(Received for publication, April 6, 1995; and in revised form, May 31, 1995)
From the
In complex with the peptidyl-prolyl isomerase cyclophilin A, the
immunosuppressive antifungal drug cyclosporin A (CsA) inhibits a
Ca/calmodulin-dependent protein phosphatase,
calcineurin, which regulates signal transduction. We isolated and
characterized cyclophilin A mutations that confer CsA resistance in a Saccharomyces cerevisiae strain whose growth is CsA-sensitive.
Three mutations (G70S, H90Y, and G102A) alter single amino acids
conserved between yeast and human cyclophilin A, which structural
analyses implicate in CsA binding to human cyclophilin A. By Western
analysis, all three mutant proteins are expressed in yeast. In
vitro, two purified mutant cyclophilins (G70S, G102A) retain
prolyl isomerase activity and have moderately reduced affinity for CsA
and calcineurin but, when bound to CsA, do bind and inhibit calcineurin
phosphatase activity. In contrast, the purified H90Y mutant cyclophilin
is dramatically decreased in prolyl isomerase activity, CsA affinity,
and calcineurin binding and inhibition. These studies identify
conserved cyclophilin A residues that participate in CsA binding and
catalysis.
The natural product cyclosporin A (CsA) ()is a potent
antifungal and immunosuppressive compound via its ability to inhibit
signal transduction (for review, see (1, 2, 3) ). CsA is a hydrophobic cyclic
peptide that binds to a family of intracellular proteins, the
cyclophilins, which are abundant, ubiquitous, highly conserved, and
found in multiple forms in different intracellular compartments. In
addition, cyclophilins are enzymes that catalyze protein folding by
cis-trans isomerization of peptidyl-prolyl bonds. Cyclophilin A, an
18-kDa cytoplasmic protein, mediates CsA actions in
yeast(4, 5, 6) . The immunosuppressive
effects of CsA are also presumed to be mediated by cyclophilin
A(7) . The target of the cyclophilin A
CsA complex is
calcineurin(8, 9) , a calcium/calmodulin-dependent
serine-threonine-specific protein phosphatase highly conserved from
yeast to man(10, 11, 12) . Calcineurin is
also the target of the macrolide FK506 bound to a different prolyl
isomerase, FKBP12(13) . Calcineurin regulates nuclear import of
the nuclear factor of activated T-cells transcription factor during
T-cell activation(14, 15, 16) , participates
in recovery of yeast cells from pheromone-induced cell cycle
arrest(4, 10, 12) , and is essential in
CsA-sensitive yeast strains(5, 17) .
A wealth of
structural information is available for cyclophilins, both free and in
complex with CsA or substrate. The x-ray crystal structures have been
solved for cyclophilin A alone (18, 19) and in complex
with CsA(20, 21) , a tetrapeptide model substrate or a
dipeptide (Ala-Pro) (22, 23, 24) , and a CsA
analog(25) . Similarly, NMR structures are available for the
cyclophilin A-bound conformation of CsA(26) , the human
cyclophilin ACsA complex(22, 27) , and for
wild-type and an F112W mutant of Escherichia coli periplasmic
cyclophilin (28, 29) . Finally, the x-ray structures
of cyclophilin B-CsA and cyclophilin C
CsA have been
solved(30, 31) . These studies reveal that CsA and
substrate bind a common hydrophobic pocket and provide a foundation for
mutagenic and biochemical studies to analyze cyclophilin functions in vivo.
Previous studies employing genetic and biochemical
analyses established the roles of specific cyclophilin residues in CsA
binding, active site function, calcineurin inhibition, and in vivo function. E. coli periplasmic cyclophilin is distantly
related to human cyclophilin A, sharing only 34% sequence identity and
having prolyl isomerase activity relatively resistant to
CsA(32, 33) . In E. coli cyclophilin, a
tryptophan residue invariant in other cyclophilins is replaced by
phenylalanine. The CsA sensitivity of an F112W mutant E. coli protein was increased 75-fold, whereas substitution of Trp by phenylalanine in human cyclophilin A decreased CsA affinity
17-fold(34) . In the x-ray and NMR structures of cyclophilin A,
Trp
lies in the middle of the
Thr
-Gly
loop in the CsA binding
pocket, near CsA residue 11 and hydrogen-bonded with CsA residue
9(21, 22) . A potential role for cysteine residues in
catalysis by cyclophilin A was excluded by isolating active mutants
lacking each of four different cysteines(32) . Based on the
x-ray crystal structure of human cyclophilin A, active site residues
(Arg
, Phe
, Phe
, and
His
) were identified by site-directed
mutagenesis(35) . Interestingly, cyclophilin mutants lacking
peptidyl-prolyl isomerase activity retained the ability to bind CsA and
inhibit calcineurin(35) . Residues surrounding the cyclophilin
A ligand pocket (Arg
, Lys
, and
Arg
) that participate in interactions between cyclophilin
A
CsA and calcineurin were identified by site-directed
mutagenesis(36) . Finally, mutations in the Drosophila NinaA cyclophilin homolog (37, 38) were found
amongst mutations that disrupt targeting of rhodopsin and distort the
fly eye(39) . This analysis revealed NinaA regions critical for
biological function, including residues within and surrounding the
ligand binding pocket and hydrophobic membrane
anchor(39, 40, 41, 42) .
Here we
report the isolation of cyclophilin A mutations that confer
CsA-resistance in a CsA-sensitive Saccharomyces cerevisiae yeast strain. These mutations identify three residues,
Gly, His
, and Gly
, which
participate in CsA binding to yeast cyclophilin A and are identical in
human cyclophilin A (Gly
, His
, and
Gly
). In the structure of human cyclophilin A, these
residues all lie within the CsA binding pocket, and, based on molecular
modeling, substitutions at these residues should perturb the
cyclophilin A ligand binding pocket. In addition, modeling suggests
that the H92Y mutation would most profoundly perturb the ligand binding
pocket, and, in vitro, the H90Y cyclophilin A mutant protein
has the most severe defect in CsA binding and prolyl isomerase
activity. The isolation of spontaneous cyclophilin A mutations that
confer CsA resistance in yeast has provided further insight into
cyclophilin A structure and corroborates and extends our view based on
structural analyses.
Yeast strains were isogenic derivatives of the
CsA-FK506 sensitive S. cerevisiae strain IL993/5c (5, 46) with the following genotypes: IL993/5c (MAT ilv5
), TB24 (MATa
ura3), TB26 (MAT
ilv5 ura3
leu2::hisG cpr1::LEU2
), TB40 (MATa ura3
leu2::hisG cpr1::LEU2
), TOC6 (MATa
ura3 CMP1-1), and TOC1 (MATa
ura3 TOC1-1).
Spontaneous
independent CsA-resistant mutants of strain TB24 were isolated on YPD
medium with 100 µg/ml CsA. Mutants resistant to CsA but not to
FK506 were identified by streaking to YPD medium with 100 µg/ml CsA
or 1 µg/ml FK506. The resulting mutants (MATa ura3
ILV) were crossed to strain IL993/5c (MAT
URA
ilv5), and
prototrophic diploids selected on YNB minimal medium and scored for CsA
resistance to determine if mutations were dominant or recessive. Each
mutant strain was transformed with the control CEN URA3 plasmid pRS316 (47) and a derivative, pTB4, bearing the
yeast CPR1 gene encoding cyclophilin A(5) .
Transformants selected on synthetic medium lacking uracil were scored
on medium containing 100 µg/ml CsA to determine if mutations were
complemented by the cyclophilin A gene. The
mitochondrial mutation and mutations conferring CsA-FK506
sensitivity prevent sporulation of diploid IL993/5c strains.
The sequences of the wild-type and CsA mutant alleles of the cyclophilin A CPR1 gene were determined using as templates PCR products amplified from genomic DNA (New England Biolabs, cycle sequencing kit) and denatured plasmid DNA of the cloned genes. Primers were as follows: JH45, 5`-GAATTCGGATCCCCGCTAATACTACCATGTCC, and JH46, 5`-AGATCTGGATCCTATTGTTCCAGGCAGAGCGG, and internal, JH111, 5`-CGGCGGTAAGTCTATCTACGG, and JH112, 5`-CTGTCGTGGTGCTTCTTGAAG. In some cases, flanking primers JH123 and JH124 described above were also used. Mutations were identified by comparison with the wild-type sequence, run in parallel, and comparison with the known gene sequence(49) .
Figure 1:
Expression of
cyclophilin A protein in CsA-resistant mutants. Protein extracts from
equal amounts of cells prepared from control and CsA-resistant strains
were fractionated by 15% SDS-PAGE and analyzed by Western blot with a
rabbit polyclonal antisera against yeast cyclophilin A. The figure
shows results for the CsA-sensitive cyclophilin A wild-type strain TB24 (WT, lane1), a mutant strain resistant to CsA and to
FK506 (TOC1-1) that expresses wild-type cyclophilin A (WT, lane2), a CsA-resistant calcineurin A
mutant strain (TOC6-1) that expresses wild-type cyclophilin A (WT, lane3), a cpr1 deletion
strain (TB26) lacking cyclophilin A (cpr1, lane4), and CsA-resistant mutant isolates number 7 (G70S, lane5), 46 (G102A, lane6), 47
(A10P, lane7), 48 (ND, lane8), 50
(ND, lane9), 141 (H90Y, lane10),
and 183 (H90R
, lane11). The arrow indicates the migration position of cyclophilin A. ND indicates cyclophilin A recessive mutants for which the site of
mutation lies outside the coding region, presumably in the
promoter.
Here we isolated and characterized 200 spontaneous independent CsA-resistant mutants of the CsA-FK506-sensitive strain TB24. 193 mutants were resistant to both CsA and to FK506 and presumably result from mutations that render calcineurin resistant to both drugs or no longer essential. The remaining seven mutants were CsA-resistant but were as FK506-sensitive as the isogenic wild-type parental strain (Table 1). When each of these mutant strains was mated to an isogenic CsA-sensitive strain of opposite mating type (strain IL993/5C), CsA-sensitivity was restored in all of the resulting diploid strains, indicating that the mutations are recessive (Table 1).
To
determine the molecular nature of the cyclophilin A mutations that
confer CsA resistance, the CPR1 gene was retrieved from
wild-type and mutant genomic DNA by PCR with oligonucleotides flanking
the CPR1 gene coding region. DNA sequence analysis of the
resulting PCR products revealed that four of the mutations (7, 46, 141,
and 47) are single-nucleotide changes that result in amino acid
substitutions, G70S, G102A, H90Y, and A10P (Table 1). In one
additional mutation (183), a single nucleotide substitution replaces
His with arginine, but a single-base deletion in the
adjacent codon renders the gene out of frame (H90R
). In two cases
(48 and 50), no changes were identified in the coding portion of the
cyclophilin A gene, even though these are clearly cyclophilin A
mutations based on failure to complement a cyclophilin A mutation and
complementation by the cloned cyclophilin A gene. Because only the
coding region of the gene was amplified and sequenced, these two
mutants may harbor promoter mutations. In accordance with this
interpretation, cells bearing these mutations do not express any
cyclophilin A protein ( Table 1and described below).
As a
first step to determine how these cyclophilin A mutations confer CsA
resistance, extracts from wild-type and mutant strains were subjected
to Western analysis with antisera specific for yeast cyclophilin A. As
shown in Fig. 1, this antisera detects wild-type cyclophilin A,
an 18-kDa protein, expressed in the wild-type CsA-sensitive strain (Fig. 1, lane1), and also in a mutant
resistant to both CsA and to FK506 (Fig. 1, lane2) and a CsA-resistant calcineurin mutant (Fig. 1, lane3), which both express wild-type cyclophilin A.
This protein is not present in extract from a cyclophilin A deletion
strain (cpr1, Fig. 1, lane4). In
extracts from the mutant strains, three express wild-type levels of
cyclophilin A (G70S, H90Y, and A10P) (Fig. 1, lanes5, 6, and 7), one expresses about
one-fourth the wild-type level (H90Y) (Fig. 1, lane10), and the remaining three do not express cyclophilin A (Fig. 1, lanes8, 9, and 11). Thus, cyclophilin A mutations that confer CsA resistance
can result from a complete lack of protein expression or expression of
normal levels of a mutant protein.
Figure 2: CsA resistance is conferred by mutations in highly conserved cyclophilin A residues. Human and yeast cyclophilin A sequences are aligned. Amino acid number designations are from yeast cyclophilin A, which is two amino acids larger than the human, and every 10th amino acid is underlined. In the alignment and consensus, identical residues are capitalized and indicated by doubledots. Cyclophilin A mutations that confer CsA resistance in yeast are indicated below the consensus.
To assess their ability to bind CsA, the purified cyclophilin A proteins were subjected to LH-20 ligand binding assays(51) . As shown in Fig. 3A, wild-type yeast cyclophilin A and the G70S and G102A mutant cyclophilins bound CsA, whereas the affinity of the H90Y mutant protein for CsA was dramatically decreased. When LH-20 assays were performed with 5-fold lower amounts of cyclophilin A proteins, CsA binding to the G102A mutant cyclophilin A was reduced in comparison with wild-type cyclophilin A (Fig. 3B). Thus, the apparent affinity for CsA by the LH-20 assay was wild-type, G70S > G102A H90Y.
Figure 3:
Binding of CsA by wild-type and mutant
cyclophilin A proteins. The ability of purified wild-type and
cyclophilin A mutant proteins to bind CsA was tested by LH-20 assays in
binding mixes containing 2.5 µM (panelA) or 500 nM (panelB)
cyclophilin A and 250 nM of [H]CsA.
Following fractionation by LH-20 hydrophobic chromatography, the free
or protein-bound [
H]CsA was detected by
scintillation counting fractions, and the percent of total cpm was
plotted. Proteins are indicated by the following symbols: wild-type
cyclophilin A (squares), G70S (diamonds), G102A (circles), and H90Y (triangles).
Figure 4: Peptidyl-prolyl isomerase activity of wild-type and mutant cyclophilins. Proline isomerase assays were performed with 20 ng of the indicated purified cyclophilin A protein, using the chromogenic substrate N-succ-Ala-Ala-Pro-Phe-p-nitroanilide as substrate as described under ``Materials and Methods.''
We next
assessed the ability of CsA to inhibit prolyl isomerase activity of the
wild-type, G70S, and G102A cyclophilin A enzymes. Prolyl isomerase
assays were conducted following preincubation of 20 ng of the different
enzymes with CsA at a range of concentrations from 0 to 1000
nM, and the initial reaction velocities were calculated and
used to determine the CsA concentration resulting in half-maximal
reaction velocity (see ``Materials and Methods''). By this
analysis, the K for inhibition of
wild-type cyclophilin A by CsA was approximately 10 nM,
whereas for the G70S and G102A, the K
was
increased approximately 10-fold, to 100 nM. Thus, the G70S and
G102A mutations result in a moderate reduction in cyclophilin A
affinity for CsA. Because the CsA minimum inhibitory concentration in
strain TB24 is approximately 25 µg/ml and these mutations confer
resistance to 100 µg/ml CsA or 4-fold higher than the MIC, a small
reduction in CsA affinity of cyclophilin A should suffice to render the
cell drug-resistant. This appears to be the case for the G70S and G102A
mutations.
Figure 5: Interaction of wild-type and mutant cyclophilins with calcineurin in vitro. Binding assays with wild-type or G70S, G102A, and H90Y mutant cyclophilin A proteins immobilized on Affi-Gel-10 were performed with equal amounts of protein extracts from a cyclophilin A-deficient cpr1 mutant strain (MH250-2C) in the absence(-) or presence (+) of 100 µM CsA. Bound proteins were eluted, fractionated by SDS-PAGE, and analyzed by Western blot with affinity-purified antisera against the calcineurin A catalytic subunit CMP1, whose migration position is indicated by an arrow. Numbers to the left indicate size and migration position of molecular weight standards.
Finally, we assessed the ability
of the wild-type and mutant cyclophilins to inhibit calcineurin protein
phosphatase activity in vitro. For this purpose, we used the
assay described by Hubbard and Klee(53) . Bovine calcineurin
was reconstituted with calmodulin and incubated with the
DLDVPIPGRFDRRVSVAAE phosphopeptide and purified wild-type yeast
cyclophilin A, or the G70S, G102A, or H90Y cyclophilin A mutant
enzymes, either in the absence or the presence of CsA. As shown in Fig. 6, wild-type yeast cyclophilin ACsA and the
G70S
CsA and G102A
CsA complexes inhibited bovine calcineurin
activity in vitro. However, in comparison with wild-type
cyclophilin, calcineurin inhibition by the G70S and G102A mutant
proteins required higher levels of cyclophilin, which is consistent
with their decreased affinity for CsA. In addition, these mutations
might also perturb nearby residues, such as Arg
(Arg
in human cyclophilin A), involved in
calcineurin binding. In contrast, the H90Y cyclophilin A mutant protein
only weakly inhibited calcineurin activity, which is consistent with
its profound CsA binding defect. These observations underscore that,
whereas the G70S and G102A mutations subtly alter cyclophilin A
properties, the H90Y mutation more dramatically alters the cyclophilin
A ligand binding pocket.
Figure 6: Inhibition of calcineurin phosphatase activity by wild-type and mutant yeast cyclophilin A-CsA complexes. Calcineurin phosphatase activity assays were conducted as described under ``Materials and Methods'' in the absence(-) or the presence of 20, 50, and 100 nM purified wild-type (WT) or G70S, G102A, and H90Y mutant cyclophilin A. Reactions contained no CsA(-) or 200 nM CsA (+). Results are expressed as percentage of the control reaction in the absence of cyclophilin A or CsA and are representative of at least two independent determinations.
Figure 7:
Cyclosporin A resistance by alteration of
the cyclophilin A drug-binding site. The -carbon tracing of human
cyclophilin A, as determined by x-ray cystallography(20) , is
depicted in stereo.
-Carbons of several residues are indicated by
single-letter amino acid abbreviation and residue number. Regions of
the protein comprising the CsA and substrate binding pocket are
indicated as filled bonds. Residues Val
, Gly
,
His
, and Gly
correspond to residues in yeast
cyclophilin A (Ala
, Gly
, His
,
and Gly
), which, when mutated, confer CsA resistance. By
molecular modeling of human cyclophilin A, G72S, H92Y, and G104A
substitutions are expected to disrupt the ligand binding site. The
-carbon tracing of human cyclophilin A was modified with
permission from Pflugl and co-workers (20) .
All four of the mutations alter single amino acids that
lie within the CsA binding site on cyclophilin A. Three mutations alter
Gly, His
, and Gly
in yeast
cyclophilin A, which correspond to Gly
, His
,
and Gly
in human cyclophilin A. By molecular modeling, (
)a G72S substitution is predicted to perturb the
conformation of the 70s loop of cyclophilin A in which Asn
and Thr
bind water molecules that pack against CsA.
A G104A substitution would probably alter the conformation of adjacent
residues Ala
, Asn
, Ala
, which
form part of the hydrophobic pocket (Ala
,
Ala
) or hydrogen bond the amino nitrogen of CsA residue
Abu2 (Asn
). The G72S and G104A substitutions could also
alter the conformation of residues required for cyclophilin A
CsA
binding to calcineurin, given our finding that the yeast G70S and G102A
cyclophilin A
CsA complexes bound to calcineurin 3-fold less well
than wild-type ( Fig. 5and Fig. 6). Finally, a H92Y
substitution would change the conformation of His
and
possibly also Trp
, which, respectively, hydrogen bond
with the carbonyl oxygens of CsA residues MeVal
and
MeLeu
. At present, it is not clear, based on molecular
modeling, how an alteration of Val
(corresponding to yeast
A10P) would alter the CsA-binding pocket.
Our studies identified yeast cyclophilin A mutant proteins
with single-amino acid substitutions at residues conserved between
yeast and human cyclophilin A. These mutant proteins were cloned,
overexpressed, purified, and characterized in vitro. These
analyses reveal that the G70S and G102A cyclophilin A mutant proteins
have moderately reduced CsA binding affinity, whereas the H90Y mutant
has a more profoundly perturbed ligand binding pocket and fails to bind
CsA or catalyze prolyl isomerization. Based on the structure of human
cyclophilin A, molecular modeling predicts that these mutations would
perturb the ligand binding pocket. None of these residues were
identified in earlier studies employing site-directed
mutagenesis(32, 34, 35, 36) , and
thus this random collection of mutations has revealed additional
residues that participate in cyclophilin A-ligand interactions. One
mutant of the Drosophila NinaA cyclophilin homolog, G96E, has
a substitution at the residue corresponding to Gly in
yeast cyclophilin A(39) . Thus, two different studies converged
to implicate this residue in cyclophilin interactions with CsA and a
presumed in vivo substrate. Further studies in yeast should
allow a more precise dissection of cyclophilin A interactions with
substrates, ligands, and calcineurin.