(Received for publication, November 15, 1994; and in revised form, May 16, 1995)
From the
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 FKBP12FK506 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 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 ()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
FKBP12
FK506 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 FKBP13FK506 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
FKBP12 FK506
calcineurin 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 FKBP12
FK506 complex
must include the Arg-42, His-87(9) , and Ile-90 (
)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 (
)(21) . We report here the separable effects of
these FKBP surface mutations on binding interactions with
peptidyl-prolyl isomerase substrates, macrolides, and calcineurin.
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--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 amp
gene and a lacI
allele of the lac repressor(23) .
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-
-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
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.
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, 0.1 mM CaCl
, 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
(31) . The quenched reaction mixtures
(0.6 ml) were applied to the columns, followed by a 0.6-ml
H
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 FKBPFK506 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
SO (0.1% (v/v))
as reactions containing FK506.
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 FKBP12
FK506 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 (A) 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 A
isoform preparations. The inhibition constant for
calcineurin by the mutant FKBP
FK506 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 mutant
FK506 complex: I/(1 - I)
=
[FK506]
[FKBP12]
/(K
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.
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
95% 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) .
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.
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 FKBP12FK506 complex(35) , enabled
identification of the probable effector region of FK506 as the
cyclohexyl ring (C
-C
) and the
C
-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. (
)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
FK506FKBP12 H87V complex, with no significant decrease in FK506
and rapamycin affinity (9) . It was later proposed by Rosen et al.(38) , that the
-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 FK506
FKBP12 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 FKBP12FK506,
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 FK506
FKBP12
R42I complex
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 FKBP13FK506 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
FK506
FKBP13 Q50R/A95H/K98I triple mutant complex has a
calcineurin affinity equivalent to that of the FK506
FKBP12
complex within experimental error (Table1). Structural analysis
of the Q50R/A95H/K98I triple mutant FKBP13
FK506 complex
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
FKBP12
FK506
calcineurin complex. Such structural and
mutagenesis information will be useful in the design of novel FKBP12
peptidyl-prolyl isomerase and calcineurin inhibitors(39) .