calcineurin, and (1 - I) is the fractional activity remaining. K and the associated standard deviation were calculated from linear regressions performed on MiniTab (Addison-Wesley). A replot of calcineurin inhibition data to the above equation is shown in Fig.3B.


RESULTS

Expression and Purification of FKBP13

Human FKBP13 is a membrane-associated protein that contains a 21-residue N-terminal signal peptide(7) . The cDNA fragment of FKBP13 that we used for mutagenesis and expression did not contain the sequence for the signal peptide and thus produced a mature form of the protein of 13 kDa molecular mass. A GST-fusion expression system allowed us to obtain expression levels of 3-12 mg of the GST-FKBP13 chimera/g of cell paste. Induction of the cell culture at higher optical density (A of 0.9-1.1) with isopropyl-1-thio-beta-D-galactopyranoside helped to increase expression level.

We used a purification scheme for FKBP13 proteins that was modified from one developed for isolation of foreign polypeptides as GST fusion proteins(23) . Affinity chromatography on a glutathione-Sepharose column allowed us to obtain fusion protein that was >50% homogeneous in a single purification step. Two major bands of mobilities corresponding to 43 and 29 kDa were visualized on the Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis (Fig.2), corresponding to the fusion protein and free GST, respectively.

The same glutathione Sepharose column was used in a second purification step after thrombin cleavage of the fusion protein. Most of the GST was retained on the column, while GST-free FKBP 13 was collected in the flow-through fractions. However, the protein obtained at this step was still slightly contaminated with free GST and other minor protein species. To remove these impurities, we used hydrophobic interaction chromatography as a final purification step. FKBP13 mutants resulting from this purification scheme were geq95% pure as determined by densitometry of SDS-polyacrylamide gel electrophoresis (Fig.2). Each FKBP12 mutant generated in this investigation was also purified to homogeneity by methods described previously(9, 22) .

Prolyl Isomerase Activity and Drug Binding of FKBP Mutants

Site-directed mutagenesis was used to make conservative mutations of FKBP12 binding pocket and surface residues (Fig.1). All FKBP12 and FKBP13 mutants produced in this investigation are active catalysts of the peptidyl-prolyl isomerase reaction. Mutation of two hydrophilic residues in the solvent-exposed interface of the binding pocket cause more significant decreases in peptidyl-prolyl isomerase activity. The D37V mutant has 10% of the catalytic efficiency of the wild-type protein. The R42I mutant has approximately 60% of the specific activity of the wild-type protein, while the other Arg-42 mutants affect activity less. Of the His-87 mutations, only H87L significantly decreases the catalytic efficiency. Specific activities of FKBP13 variants range from 39 to 110% of the wild-type protein (Table1). Only a few of these mutations, however, cause greater than 2-fold decreases in the catalytic efficiency (k/K) of this reaction.



The D37V surface residue mutant has an affinity for FK506 and rapamycin 580- and 180-fold lower, respectively, than the wild-type protein (the most dramatic change in drug affinity caused by a point mutation that we have observed). This effect is likely due to disruption of the electrostatic interaction formed by Asp-37 carboxylate oxygen and the Arg-42 guanidino NH in the FK506 and rapamycin complexes(15, 16) . The R42I and R42K mutants have unaltered affinities for FK506, while R42I has a 20-fold lower affinity for rapamycin than the wild-type protein. However, mutation of this residue to Gln, the residue found at the homologous position in FKBP13, causes a greater loss in affinity for these ligands (Table1). Mutation of the His-87 surface residue to either Phe, Val, or Ala has little effect on the specific activity or affinity of FKBP12 for ligands. However, mutation of this residue to Leu decreases FK506 affinity 40-fold and rapamycin affinity 100-fold. Mutations at the Ile-90 surface residue of FKBP12 have minimal effects on drug binding.

Calcineurin Binding Determinants

We have reported that mutation of FKBP12 surface residue His-87 to Val decreases calcineurin affinity 4-fold(9) . Subsequently we generated three more mutations at this residue and observed that each of them decreases calcineurin binding by no more than 2-fold (Table1). We do not observe inhibition of calcineurin by the wild-type FKBP13bulletFK506 complex at concentrations up to 10 µM. To determine whether mutation of the corresponding residue of FKBP13 to His confers calcineurin affinity to that protein, we expressed and purified the single FKBP13 A95H mutant. Its FK506 complex does not inhibit calcineurin. However, we determined that mutation of two other FKBP13 residues to their FKBP12 counterparts (Q50R and K98I) generate potent inhibitors of calcineurin when complexed with FK506 (K values of 100 and 23 nM, respectively), representing a gain of at least 3 orders of magnitude in calcineurin affinity relative to the wild-type protein. The reverse mutation of FKBP12, I90K, results in 2600-fold loss in calcineurin affinity without affecting affinity of this mutant to either FK506 or rapamycin. Combination of the two FKBP13 mutations in the double mutant Q50R/K98I results in even tighter binding of the FK506 complex to calcineurin (K = 16 nM). It is interesting to note that combination of the mutation A95H with either Q50R or with the double Q50R/K98I mutant, increases calcineurin affinity in each case. The FK506 complex of the triple mutant Q50R/A95H/K98I FKBP13 binds calcineurin as tightly (within experimental error) as does wild-type FKBP12.


DISCUSSION

Structure-activity data with FK506 analogs in immunosuppression assays (19, 34) led to the proposal that the macrolide contained effector elements that are in direct contact with a target protein and responsible for inhibition or antagonism of this protein. Such data, along with the three-dimensional structure of the FKBP12bulletFK506 complex(35) , enabled identification of the probable effector region of FK506 as the cyclohexyl ring (C-C) and the C(18)-C solvent-exposed portion encompassing the C allyl group. According to the original proposal, FKBP12 acts as a ``presenter'' of the immunophilin ligand(36) .

Based on our preliminary analysis of FKBP12 mutants as calcineurin inhibitors, we concluded that the actual effector face was a composite of the protein and ligand and must include several FKBP12 protein side chains(9) . This modified hypothesis was later supported and extended (18, 19) , and there is now consensus that the so-called ``80s loop'' (residues 84 - 91) of FKBP12 is an important binding determinant for calcineurin(1, 37) . Mutation of the homologous Lys-98 residue of FKBP13 to Ile imparts very high calcineurin affinity to the FK506 complex of that protein. A similar result was reported by Rosen et al.(38) , who argued for the requirement of a simultaneous Pro-97 to Gly mutation to impart high affinity, due to a predicted distortion in the 80s loop of FKBP13. We show here that mutation of Lys-98 alone generates a higher affinity calcineurin inhibitor (K = 23 nM) than the double P97G/K98I FKBP13 mutant reported in that work (K = 44 nM). A high resolution structure of the K98I mutation in the FKBP13 Q50R/A95H/K98I triple mutant indicates that no such loop distortion is present in the FK506 complex. (^4)We have also determined that a single mutation of the corresponding Lys-121 residue of FKBP52 to Ile, generates a protein whose FK506 complex has high calcineurin affinity (K = 90 nM), whereas the co-complex of wild-type FKBP52 has no endogenous calcineurin affinity(13) .

The mutagenesis results reported herein demonstrate that the peptidyl-prolyl isomerase catalytic efficiency of FKBP12 is relatively insensitive to mutation at the active site. All mutants generated in this investigation are catalytically active. We reported previously a 4-fold decrease in calcineurin affinity of the FK506bulletFKBP12 H87V complex, with no significant decrease in FK506 and rapamycin affinity (9) . It was later proposed by Rosen et al.(38) , that the beta-branching of the Val side chain should force this residue to occupy a conformation that would introduce strain into the 80s loop. We show here that the FK506 complex of the H87V FKBP12 mutant indeed suffers a greater decrease in calcineurin binding affinity than the FK506 complexes of H87L, H87F, or H87A. Structural analysis of the FK506bulletFKBP12 H87V complex by x-ray crystallography (21) confirms that the branched Val side chain makes van der Waals' contact with Tyr-82, distorting its position relative to that observed in the wild-type protein. This in turn shifts the position of the 80s loop away from its wild-type position. In concert with the R42K mutation, the H87V mutation can also propagate a change in the FK506 backbone conformation near C, as well as reorienting the 13- and 15-MeOH groups, as observed in solution by NMR(20) .

A second region important for calcineurin binding is composed of residues 40-44 of FKBP12(9) . Becker et al.(19) argued for the importance of this region in the effector face of FKBP12bulletFK506, based on sequence homologies among FKBP12 from different species and structural analysis of an inactive FK506 analog. We showed that mutation of Arg-42 to Lys, Ile, or Gln significantly decreases calcineurin affinity of the FK506 complex, with negligible effects on FK506 affinity. Furthermore, the structure of the FK506bulletFKBP12 R42I complex^3 provides direct evidence that this substitution causes no perturbation in global protein conformation or in FK506 conformation, although higher conformational disorder of the polypeptide backbone is observed in the region of the mutation. However, Yang et al.(18) , who characterized FKBP12/13 chimeras, interpreted their data by claiming that residues within this region were unlikely to play a role in direct interactions with calcineurin, and were only important for conformational effects on other parts of the protein. It has been suggested that these two conflicting hypotheses needed to be resolved(37) .

The evidence provided by the mutation of FKBP13 residue Gln-50 to Arg is compelling. This mutation imparts substantial calcineurin affinity to the FKBP13bulletFK506 complex (Table1). The same mutation on an FKBP13 A95H or A95H/K98I double mutant background also increases calcineurin affinity of these FK506 complexes. In fact the resulting FK506bulletFKBP13 Q50R/A95H/K98I triple mutant complex has a calcineurin affinity equivalent to that of the FK506bulletFKBP12 complex within experimental error (Table1). Structural analysis of the Q50R/A95H/K98I triple mutant FKBP13bulletFK506 complex^4 suggests multiple FK506 conformations but a striking overlap of the effector face residues of the triple mutant FKBP13 and wild-type FKBP12. These structural observations support the hypothesis that the 40s loop of FKBP12, and Arg-42 in particular, is part of the calcineurin effector surface of FKBP12. The definitive evidence would be provided, of course, by a high resolution structure of the FKBP12bulletFK506bulletcalcineurin complex. Such structural and mutagenesis information will be useful in the design of novel FKBP12 peptidyl-prolyl isomerase and calcineurin inhibitors(39) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Vertex Pharmaceuticals Inc., 40 Allston St., Cambridge, MA 02139-4211. Tel.: 617-499-2485; Fax: 617-499-2437; Livingston{at}vpharm.com.

^1
The abbreviations used are: FKBP12, the 12-kDa FK506 binding protein; FKBP13, the 13-kDa FK506 binding protein; GST, glutathione S-transferase.

^2
D. J. Livingston, R. A. Aldape, O. Futer, M. T. DeCenzo, B. P. Jarrett, and M. A. Murcko, poster presented at New York Academy of Sciences Conference on ``Immunomodulating Drugs,'' June 27, 1992.

^3
S. Itoh and M. A. Navia, submitted for publication.

^4
J. P. Griffith, K. P. Wilson, O. Futer, D. J. Livingston, and M. A. Navia, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Mark Fleming for oligonucleotide synthesis. We also thank Drs. D. Armistead, C. Lepre, M. Navia, J. Saunders, and K. Wilson for discussions and critical reading of the manuscript.


REFERENCES

  1. Armistead, D. M., and Harding, M. W. (1993) Annu. Rep. Med. Chem. 28,207-215
  2. Fruman, D. A., Burakoff, S. J., and Bierer, B. E. (1994) FASEB J. 8,391-400 [Abstract/Free Full Text]
  3. Galat, A. (1993) Eur. J. Biochem. 216,689-707 [Abstract]
  4. DiLella, A. G., and Craig, R. J. (1991) Biochemistry 30,8512-8517 [Medline] [Order article via Infotrieve]
  5. Hendrickson B. A., Zhang, W., Craig, R. J., Jin, Y.-J., Bierer B. E., Burakoff S., and DiLella A. G. (1993) Gene (Amst.) 134,271-275 [Medline] [Order article via Infotrieve]
  6. Peattie, D. A., Hsaio, K., Benasutti, M., and Lipke, J. A. (1994) Gene (Amst.) 150,251-257 [Medline] [Order article via Infotrieve]
  7. Jin, Y.-J., Albers, M. W., Lane, W. S., Bierer, B. E., Schreiber, S. L., and Burakoff, S. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,6677-6681 [Abstract]
  8. Sigal, N. H., and Dumont, F. (1992) Annu. Rev. Immunol. 10,519-560 [CrossRef][Medline] [Order article via Infotrieve]
  9. Aldape, R. A., Futer, O., DeCenzo, M. T., Jarrett, B. P., Murcko, M. A., and Livingston, D. J. (1992) J. Biol. Chem. 267,16029-16032 [Abstract/Free Full Text]
  10. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Cell 66,807-815 [Medline] [Order article via Infotrieve]
  11. Partaledis, J. A., and Berlin, V. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,5450-5454 [Abstract]
  12. Nigam, S. K., Jin, Y.-J., Jin, M.-J., Bush, K. T., Bierer, B. E., and Burakoff, S. J. (1993) Biochem. J. 294,511-515 [Medline] [Order article via Infotrieve]
  13. Peattie, D. A., Harding, M. W., Fleming, M. A., DeCenzo, M. T., Lippke, J. A., Livingston, D. J., and Benasutti, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,10974-10978 [Abstract]
  14. Moore, J. M., Peattie, D. A., Fitzgibbon, M. J., and Thomson, J. A. (1991) Nature 351,248-250 [CrossRef][Medline] [Order article via Infotrieve]
  15. Wilson, K. P., Yamashita, M. M., Sintchak, M. D., Rotstein, S. H., Murcko, M. A., Boger, J., Thomson, J. A., Fitzgibbon, M. J., Black, J. R., and Navia, M. N. (1995) Acta Crystallogr., in press
  16. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1993) J. Mol. Biol. 229,105-124 [CrossRef][Medline] [Order article via Infotrieve]
  17. Schultz, L. W., Martin, P. K., Liang, J., Schreiber, S. L., and Clardy, J. (1994) J. Am. Chem. Soc. 116,3129-3130
  18. Yang, D., Rosen, M. K., and Schreiber S. L. (1993) J. Am. Chem. Soc. 115,819-820
  19. Becker, J. W., Rotonda, J., McKeever, B. M., Chan, H. K., Marcy, A. I., Wiederrecht, G., Hermes, J. D., and Springer, J. P. (1993) J. Biol. Chem. 268,11335-11339 [Abstract/Free Full Text]
  20. Lepre, C. A., Pearlman, D. A., Cheng, J.-W., DeCenzo, M. T., Livingston, D. J., and Moore, J. M. (1994) Biochemistry 33,13571-13580 [Medline] [Order article via Infotrieve]
  21. Itoh, S., DeCenzo, M. T., Livingston, D. J., Pearlman, D. A., and Navia, M. N. (1995) Bioorg. Med. Chem. Lett. 5,in press
  22. Park, S. T., Aldape, R. A., Futer, O., DeCenzo, M. T., and Livingston, D. J. (1992) J. Biol. Chem. 267,3316-3324 [Abstract/Free Full Text]
  23. Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67,31-40 [CrossRef][Medline] [Order article via Infotrieve]
  24. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,488-492 [Abstract]
  25. Kunkel, T. A., Roberts, J., and Zakour, R. (1987) Methods Enzymol. 154,367-382 [Medline] [Order article via Infotrieve]
  26. Zoller, M. J., and Smith, M. (1982) Nucleic Acids Res. 10,6487-6500 [Abstract]
  27. Zoller, M. J., and Smith, M. (1983) Methods Enzymol. 100,468-500 [Medline] [Order article via Infotrieve]
  28. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463-5467 [Abstract]
  29. Harrison, R. K., and Stein, R. L. (1990a) Biochemistry 29,1684-1689 [Medline] [Order article via Infotrieve]
  30. Klee, C. B., and Cohen, P. (1988) Mol. Aspects Cell Regul. 5,225-248
  31. Hubbard, M. J., and Klee, C. B. (1991) in Molecular Neurobiology: A Practical Approach (Chad, J., and Wheal, H., eds) pp. 135-149, Oxford University Press, Oxford, United Kingdom
  32. Morrison, J. F. (1969) Biochim. Biophys. Acta 185,269-286 [Medline] [Order article via Infotrieve]
  33. Liu, J., Albers, M. W., Wandless, T. J., Luan, S., Alberg, D. G., Belshaw, P. J., Cohen, P., MacKintosh, C., Klee, C. B., and Schreiber, S. L. (1992) Biochemistry 31,3896-3901 [Medline] [Order article via Infotrieve]
  34. Bierer, B. E., Somers, P. K., Wandless, T. J., Burakoff, S. J., and Schreiber, S. L. (1990) Science 250,556-559 [Medline] [Order article via Infotrieve]
  35. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1991) Science 252,839-842 [Medline] [Order article via Infotrieve]
  36. Schreiber, S. L. (1992) Science 251,283-287
  37. Clardy, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,56-61 [Abstract]
  38. Rosen, M. K., Yang, D., Martin, P. K., and Schreiber, S. L. (1993) J. Am. Chem. Soc. 115,821-822
  39. Armistead, D. M., Badia, M. C., Deininger, D. D., Duffy, J. P., Saunders, J. O., Tung, R. D., Thomson, J. A., Futer, O., DeCenzo, M. T., Livingston, D. J., Murcko, M. A., Yamashita, M. M., and Navia, M. A. (1995) Acta Crystallogr., in press

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