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Natural Products as Probes of Cellular Function Studies of Immunophilins.

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Natural Products as Probes of Cellular Function : Studies of Immunophilins
By Michael K. Rosen and Stuart L. Schreiber"
One of the great mysteries of cell biology remains the mechanism of information transfer, or
signaling, through the cytoplasm of the cell. Natural products that inhibit this process offer a
unique window into fundamental aspects of cytoplasmic signal transduction, the means by
which extracellular molecules influence intracellular events. Thus, natural products chemistry,
including organic synthesis, conformational analysis, and methods of structure elucidation, is
a powerful tool in the study of cell function. This article traces our understanding of a group
of natural products from the finding that they inhibit cytoplasmic signaling to their current
recognition as mediators of the interaction between widely distributed protein targets. The
emphasis of the discussion is primarily structural. The interactions between the naturalproduct ligands and their protein receptors are analyzed at a molecular level in order to shed
light on the molecular mechanisms of the biological functions of these compounds. In the
process we hope to illustrate the power of chemical analysis as applied to biological systems.
Through chemistry we can understand the molecular basis of biological phenomena.
1. Introduction
Signal transduction refers to the process by which extracellular molecules influence intracellular events. For example, the actions of the hormone insulin are initiated at the cell
surface, when extracellular insulin binds to the membranebound insulin receptor. This binding triggers a series of
membrane-associated events, followed by a cascade of poorly defined intracellular events that result in the transcription
of genes that encode metabolic enzymes. Thus, a signal that
originates outside the cell is transmitted through the cell
membrane, along intermediate carriers in the cytoplasm, and
into the nucleus, causing a change in the status of the cell. In
recent years, much has been learned about the mechanisms
of signal transduction at the membrane and in the nucleus of
the cell. In contrast, very little is known about the mechanisms of signal transduction through the cytoplasm of the
cell. Despite the importance of such processes, the detailed
mechanisms of cytoplasmic signaling remain among the
great mysteries of cell biology, a fact that has led the cytoplasm to be referred to as the "black box" of signal transduction.
Natural products chemistry has long played an important
role in the elucidation of biological mechanisms. Pioneering
synthetic and mechanistic studies of molecules such as
steroids, prostaglandins, and porphyrins, to name only a
few, have led to fundamental insights regarding the biological functions of these important classes of compounds. Certain natural products are also uniquely suited for the study
of the mysterious processes of the cell, including cytoplasmic
signaling, due to their interference with these processes. By
studying inhibitory natural products bound to their biological receptors, we may gain a detailed understanding of the
function of these receptors. Research in this area relies on a
combination of the powerful, complementary tools of synthetic chemistry, molecular biology, cell biology, and methods of structure elucidation, including both NMR spectroscopy and X-ray crystallography. This review highlights
recent advances in the structural and mechanistic under-
standing of cytoplasmic signaling that have arisen through
application of these techniques to the study of the immunophilins, a family of cytosolic proteins that bind natural products. These advances have culminated in the identification
and characterization of pentameric complexes composed of
two normally noninteracting protein constituents and a natural product "glue" that binds the proteins together in a
biologically significant manner. In the process, it has been
discovered that a protein phosphatase is a key cytoplasmic
component of a family of signal transduction pathways.
2. Background
FK506". and cyclosporin A[31(CsA) are fungal natural
products (Fig. 1) that inhibit CaZ+-dependent[**]signaling
pathways in a variety of cell types.[4*51 In T cells, both agents
inhibit the transcription of a number of genes, including that
encoding interleukin-2 (TL-2), which are normally activated
by stimulation of (by binding of certain extracellular molecules to) the T cell receptor (TCR).r61In mast cells they
inhibit the exocytosis (i.e. movement to the cell surface and
fusion with the cell membrane) of secretory vesicles that
normally results from stimulation of the IgE re~ept0r.L~~
variety of biochemical and biological data indicate that in
both cases inhibition occurs within the cytoplasm and not at
the cell surface or in the nucleus.
On a biochemical level FK506 and CsA are also quite
similar. Through synthesis of radiolabeled and immobilized
derivatives of these agents for use in protein purification, it
was discovered that both bind with high affinity to soluble,
cytoplasmic receptor proteinsta1 (immunophilins;[8*91 this
term is used to denote immunosuppressant binding proteins,
as both FK506 and CsA are immunosuppressive
drugs['* 'I). The FK506 receptor has been named FKBP,["]
and the CsA receptor has been named cyclophilinrlz](CyP).
Both proteins catalyze the isomerization of cis and trans
amide-bond rotamers of peptide and protein substrates."
[*] Prof. Dr. S. L. Schreiber, M. K. Rosen
Department of Chemistry, Harvard University
Cambridge, MA 02138 (USA)
VCH Verlugsgesellschqfl mhH, W-6940 Weinhein?,1992
The terms Ca'+-dependent and -independent in this context refer to
signaling pathways that are characterized by the presence or absence,
respectively, of an immediate rise in the concentration of intracellular
cytoplasmic Caz following binding to the cell surface receptor.
$3.50+ ,2510
Angew. Chrm. Int. Ed. Engl. 3f (1992) 384-400
The enzymatic activity of FKBP is potently inhibited by
binding of FK506 (Ki = 0.4 nM), but not CsA,[”] and the
activity of CyP is inhibited by CsA (Ki = 6 nM),[”] but not
Rapam ycin
D-Ala 8
Cyclosporin A (CsA)
Fig. 1. Chemical formulas of the immunophilin ligands.
Although FK506 is approximately 100 times more effective than CsA in cellular assays, in virtually all other respects
the two molecules behave identically in both T cells and mast
cells. This similarity in biological function, coupled with the
discovery that the molecules inhibit two distinct rotamase
enzymes,[*] led to speculation that the biological effects of
these agents were due to their rotamase inhibition. It was
hypothesized that a necessary step in activation of unknown
proteins required for IL-2 transcription in T cells was rotamase-catalyzed isomerization of a peptidyl-prolyl amide
bond. Inhibition of this catalysis by FK506 or CsA would
then result in inhibition of IL-2 transcription.“ 31
Strong evidence against this “rotamase hypothesis” came
from biochemical and biological studies of two structurally
related molecules: rapamy~in,“~.5 1 an immunosuppressive
fungal natural product and FKBP ligand, and 506BD,[’” a
synthetic FKBP ligand (Fig. 1). Given the structural similarities between the three ligands, it is not surprising that, like
FK506, rapamycin and 506BD also bind tightly to FKBP
and inhibit the rotamase activity of the enzyme (Ki(rapamycin) = 0.2 nM; Ki(506BD) = 5 nM).[lsx In fact, NMR
and crystallographic studies have demonstrated that FK506
and rapamycin interact with a common domain of FKBP
through their common structural elements.[”] Fascinatingly,
although FK506 and rapamycin both bind to FKBP and
inhibit its rotamase activity, rapamycin does not inhibit the
same TCR-mediated signaling pathway that is affected by
FK506 and CsA. Rather, it blocks a later Caz+-independent
pathway associated with T cell activation, which is mediated
by the IL-2 receptor (IL-2R)[15318,
(Fig. 2). Even more
striking is the fact that 506BD has no inhibitory effect on
either of the signaling pathways inhibited by FK506 or rapamycin. Furthermore, it was shown that FK506, rapamycin, and 506BD can all block the actions of the others,
The expression rotamase has the same meaning as PPIase (for peptidyl
prolyl cis-trans isomerase) an acronym used by others for the description of
proteins that catalyze the isomerization of cis and fruns amide-bond
rotamers of peptides and proteins.
Stuart L. Schreiber was born in 1956 and was raised in Virginia ( U S A ) . He studied as an
undergraduate in Charlottsville, Virginia, where he obtained a B.A. in chemistry in 1977 at the
University of Virginia. Following Ph.D studies under the direction of R . B. Woodward and
Yoshito Kishi at Harvard University, he began his independent career in 1981 in the Department
of Chemistry at Yale University. In 1988, he accepted a Professorship at the Department of
Chemistry at Harvard. His group is engaged in the study of biological processes through the
combined application of synthetic organic chemistry and molecular and structural biology.
Michael Rosen was born in 1965 in Philadelphia, Pennsylvania ( U S A ) . He received B.S. degrees
in chemistry and chemical engineeringfrom the University of Michigan in 1987. A s a Winston
Churchill Scholar during 1987-1988 he earned a C.P.G.S. in the Natural Sciences from the
University of Cambridge (England) under the direction of Professor Alan R . Battersby. Since
that time he has been a graduate student in the research group of Projessor Stuart L. Schreiber
at Harvard University. His primary interests lie in the structural analysis ofproteins andproteinligand complexes by N M R .
Angew. Chem. Inf. Ed. Engl. 31 (1992) 384-400
presumably through competitive binding to a common receptor, FKBP. In contrast, the actions of FK506 and rapamycin are unaffected by CsA, which does not bind FKBP;
the actions of CsA are also unaffected by FK506, rapamycin,
and 506BD, which do not bind C Y P . [ ' ~ , '19,201
~ , If the biological properties of FK506 and rapamycin were simply due
to inhibition of the rotamase activity of FKBP, then they
should both interfere with the same pathways. 506BD should
upon binding to CyP a yet-to-be discovered CyP ligand may
provide a complex that has properties indistinguishable from
those of the FKBP-rapamycin complex.
One obvious, if seemingly unlikely prediction from the
above biological data is that the structurally unrelated
FKBP-FK506 and CyP-CsA complexes should act, either
directly or indirectly (through other proteins), upon a common target molecule that is distinct from the target acted
upon by the FKBP-rapamycin complex. The FKBP-FK506
and CyP-CsA target should be a component of Ca2+-dependent signaling pathways such as TCR-mediated transcription in T cells and IgE receptor-mediated exocytosis in mast
cells; the FKBP-rapamycin target molecule should be a
component of CaZ+-independent signaling pathways such as
IL-2R-mediated proliferation in T cells. In both cases, the
target should be unaffected by either protein or ligand alone,
since only the immunophilin-ligand complexes are active
inhibitors (Fig. 3 top).
In fact, we have recently demonstrated that calcineurin
(CN), also referred to as protein phosphatase 2B, a calmoddin-dependent serine/threonine protein ph~sphatase,['~]
possesses all the predicted biochemical properties of a target
common to both FKBP-FK506 and C ~ P - C S A . [ CN
~ ~ ] is a
heterodimeric protein composed of two subunits, calcineurin
A (CNA), which contains the calmodulin-binding and phos-
Fig. 2. FK506 and CsA inhibit Ca2+-dependentsignaling pathways, while rapamycin inhibits Ca'+-independent pathways. These are illustrated in the T cell
by the pathways emanating from the T cell receptor and the IL-2 receptor,
respectively, but include signaling pathways in a variety of cell types, including
mast cells and neurons.
also inhibit these same pathways and thus be biologically
active. Because this is not the case, it implies that FK506 and
rapamycin do not act by eliminating a function of FKBP;
rather they act by adding a function to the protein. In a sense,
FK506 and rapamycin are prodrugs that are activated by
binding to FKBP. CsA is similarly inactive until it binds
CyP. This explanation of the immunophilin-ligand complexes
as the species responsible for signal inhibition has been termed
the "active complex" hypothesis.[*,9, 15, 1 6 , 'I Genetic studies have also provided strong support for this idea by demonstrating that FKBP is both necessary and sufficient to mediate the actions of rapamycin in yeast,[z2~231
and that CyP
mediates the actions of CsA in this organism.[21,241 Paradoxically, although FK506 and rapamycin are structurally similar and bind to a common protein, FKBP, their FKBP complexes have different biological actions. Furthermore, while
CsA is structurally dissimilar to FK506 and binds to CyP,
which is unrelated to FKBP, the biological actions of the
CyP-CsA complex are indistinguishable from those of the
FKBP-FK506 complex. It is interesting to speculate that
Fig. 3. Top: The CyP-CsA and FKBP-FK506 complexes act on a common
target molecule that is not affected by FKBP or CyP alone, or by the FKBPrapamycin or FKBP-506BD complexes. Bottom: Formation of the pentameric
complex (see text).
Angew. Chem. Int. Ed. Engl. 31
(f992) 384-400
phatase active sites, and calcineurin B (CNB), which is a
Ca2+-bindingprotein with, as yet, unknown function. The
binding of calmodulin to CNA results in a tenfold increase
in the phosphatase activity of the enzyme. In direct binding
assays, C N binds to both the FKBP-FK506 and CyP-CsA
complexes, but not to FKBP or CyP alone, or to the FKBPrapamycin complex. In addition, the binding of CN to
FKBP-FK506 is inhibited by CyP-CsA (but not by CyP or
CsA alone), suggesting that the two complexes compete for
the same, or two interacting binding sites. The phosphatase
activity of calcineurin, when measured with a phosphopeptide substrate, is potently inhibited by the FKBP-FK506
and CyP-CsA complexes, but is unaffected by FKBP, CyP,
FK506, rapamycin, 506BD, or the FKBP-rapamycin complex. Interestingly, it was recently reported that the translocation of a cytosolic component of the transcription factor
NF-AT, which regulates IL-2 transcription and is sensitive to
FK506, into the cell nucleus due to a rise in intracellular
calcium levels is blocked by both FK506 and CSA.['~]The
common dependence on intracellular calcium, location in
the cytosol, and sensitivity to FK506 and CsA have led us
and others to speculate that this component of NF-AT may
be a substrate for CN, and that its location in the cell may be
dependent on its phosphorylation state.[', 28, 291 Thus, the
inhibition of IL-2 transcription by FK506 and CsA may be
the result of their indirect inhibition of the dephosphorylation of a component of NF-AT.
An important question arises from this body of data: How
does the binding of FK506 and rapamycin to FKBP and of
CsA to CyP change both the ligands and the receptors, enabling the complexes to perform functions the individual
components are incapable of performing alone? The remainder of this review will focus on the aspects of ligand-receptor
interactions most relevant to this question. In particular, we
will discuss the structures of both the natural products and
the proteins, and the changes that occur to each upon
binding. The pentameric immunophilin-drug-CNA-CNBcalmodulin complexes (Fig. 3 bottom) will then be
analyzed in light of the known biochemical data on the interactions between the immunophilin-ligand complexes and
3. Structural Studies of FKBP
and its Complexes with FK506 and Rapamycin
3.1. Initial Work
Early structural studies of the FKBP-FK506 and CyPCsA complexes centered on analysis of the interactions between ligand and receptor. These studies were motivated, in
part, by two conflicting proposed mechanisms of rotamase
catalysis, and hence of rotamase inhibition by the ligandsL3'I
(Fig. 4). One mechanism involved initial formation of a tetrahedral enzyme-substrate adduct similar to that formed
during the hydrolysis of the amide bond by serine or cysteine
proteases. Rotation about the C-N bond in the adduct, followed by expulsion of the enzyme nucleophile would result
in amide-bond isomerization. This mechanism was supported by studies of Fischer et al.,[lZblwho showed that in CyP
modification of a cysteine in the active site with p-hyAngew. Chem. Int. Ed. Engl. 31 (1992) 384-400
Fig. 4. Two proposed mechanisms of rotamase catalysis. Left: Initial formation
of a tetrahedral intermediate. Right: Catalysis by stabilization of a twisted
amide bond. enz = enzyme.
droxymercuribenzoic acid eliminated the rotamase activity
of the enzyme. Fischer et al.13'] also found an inverse secondary deuterium-isotope effect with substrates containing
indicating a change in hybridization
(sp' 3 s p 3 ) and thus formation of a covalent bond in the
transition state of the reaction. These kinetic data were, however, disputed by Harrison and Stein,["] who found a normal secondary deuterium-isotope effect using the same substrates. Site-directed mutagenesis studies further demonstrated that none of the cysteine residues in CyP are required
for catalysis.1331
These results, coupled with measurement of
thermodynamic activation parameters for C Y P ' ~ ' and
FKBP,'34. "I led to an alternative mechanism of rotamase
activity involving binding of a transition-state structure that
contains a twisted, or distorted amide bond. In this view, the
energy needed to overcome amide bond resonance and isomerize the C-N bond would come from favorable noncovalent interactions between the enzyme and a peptide substrate.
The mechanism involving a tetrahedral intermediate led to
the consideration that FK506 and rapamycin might bind to
FKBP through nucleophilic attack of a side chain of the
enzyme on one of the two electrophilic carbonyl carbon
NMR studies of the
atoms of the ligand, C8 or C9.
complex of fully synthetic [8,9-13C]FK506[361and recombinant human FKBP,1371however, showed no evidence for
formation of a tetrahedral add~ct.'~']These studies also
demonstrated that FK506 binds to FKBP in a single conformation, although the unbound ligand exists in organic solution as a 2: 1 mixture of cis and trans amide-bond rotamers.
The finding that FK506 and, by inference, rapamycin do
not bind covalently to FKBP suggested a possible explanation for the strong interaction between the ligands and their
receptor. In the solid state, both FK506 and rapamycin possess a dihedral angle of approximately 90" about the C8 -C9
bond. This conformation is also maintained in the complexes
of both ligands with FKBP. A dihedral angle of 90" between
C8 and C9 and a planar N7-C8 amide group place the keto
carbonyl roughly perpendicular to the plane of the
pipecolinyl ring (pipecoline = methylpiperidine). Because
the pipecolinyl ring most probably mimics the proline ring in
natural peptide substrates, the keto carbonyl of FK506 or
rapamycin is in the same position as would be a twisted
amide carbonyl group of a peptide undergoing rotamase
catalysis (Fig. 5). Thus, the perpendicular keto carbonyl
groups of FK506 and rapamycin allow the ligands to mimic
3.2. Three-Dimensional Structures
Fig. 5. FK506 and rapamycin may mimic a twisted peptido-prolyl amide bond
in a peptide substrate. Left: Model of a twisted amide bond in a peptide
substrate. Right: Portion of the crystal structure of free FK506. The analogous
carbonyl groups in the two structures are colored red.
a transition-state structure involving a twisted amide
The view of FK506 and rapamycin as twisted amide peptidomimetics was further extended by studies of the substrate
specificity of FKBP.[341In the peptide series succinyl-AlaXaa-Pro-Phe-(p-nitro)anilide, it was found that peptides
with branched hydrophobic amino acids Leu, Ile, and Val as
Xaa were greatly favored (up to 1000-fold over peptides with
Xaa amino acids with charged side chains as determined
by measurement of K c a , K i l values) with Leu > Ile
> Val. Analysis of the structures of FK506 and rapamycin
suggested a possible rationale for these observations. Beginning with the pipecolinic acid moiety and proceeding in the
“N-terminal” direction, both ligands possess a dicarbonyl
group, a tertiary hydroxyl group, and a branched aliphatic
chain. As illustrated in Figure 6, these can be mapped onto
the amide carbonyl group, amide nitrogen atom, and the side
chain of a branched aliphatic amino acid residue N-terminal
to a proline. Thus, the binding of FK506 and rapamycin to
FKBP was proposed to result from the ability of the ligands
to mimic a Leu-Pro dipeptide with a twisted amide bond.r381
Fig. 6. FK506 (left) and rapamycin may mimic a leucine-proline substrate with
a twisted amide bond (right). Analogous atoms are colored alike. Leu is the
preferred P1 residue.
In order to further explain the enzymatic, ligand-binding
and biological properties of the immunophilins, we undertook the structure determination of free human FKBP by
Our success in this effort was due significantly
to the cooperative nature of FKBP, which proved to be
soluble and stable, and gave beautiful NMR spectra. Structural studies of the human FKBP-ligand complexes were
also undertaken in a fruitful collaboration with Professor
Jon Clardy’s group at Cornell University. This work resulted
in high-resolution crystal structures of both the FKBPFK506 and FKBP-rapamycin complexes.[42- 441 Analyses
of the three FKBP structures, showing both the bound and
free forms of the protein, have yielded new insights into
many aspects of immunophilin function.
3.2.1. Overview of the Structures
All three FKBP structures show the same fold of the
protein (Fig. 7 left). The structure is characterized by a fivestranded antiparallel jsheet with a novel + 3, + 1, - 3, + 1
loop topology. The strands of the sheet, which run roughly
Fig. 7. Richardson diagrams of FKBP (left) and CyP (right). A ball-and-stick
model of FK506 is positioned in the ligand-binding site of FKBP[42,44]. A
similar model of CsA[69,70]is used to indicate schematicully the location of the
ligand-binding site in CyP[67,68]. We prepared an approximate model of the
CyP-CsA complex by docking the structure of bound CsA(691into the structure of free CyP[68] using reported intennolecular NOES observed between
MeLeu 9 of CsA and Trp 121 of CyP[73]. We thank Professor Ke for providing
the CyP coordinates.
perpendicular to the long axis of the molecule, are composed
of residues 2-8, 21-30, 35-38 with 46-49 (this strand is
interrupted by a loop at residues 39-45), 71 -76, and 97106. A short amphipathic a helix containing residues 57-63 is
aligned with the long axis of the protein and lies against the
sheet, forming a tightly packed hydrophobic core. The core
is composed entirely of aliphatic and aromatic residues, with
all but one of the aromatic residues clustered at one end of
the molecule. The conserved aromatic and aliphatic side
chains of Tyr 26, Phe 36, Phe46, Val 55, Ile 56, Trp 59, Tyr 82,
and Phe99 line a shallow cleft at the N-terminus of the a
helix, forming the FK506 and rapamycin binding site. The
side chains of these residues are well defined in both the
Angew. Chem. Int. Ed. Engl. 31 (1992) 384-400
Fig. 8. Stereoview of selected residues in the ligand-binding pocket of FKBP.
An overlay of 15 structures of the free protein generated by NOE-restrained
molecular dynamics simulation is shown. The orientation is approximately
perpendicular to that in Figure 7. (Note the a helix at the upper right and the
/I strands lining the lower left.) Residues are identified by the one-letter amino
acid code. Side chains are colored as follows: Phe = red; Tyr = green; Arg,
Asp, Trp = white; Ile, Val = purple. The loops Ser38-Pro45 and Gly83His94 have been removed for clarity.
ligated and unligated forms of FKBP (Figs. 8 and 9). Both
ligands fit tightly into the binding pocket and have a number
of similar hydrophobic and hydrogen-bonding interactions
with the protein. The FKBP-binding domain of FK506
extends from the C24 hydroxyl group, through the diketo
pipecolinyl moiety and the pyranose ring, to the C15
methoxy group. This finding was anticipated on the basis of
structural analyses of FK506 and rapamycin and on the
biological properties of the two molecules, and led to the
design of the FKBP ligand 506BD (see Fig. 1).Ii6] Most of
the trisubstituted cyclohexyl ring (C26-C34) of FKS06 and
Fig. 9. Stereoview of selected residues in the binding pocket of the FKBPFK506 complex. Orientation and colors of the side chains are the same as in
Figure 8. Bound FK506 is yellow. Hydrogen bonds (see text) are white.
Anyew. Chem. I n t . Ed. Enpl. 31 (1992) 384-400
carbon atoms C18-C23 of its macrocycle, including the ally1
group, do not contact FKBP and are exposed to solvent.
Similarly, the binding domain of rapamycin consists of the
region from the C28 hydroxyl group through the pyranose
ring; carbon atoms C15-C27 are exposed to solvent. For
rapamycin the orientation of the cyclohexyl ring (C34-C42)
is different from that in FK506, and makes several contacts
to FKBP. The pipecolinyl ring is the most deeply imbedded
portion of both ligands (Fig. 9). This moiety is in van der
Waals contact with the indole ring of Trp59 at the back of
the binding pocket, and with the side chains of Tyr26,
Phe 46, Val 55, rle 56, and Phe 99 at the sides of the pocket. In
the NMR spectra of the FKBP-FK506 complex the extremely high-field chemical shifts (6 = - 2 to 0) of the
pipecolinyl methylene hydrogens, and several NOE crosspeaks in the aromatic region also reflect the proximity of this
group to aromatic residues of the protein."'] A number of
additional hydrophobic contacts are made between the ligands and the protein. These include interactions between the
pyranose ring and residues Tyr 26, Asp 37, Tyr 82, His 87, and
Ile90, and between the region C24-C26 of FK506, and
C28-C32 of rapamycin, and residues Phe46 and Glu 54 of
FKBP. Thus, the nanomolar binding constants of FK506
and rapamycin may be due, in part, to the complementary
hydrophobic surface of FKBP.
The favorable van der Waals interactions between FKBP
and both FK506 and rapamycin are complemented by similar arrays of intermolecular hydrogen bonds. Both ligands
have identical hydrogen bonds from the C1 ester carbonyl
group to Ile56 NH, the C8 amide carbonyl group to the
Tyr 82 phenolic OH group, and the C10 hemiketal OH group
to the Asp 37 carboxylate. Despite the differences between
the C24-C26 region of FKS06 and the C28-C33 region of
rapamycin, both also make analogous hydrogen bonds to
Glu54 CO, FK506 from the C24 hydroxyl group, and rapamycin from the C28 hydroxyl group. Rapamycin makes
one additional hydrogen bond not seen in the FK506 complex, from the C40 hydroxyl group to Glu 53 side chain CO
group. The pattern of hydrogen bonds involving Ile 56 NH,
Glu 54 CO, and Gln 53 CO is reminiscent of the antiparallel
P-sheet-like contacts often seen between proteins and peptide ligands. These hydrogen bonds then extend the portions
of FK506 and rapamycin that may be described as peptidomimetic to include the region between the C1 ester carbony1 group and the C24 (C28 for rapamycin) hydroxyl
group. The structural analogy between the ligands and a
tetrapeptide is illustrated in Figure 10. The pipecolinyl ester
carbonyl group acts as the proline amide carbonyl group in
a Leu-Pro peptide fragment. The region from 01 to C25
(C29 for rapamycin), including the cyclohexyl ring, is roughly
equivalent to an aromatic amino acid residue. The C24 hydroxyl group (C28-OH for rapamycin) then represents the
amide NH group of the following residue in the chain. Thus,
FK506 and rapamycin may bind to FKBP by acting as transition-state analogues of a Leu(or Val)-Pro-Xaa-Yaa peptide
substrate. We are currently investigating this hypothesis
through synthesis of peptides and peptide analogues designed based on the FKBP-FK506 and FKBP-rapamycin
complex structures.[451Biochemical analyses of these molecules, coupled with structural analysis of their complexes
with FKBP (through X-ray crystallography and/or NMR
Fig. 10. Schematic illustration of the bonding between FKBP and the ligands rapamycin Oeft) and FK506 (middle) and a peptide (right). Analogous atoms have the
same color.
spectroscopy) will probe the energetic contributions of the
various receptor-ligand interactions.
The hypothesis that FK506 and rapamycin are transitionstate analogues of an FKBP substrate predicts that in addition to the interactions discussed above there should be significant contacts between the protein and the keto carbonyl
groups of the ligands. These same contacts would then stabilize a twisted amide bond in the transition state for rotamase
catalysis. Initially we had expected, based on the many known
structures of protein-ligand complexes, that the keto carbony1 group (at C9) would be bound by a network of hydrogen bonds or typical electrostatic interactions with charged
residues. However, the structures of the two receptor-ligand
complexes clearly show that the keto carbonyl group does
not make any hydrogen bonds to the protein, nor does it
contact any charged residues of the protein. Instead, it is in
close proximity to the C'H atoms at the edges of three aromatic rings, Tyr26, Phe36, and Phe99 (Fig. 9). Such
C-H . . . O interactions have been observed in small organic
m o l e c ~ l e and
s ~ ~proteins,[471
and it has been proposed that
these contribute to the stability of such systems. Theoretical
calculations, too, support the notion that C-H . . .O interactions can stabilize small molecules as well as proteins.[481
However, to our knowledge the FKBP complexes would be
the first examples in which this type of interaction contributes significantly to the binding of a ligand to its receptor. It would also represent a novel mechanism of catalysis,
wherein transition-state stabilization is achieved through
arene-carbonyl interaction.
3.2.2. Structural Consequences of Binding
Since neither FKBP nor FK506 or rapamycin are able to
bind to or inhibit the phosphatase activity of CN, it is important to analyze the differences between the free and bound
forms of both protein and ligands in order to understand the
biological actions of the two complexes. How does binding
activate FKBP and its ligands, allowing FKBP-FK506 to
recognize CN, and FKBP-rapamycin to recognize a different target protein? The existance of both free and bound
structures of all components of these complexes now allows
this question to be addressed. Comparison of free and bound FKBP
Comparison of the structure of unbound FKBP with
those of either of the FKBP-ligand complexes reveals that
the majority of residues in the protein binding pocket are not
greatly perturbed by ligand binding. In particular, the aromatic and aliphatic residues making direct contact to ligand
(Tyr 26, Phe 46, Val 55, Ile 56, Trp 59, and Phe99) show only
small conformational differences between the bound and
free structures (compare Figs. 8 and 9). In addition, regions
of the protein that are not directly involved in binding (for
example, the bulk of the a helix and the p sheet) are also not
dramatically different in the bound and free structures. Subtle changes in these regions may prove to be significant, but
as will be discussed later in Section 5, this seems unlikely.
There are several loops in FKBP, however, that appear to
undergo both structural and dynamic changes on ligand
binding. Not surprisingly, these loops are located at the rim
of the binding pocket. In the structure of free FKBP, the
seven residue bulge, Ser 39 to Pro 45, that occurs in the middle of the fifth strand of the fi sheet is only poorly characterized by the NMR data (Fig. 11). A lack of medium- and
long-range NOES in this loop, coupled with the relatively
weak sequential and intraresidue NOES further suggest that
this region may be disordered, sampling a wide range of
conformations. In the crystal structure of the FKBP-FK506
complex this loop is well defined by the electron density. This
structure is stabilized by a large number of backbone/backbone and backbonelside-chain hydrogen bonds despite its
rather unusual conformation. A similar set of hydrogen
bonds is seen in the FKBP-rapamycin complex. However,
the high thermal factors observed for residues 40 to 43 in this
structure indicate greater mobility or disorder than in the
Angew. Chem. Inl. Ed. Engl. 31 (1992) 384-400
FK506-FK506 complex. This difference probably reflects
different numbers of intermolecular contacts in this region in
the two crystal forms.[441Many of the hydrogen bonds in the
region appear to be facilitated by an Asp-Arg-Tyr triadL4']
involving the side chains of Asp37, Arg42, and Tyr26
(Fig. 9). This interaction bends the backbone of the loop
toward the body of the protein, enabling many hydrogenbond donors and acceptors to come together. In free FKBP
no evidence for these interactions with Arg42 has yet been
observed in the NMR data. Further, preliminary NMR
analyses of the FKBP-FK506 complex have shown that the
chemical shifts of the amide hydrogens of several residues
between Ser 39 and Pro 45 change substantially on binding.
Thus, ligand-binding may increase the propensities of
residues in this region to form hydrogen bonds, resulting in
changes in conformation and/or mobility of the loop. Interestingly, the region from Lys 34 to Lys 44 is one of the most
highly charged (seven of eleven residues are charged) and
most basic (net charge 3) sequences ofFKBP (see hFKBP12
sequence in Fig. 12). The proximity to the bound ligand,
structural changes on ligand binding, and high charge density all suggest that this region may be important in contacting
CN in the pentameric complex.
A second region of FKBP that appears to change significantly on ligand binding is the segment from Tyr 82 to His 94
(green in Fig' '*). This loop 's poorly defined in free FKBP'
Like the basic loop described above, this segment shows few
Fig. 11. a Carbon overlay of ten structures of free FKBP generated by NOErestrained molecular dynamics simulation. The view is similar to that in Figures 8 and 9. The a helix is colored red (as in Fig. 7). The basic loop Ser39Pro45 is blue. The loop Gly83-His94 is green.
Mustrans2 16
PaORF 117
CtMIP 51
LpMIP 117
bFKBP30 92
0 .
B B B B B P B B B B 11 P P B
Mustrans2 76
PaORF 174
CtMIP 116
LpMIP 176
hFKBP13 59
bFKBP30 159
bFKBP12 51
hFKBP12 51
aaall 2 2
p @ p @ B p 311
i x q p n x L IF E
Fig. 12. Manual sequence alignments of known FKBPs. Only the homologous region of each protein is shown. Numbers at the ends of each row are the position of
the nearest amino acid in the full protein. Gaps inserted in the sequences are represented by hyphens, C-termini are marked with stars, and uncertain assignments are
in lowercase. Residues in boldface correspond to residues of hFKBP1Z that contact FK506 in the X-ray structure of the complex[42,44]. Degrees of conservation are
indicated by solid and open circles above the sequence as follows: 0 completely conserved; o strong preference for one amino acid (at least seven occurrences);
conservative substitutions; o mostly similar amino acids. Below the sequences, the secondary structure of hFKBP12 in the complex with FK506 is shown; a helix
is marked with a, antiparallel strand with @, and p turns with numbers corresponding to type beneath the second and third positions. The following sequences are
included: human FKBP12[37], bovine FKBP12[57a], bovine FKBPl2[57a], bovine FKBP25[57 b], human FKBP13[56], NcFKBP (Neurosporu crassa)[57c], ScFKBP
(Saccharomyces cerevisiae)[57 d], NmFKBP (Neisseria meningiridis)[57e],LpMIP (Legionella pneumophilu macrophage infectivity potentiator)[57fl, CtMIP (Chlumydia trurhomatis)[57 g], PaORF (open reading frame in the Pseudonionas ueruginosu algR2 gene)[57 h], and MustransZ (homologue of mouse transition protein 2)[57 i].
A n p w . Chem. Int. Ed. En,ql. 31 (1992) 384-400
39 1
long-range NOEs, and only weak sequential and intraresidue
NOEs. In addition, TOCSY and NOESY NMR spectra
provide evidence for two distinct conformations of this loop
that interconvert slowly on the NMR time
Although the exact nature of this interconversion has not yet
been determined, chemical shift perturbations show that it
affects several residues between Tyr82 and His 87.ISo1Thus,
in the absence of ligand the loop Tyr 82 to His 94 appears to
sample at least two, and possibly more, different conformations. In contrast, in both FKBP-ligand complexes this loop
shows a single, well-defined conformation that is stabilized
by several intraprotein hydrogen bonds and a number of van
der Waals contacts to the ligand. In the free protein these
contacts would be absent, allowing the loop greater flexibility. These changes suggest that this loop, like that from
Ser 39 to Pro 45, may be involved in CN recognition. If this
proves to be the case, then the CN binding site could span up
to almost 20 A, and contain regions of both high positive
charge density (from the protein) and high hydrophobicity
(from the effector domains of the ligands). Mutagenesis
studies are currently underway to identify definitively the
regions of FKBP that are important in the formation of the
ternary complex with CN. Comparison of Free and Bound FKBP Ligands
The differences between bound and free FK506 and
rapamycin are also revealing. Free FK506 has been examined with a variety of techniques, including X-ray crystallography,"] NMR spectroscopy,151."I and theoretical calculation~.['~]
The structure of the molecule crystallized from
organic solvent['] is shown in Figure 13. In the crystal,
FK506 exists in a single conformation with a cis amide bond.
In contrast, in organic solution it exists as a 2: 1 mixture of
cis:trans amide-bond rotamers.["
"I The structures of
both rotamers were recently determined by NOE-restrained
molecular dynamics calculations.[". "I Although similar to
the crystal structure in some respects, both conformations
are significantly different in others. In the FKBP binding
domain, the cis structure in solution is quite similar to the
solid-state structure. However, the two differ in the region
surrounding C23, with large deviations in the torsion angles
about C20-C21, C21-C22, C23-C24, C24-C25, C25-C26,
and C26-C27. Most notably, the cyclohexyl residue extends
away from the macrocycle in the solid state, while in solution
it is wrapped under the macrocycle due to a C25-C26-C27C28 dihedral angle of 91". It is interesting that the trans
conformer in solution differs from the crystal structure in the
FKBP binding domain but is relatively similar in the remainder of the molecule. Other than changes to the torsion angles
about CI-C2 and C9-CI0, which compensate for the trans
versus cis amide bond, the only other differences between the
two structures are in the torsion angles from C16 to C19. An
important common feature (see below) of all three unbound
conformers is the orientation of the pyranose moiety outside
of the macrocyclic ring. In this orientation, the pyranose ring
does not make any intramolecular contacts.
Free FK506 has also been studied t h e ~ r e t i c a l l y . Calcu~'~~
lations employing a Monte Carlo search of torsion angles in
the macrocycle of the molecule have revealed the presence of
Fig. 13. Stereoviews of the structures of free FK506 (top), bound FK506 (middle), and bound rapamycin (bottom) in the solid state. In middle and bottom
pictures, atoms in close contact ( < 4 A) with heavy atoms of FKBP12 are
shaded red[42-44].
21 distinct low-energy conformations (within 12 kcalmolof the lowest energy structure)[s31including both the cis and
trans amide-bond isomers. Molecular dynamics simulations
have also been used to study the structure and dynamics of
FK506 in water['3] (the insolubility of the molecule in
aqueous media has prevented direct structural analysis in
this environment). Over the course of a simulation of 200 ps,
beginning with the energy-minimized solid-state structure,
FK506 adopted two stable conformations that differed primarily in the torsion angles about C17-CI8 and C18-Cl9.
Flexibility was also observed about the C1-C2 and C8-C9
torsions. In general, however, the structure did not deviate
significantly from the solid-state conformation during the
200 ps.
The structure of bound FK506[42,44''41 (Fig. 13 middle)
is dramatically different from the known structures of the
free ligand[42,441
(A necessary caveat at this point is that the
structure of FK506 in aqueous solution has not yet been
experimentally determined. Thus, the differences between
the free and bound forms of the molecule described below do
Angew Chem. Znr. Ed. Enxl. 31 ( 1992) 384 -400
not necessarily reflect effects of binding, and thus should
only cautiously be interpreted as such). This is true of both
the solution and solid-state conformations. The most notable change between the crystalline forms is that the amide
bond of bound FK506 is trans, while in the crystal structure
of the free molecule it is cis. This change is compensated for
by large rotations about the C1 -C2, C14-C15, C15-CI6,
and C16-Cl7 bonds. Significant changes are also seen in
these same torsion angles when either of the free forms in
solution is compared to the bound form. The differences in
the torsion angles flanking the pyranose ring, C14-Cl5 and
either the amide torsion angle (for the cis conformations) or
C9-ClO (for the trans conformation), result in a rotation of
this moiety under the macrocycle in the bound structure.
This gives the binding domain a more compact shape and
forms a hydrophobic core of the ligand. Overall, these changes
result in only a 3 kcal mol- increase in energy due to unfavorable steric interactions on binding (comparing crystal
structures in both the free and bound forms).[421However,
the approximately 150" rotation about the C14-CI5, bond
does result in steric interaction between the methyl substituents of the C13 and C15 methoxy groups. These groups
are roughly anti to each other in all forms of the free ligand.
But in the bound structure of FK506, the C15 methoxy
group is rotated to the outer side of the macrocyclic ring and
adopts a gauche /gauche - orientation with respect to
the C13 methoxy group. The energetic consequence of this
interaction, which is not present in rapamycin, may be in
part responsible for the ligand specificity observed in other
FKBPs (see Section 6).
In contrast to FK506, the free and bound conformations
of rapamycin are highly
441 The root-mean-square
deviation (rmsd) between the bound and free structures is
0.48 A. Like bound FK506, rapamycin has a trans amide
bond in the solid state (in organic solution it exists as a 4: 1
mixture of trans:cis amide-bond rotamers). It also has the
pyranose ring folded under the macrocycle, and the cyclohexyl moiety extended outward from the body of the molecule. Interestingly, the conformation of the FKBP-binding
domain of bound FK506 is virtually identical to that of
rapamycin. The rmsd between the two bound ligands from
C1 to C13 is 0.12 A. Thus, the FKBP binding pocket interacts strongly with only one conformation of the two ligands.
The fact that rapamycin's FKBP-affinity is twice that of
FK506 may, in part, reflect its greater propensity to adopt this
Although the conformation of FK506 may be significantly altered by binding to FKBP, the same can probably not be
said of rapamycin. This is true of both domains of the
molecules. While ligation may serve to alter the conformation of FK506, better enabling it to contact CN in the pentameric complex, for rapamycin it appears that this may simply serve to surround the ligand with residues of the protein.
Binding may also aid in the transfer of both molecules from
the lipid membrane into the aqueous environment of the
cytoplasm. It seems likely that since FK506 and rapamycin
bind to the same site in FKBP, the selectivity of the two
FKBP-ligand complexes arises from the different chemical
compositions of the two ligands. These result in two different
composite protein-ligand surfaces, enabling the complexes
to bind two different targets.
Angew Chem. I n f . Ed. EngI. 31 11992) 384-400
3.3. 506BD
The synthetic FKBP ligand 506BD['61 has proven to be
extremely useful in increasing our understanding of the
structural basis for the biological actions of FK506 and rapamycin. This molecule contains the common FKBP-binding domain of FK506 and rapamycin, but lacks the effector
domain of either ligand.[161Instead, the geometry of the
binding domain is maintained by a simple (both structurally
and synthetically) trans enoate spacer. The ability of 506BD
to bind FKBP and inhibit the biological actions of FK506
and rapamycin, coupled with the inability of the ligand to
inhibit signal transmission pathways clearly defined the twodomain (binding and effector) nature of FK506 and rapamycin. In retrospect, however, the success of 506BD as an
FKBP ligand was somewhat fortuitous, for the molecule was
designed based on the crystal structure of unbound FK506.
When 506DB was designed, the conformation of bound
FK506 would not be known for almost two years. Because
the free and bound structures of FK506 are very different, it
is surprising that a linker designed to bridge two points in the
free molecule would be able to bridge the same two points in
the bound. This is in fact the case. The enoate spacer in
506BD was designed to connect from C16 to C25, a distance
of 5.2 A in unbound FK506.['] A trans enoate was employed
in order to accomodate both the cis and a possible trans
amide-bond rotamer. The regions flanking the spacer were
also designed to maintain the local conformational preferences seen in the C14-C26 region of FK506. Inspection of
bound FK506 revealed that despite the many unanticipated
changes detailed above, C16 and C25 are 4.2 8, apart in this
conformation. Thus, the same trans enoate linker can join
these points together without significantly perturbing the
bound structure of the binding domain.['61
3.4. Insights from Protein Sequence
FKBP is only one member of a growing family of FK506and rapamycin-binding proteins. At least four other proteins
that bind the two ligands have been observed and partially
characterized in human T cells.[55. In addition, a number
of proteins with high homology to FKBP have been identified in various organisms, including other mammals, yeast,
and bacteria.[571Cyclophilins, too, have been identified in a
variety of cell types and different organisms.[s81The known
FKBPs range in molecular weight from 12 kDa for the
archetypal protein (hereafter referred to as FKBPl2) to
59 kDa for the phosphorylated FKBP59 found in T cells.[551
Despite their variations in size, all members of the FKBP
family that have been sequenced to date (either directly
through automated Edman degradation, or indirectly through
cloning and sequencing of their complementary DNA
(cDNA)) show a common FKBPl2-like domain of approximately 12 kDa. An alignment of the sequences of some of
these proteins is shown in Figure 12 (see Section
Several features of this alignment are striking. First, although the number of conserved amino acids from approximately residue 25 through the C-terminus (residue numbers
referenced relative to hFKBP12) is fairly high, the N-terminus shows very little homology among the various proteins.
Interestingly, in hFKBP12 residues 1 to 13 and 13 to 29
correspond to the first two of four exons in the genomic
DNA encoding the protein.[s91These exons are separated
from the remaining two exons, which encode residues 29 to
66 and 67 to 107, by a large intron of 16000 DNA bases. The
other introns are much smaller and contain only 109 and
3300 bases. This gene structure suggests that the N-terminal
regions of the FKBPs may have developed through the recruitment of unrelated exons by the large intron preceeding
the common C-terminal domain. Thus, analysis of the
FKBP12 gene provides an evolutionary explanation for the
high variability seen in the N-terminal peptide of members of
the FKBP
Examination of the sequence alignment in light of the
structures of unbound FKBP12 and of the two FKBP12ligand complexes also provides insight into the requirements
for stability, ligand binding, and rotamase catalysis of these
proteins. A total of 13 residues are exactly conserved in all
known FKBPs. Six of these, Gly33, Gly69, Gly83, Gly28,
Leu 97, and Leu 103, are important to the structural integrity
of the protein.[s7b1The remaining seven, Tyr 26, Phe 36,
Asp37, Val55, Ile56, Tyr82, and Phe99, line the binding
pocket or make important contacts to ligand. Trp 59, which
also appears critical for maintaining the structure of the
binding pocket is conserved in all but one sequence, where it
is replaced by Phe. The high conservation of residues in the
FKBP binding pocket suggests that all proteins in the family
will bind ligands in a similar manner. In all cases, binding
will involve a large number of van der Waals interactions
with aromatic and aliphatic residues of the protein, plus several specific hydrogen bonds from the ligand to the protein.
The sequence conservation coupled with a view of FK506
and rapamycin as transition-state analogues also suggests
that these types of interactions may be necessary for rotamase catalysis. The conserved residues Tyr26, Phe 36, and
Phe99 may be important for stabilizing the carbonyl group
of a twisted amide in the rotamase transition state. The potential of the Tyr 82 phenolic hydroxyl group to form a hydrogen bond and the capacity of the Asp37 carboxylate
group to form a salt bridge may be important as well. Furthermore, it has been suggested that the hydrophobicity of
the rotamase active sites of both FKBP and CyP may play a
significant role in the enzymatic mechanism of the isomerization reaction.[35.44, 6oJ Vapor pressure measurements have
shown that peptide bonds are highly solvated in aqueous
media.L6'1 Wolfenden and Radzickar6'I have argued that
bound water molecules will inhibit isomerization of the
amide bond since the presumably non-resonance-stabilized
transition-state structure will be less polar than the starting
or product structures. An enzyme that promotes desolvation
by providing a hydrophobic binding site will then facilitate
isomerization. In support of this hypothesis for catalysis by
desolvation[62]are NMR data demonstrating that the isomerization of N,N-dimethylacetamide is significantly more
rapid in methylene chloride than in water.f631Kofron et al.
have discussed a similar mechanism[3s1on the basis of theoretical calculations indicating that interactions with water
reduce the flexibility of amide bonds.[641Finally, Van Duyne
have noted calculations showing that the dipole
along the C-N bond is decreased in a twisted amide relative
to the planar
and that the stability of dipolar
species is decreased in low-dielectric environments.[661Thus,
they postulate that the hydrophobic ligand-binding site of
FKBP may promote isomerization by destabilizing the
strongly dipolar amide bond in a peptide substrate.
4. Structural Studies of Cyclophilin (CyP)
and its Complex with Cyclosporin A (CsA)
4.1. Cyclophilin (CyP)
Although the structure of the CyP-CsA complex remains
unknown, Kallen et al. and Ke et al. have recently determined the structures of a CyP-tetrapeptide complex and
unbound CyP by X-ray crystallography.[67* In addition,
Weber et al. and Fesik et al. have also determined the structure of CsA bound to CyP by isotope-edited NMR spect r o ~ c o p y .701
~ ~The
~ . results of these studies provide many
clues regarding the structure and biological function of the
CyP-CsA complex. As can be seen in Figure 7, the structure
of CyP bears no obvious resemblance to that of FKBP12.
CyP is composed of an eight-stranded antiparallel ,!? barrel
capped at each end by an ix helix. The topology of the jl
strandsis + I , -3, -1, -2, + I , -2, -3,whichresultsin
an unusual loop crossing reminiscent of that seen in
FKBP12. The eight-stranded P-barrel structure of CyP is
related to the structures of several members of the ,!?-protein
family,17'1 including bilin-binding
retinol-binding protein,r72b1
and in~ecticyanin.['~~]
Like CyP, these proteins also bind small hydrophobic ligands. The ligand-binding pocket of ,!?-proteinsis in a cleft
between the two sheets forming the
In CyP,
however, intermolecular NOES, chemical shift changes on
binding, and overall geometry of the protein indicate that the
CsA-binding pocket is not located inside the central fi barre1.[673 Rather, it is in a cleft at the protein surface formed
by one sheet of the barrel and the loop from Lys 118 to
His126. Previous NMR studies indicated that bound CsA
contacts the side chains of the single tryptophan residue of
CyP, Trp 121, and an unidentified phenylalanine residue.[731
The CyP structure corroborates this data, since Trp 121 sits
at one end of the binding pocket, and Phe 60 and Phe 113 are
located at its base. The structure also provides an explanation of the inhibition of CyP rotamase activity and CsA
binding by modification of cysteine residues with p-hydroxymercuribenzoic acid,[12b1as CysllS and Cys62 are both
located in the binding pocket. The roles of other residues in
the binding cleft, including His 54, Arg 55, Ile 57, Met 61,
Asnl02, Gln111, Leu122, and His126 are still unclear.
However, the generally high conservation of these residues in
the various cyclophilins suggests that they make important
contacts to the ligand, and may also be involved in rotamase
catalysis. In this regard, it is curious that there are no obvious similarities between the active sites of FKBP12 and CyP.
4.2. Cyclosporin A (CsA)
In the absence of a complete structure of the CyP-CsA
complex it is difficult to identify differences between bound
and unbound CyP that may be important for interaction
Angew. Chem. Int. Ed. Engl. 31 (1992) 384-400
Fig. 14. Stereoviews of free (top) and
bound (bottom) CsA.In the structure of
bound CsA, atoms with attached hydrogens that show NOE crosspeaks to CyP
in a NOESY spectrum (50ms) of the
CyP-CsA complex are shaded red[70].
with CN. However, the existance of both free1741and
bound[69.701CsA structures (Fig. 14) does allow such an
analysis to be made for the ligand portion of the complex.
The conformation of bound CsA is strikingly different from
the conformation of the free ligand in the crystalline state
and in organic solvents. (The crystal and solution structures
of CsA in organic media, for example methylene chloride,
are extremely similar, and hence will be referred to jointly as
the conformation of the “free” ligand. The insolubility of
CsA in aqueous media has prevented detailed structure determination in more biological environments (however, see
Quesniaux et al.[75]).Thus, as with the FKBP ligands, comparisons between free and bound CyP ligands should be
interpreted cautiously,) Free CsA can be divided into two
overlapping domains. The first consists of residues 11-3 and
4-7, which form two strands of an antiparallel fl sheet joined
by a type b11‘ turn at residues 3 and 4. There are hydrogen
bonds between Abu 2 NH and Val 5 CO, Val 5 NH and Abu 2
CO, and Ala 7 NH and MeVal 11 CO stabilizing the structure. Notably, all the N-methyl groups in this region (11, 2,
4, 6) are directed outside the macrocyclic ring. The second
domain consists of residues 7-11, which form an irregular
loop stabilized by a hydrogen bond from D-Ala8 NH to
MeLeu6 CO and a number of van der Waals contacts, including several to the N-methyl group of MeVal 3 3 , which is
directed into the center of the macrocycle. All amide bonds
in the structure are trans, except that between MeLeu 9 and
MeLeu 10, which is cis.
In contrast to free CSA, the bound molecule has no elements of regular secondary structure. In addition, there are
no intramolecular hydrogen bonds. Of the seven N-methyl
groups, only two, those of MeVal 11 and MeLeu9, are exposed. The remainder are directed into the macrocycle and
make a number of intramolecular van der Waals contacts.
Correspondingly, all four amide N H groups and ten of
eleven amide CO groups are at least partly exposed. On the
basis of H/D-exchange rates it was proposed that one amide
Angew. Chem I n f . Ed. EnzI. 31 (1992) 384-400
NH group, that of Abu 2, makes an intermolecular hydrogen
bond to CyP. Strikingly, all amide bonds in the bound
conformation are trans, indicating a high energy barrier to
In addition to determining the structure of bound CsA,
Weber et al. and Fesik et al. also identified a number of
intermolecular NOES between ligand and receptor!693 701 In
particular, many NOES were observed from aromatic and
aliphatic residues of CyP, including the sole tryptophan,
Trp 121. These NOES indicate, as had been previously sugg e ~ t e d ,that
~ ~ ~CsA
] binds in a hydrophobic pocket of the
protein. The patterns and locations of protein-ligand NOES
also allowed both groups to determine the regions of CsA
that contact CyP in the complex. As indicated in Figure 14
bottom, the amino acid residues 9 through 3 all show a
number of intermolecular contacts. Hence, these residues
constitute the binding domain of CsA. In contrast, residues
4 through 8 show only intramolecular NOES, indicating they
do not strongly interact with the protein. This suggests that
these residues may constitute the region of CsA that is in
contact with calcineurin (CN) when simultaneously bound
to CyP.
An important tenet of the active complex hypothesis is
that the only necessary requirement for signal inhibition by
a drug is binding of the immunophilin-drug complex to its
relevent biological target (CN in the case of FK506 and
CsA). The tight binding of drug to immunophilin is not
necessary per se, and as demonstrated with 506BD is also not
sufficient to cause inhibition. With this in mind, the identification of distinct binding and possible effector domains of
CsA allows explanation of the biochemical and biological
data on a wide range of CsA analogues. In most but not all
cases there is a direct correlation between CyP binding and
inhibitory activity measured in T-cell activation assays.1773
Not surprisingly, most of the analogues that do not bind
CyP have been modified in the regions of the drug that
contact the protein. For example, the following analogues all
have > 80 YOdecrease in CyP binding and activity in T-cell
activation assays relative to CsA: MeAla9, MeAla 10,
MeAla 11, MeLeu 11, cc-amino-y-fluorobutyrate 2.f7’] A
number of modifications of the MeBmtl side chain have
similar effects. There are, however, several CsA analogues
that do not show a correlation between CyP binding and
signal inhibition. These can be divided into two categories :
1) those that do not bind CyP tightly, but are inhibitory and
2) those that do bind CyP, but are not inhibitory. The existence of such compounds has led to speculation that CyP
may not be the relevant biological receptor of CsA (that is,
not the receptor responsible for the biological actions of the
drug).[’*] The active complex hypothesis provides an interesting alternative explanation of this data. In the first category there is only one reported molecule, dimethyl-Bmt-CsA
(Fig. 15). This compound binds CyP roughly a hundredth as
Dimethyl-Bmt 1
MeAla 6
Me-%a-Bmt 1
TPc-o-Om 8
Fig. 15. Modified side chains of CsA analogues whose immunosuppressive
activity does not correlate with affinity for CyP. Numbers refer to amino acid
positions in CsA.
tightly as CsA, but retains 35 YOof the inhibitory activity of
CsA in T-cell activation assays. It is possible that although
dimethyl-Bmt-CsA binds CyP weakly on its own, its binding
may be enhanced on binding of the ligand-receptor complex
to CN. That is, the additional protein-protein and proteinligand contacts in the ternary complex may be sufficient to
overcome the energetic penalty incurred by the addition of a
methyl group to CsA. Investigations of the CyP-dimethylBmt-CsA complex support this possibility and indeed resolve the paradox surrounding this molecule.t791The second
category of CsA analogues consists of compounds with
modifications to three residues: MeBmt 1, MeLeu6, and DAla 8. These compounds include, among others, MeAla 6,
Me-Thia-Bmt 1, and TPc-D-Orn 8 (Fig. 15). The properties
of these molecules are reminiscent of those of 506BD, which
binds FKBP but does not interfere with signal transduction
in T cells or mast cells. By analogy with 506BD, these molecules contain the elements necessary to interact with CyP,
but do not have the proper structure to enable their ligandreceptor complexes to bind to CN. This explanation is consistent with the location of residues 6 and 8 on the effector
domain of CsA. In addition, the MeBmt 1 derivatives in the
second category all possess modifications of the MeBmt side
chain at locations near the side chain of MeLeu 6 on one face
of the bound CsA macrocycle. Thus, MeBmt1 may be important for contacting both CyP and CN in the pentameric
complex. In support of these explanations of the various
synthetic analogues, results in our laboratory illustrate that
for a number of analogues of both FK506 and CsA, inhibition of signal transduction correlates exactly with the ability
of complexes of these molecules with their cognate immunophilin receptors to inhibit the phosphatase activity of
The conformation of the CsA effector domain also suggests a possible mode of interaction of the CsA portion of the
CyP-CsA complex with CN. The CsA backbone from the
carbonyl group of Sar3 through the amide NH group of
Ala7, with the exception of MeLeu4, is reminiscent of one
strand of a pleated sheet. This suggests that the interactions between bound CsA and CN may resemble those observed between strands of a p sheet. The carbonyl groups of
residues 3 and 5 , and the NH group of residue 7 all lie in the
plane of the CsA macrocycle, and are directed outward. The
amide plane containing Val 5 NH and MeLeu4 CO is angled
relative to the plane of the macrocycle, although Val 5 NH
could still be accessible for hydrogen-bonding interactions
with CN. Note that the N-methyl groups of residues 4 and 6,
which might interfere sterically with a regular pattern of
hydrogen bonds, are directed into the macrocyclic ring and
thus remain unobtrusive. Finally, the side chains of residues
4, 5, and 6 alternate above and below the plane of the CsA
macrocycle in a fashion similar to that seen in p sheets. This
enables the side chains of MeLeu4 and MeLeu6 to participate in stabilizing contacts with the side chains of MeLeu 10
and MeBmt 1 on one face of the macrocycle. These features
together result in a regular, accessible surface composed of
the side chains of 4 5 , and 6, and the backbone amide groups
of residues 3 through 7 that would be able to make a number
of hydrophobic and hydrogen-bonding contacts with CN.
The structure of this surface suggests that the CsA portion of
the CyP-CsA complex may interact with CN in a p-sheetlike structure.
5. Structures of the Pentameric ImmunophilinDrug-CNA-CNB-Calmodulin Complexes
5.1. Contacts between the Immunophilin-Ligand
Complexes and CN
It interesting to speculate on which regions of the various
immunophilin-ligand complexes are important for contacting their biological targets and which regions impart specificity to the complexes, allowing them to interact with different
partners. Is it possible that only the ligand portions of the
complexes contact the partner, and that the sole function of
the protein is to correctly position the ligand and mold it into
the proper conformation for binding? Or can it be that only
the protein is important for binding, and that the function of
the ligands is to induce or stabilize particular protein conformations that are then able to interact with different biological partners? Or is it possible that a composite surface formed
by both ligand and receptor is required for binding, and that
the ligands and receptors mold each other cooperatively? A
variety of evidence suggests that, at least for the FKBP12ligand complexes, the last possibility is the most likely. The
free and bound structures of rapamycin are virtually identical. Thus, rapamycin does not need to interact with FKBP12
Angew. Chem. lnr. Ed. Engl. 31 (1992) 384-400
in order to take on its biologically active conformation. Yet
it is inactive on its
231 Comparison of the structure of
free FKBP12 with those of its FK506 and rapamycin complexes reveals that regions of the protein that do not contact
ligand are not changed significantly by complexation. Thus,
the ligands do not exert large allosteric effects on the protein
that might facilitate binding to target. This is not surprising,
given the relatively small size of FKBP12. More significantly, comparison of the two complexes shows that even in the
regions of the structure surrounding the ligands, the two
proteins are extremely similar. (The differences observed in
the region Gly28-Gly33 are most likely artifacts of crystal
packing and therefore are probably not ~ignificant.'~~])
Given the distinct biological effects of the complexes, it then
seems unlikely that only the protein contacts the biological
target molecules. This analysis suggests a ternary complex in
which both immunophilin and ligand make contacts with the
m a
5.2. Structure of the FKBP-FK506 and CyP-CsA
Binding Site of Calcineurin
Phosphatase inhibition and binding dataLz6]on the immunophilin-ligand complexes provide insight into the structures of the pentameric complexes. The ability of CyP-CsA
to displace calcineurin and calmodulin from an immobilized
FKBP-FK506 reagent demonstrates that the two complexes
share either a common binding site or two interacting binding sites on CN.IZ6]It is not apparent from the available
structural data how the two complexes might interact with a
common site. The resolution of this issue may require further
information on the regions of the two proteins (FKBP12 and
CyP) that are involved in C N recognition. The inability of
calmodulin or the CNB subunit to bind immunophilin-drug
complexes suggests that the binding site(s) probably resides
in the CNA subunit.1791The increased binding of FKBPFK506 and CyP-CsA to CN in the presence of calmodulin
also suggests that the calmodulin- and immunophilinligand-binding sites are not independent.["] The interaction
between these sites may be transmitted indirectly, through
changes to CN, or more directly, through stabilizing interactions between calrnodulin and the immunophilin-ligand
complex when both are bound to CNA. A more detailed
picture of the immunophilin-ligand binding site may be
derived from the substrate-dependence of CN inhibition.
The dephosphorylation of a 20-residue phosphopeptide by
CN is strongly inhibited by both FKBP-FK506 and CyPCsA (Ki = ca. 30 nM when measured with bovine CN and
human i r n m ~ n o p h i l i n s ~However,
~ ~ ~ ) . dephosphorylation of
p-nitrophenyl phosphate is actually enhanced by approximately a factor of 3 at similar concentrations of the two
irnmunophilin-drug complexes.1261These data indicate that
the binding site for the complex is not the enzyme active site,
but the two sites are probably spatially close to each other.
A similar relationship between binding and active sites is
seen in the recently solved crystal structure of cyclic AMPdependent protein kinase bound to a peptide inhibitor.["] A
schematic illustration of this description of the immunophilin-ligand binding site is presented in Figure 16.
Thus, our current model of pentameric complex formation
Angrw. Chem. l n t . Ed. Engl. 31 (1992) 384-4000
Fig. 16. Model of the binding site of CN in the immunophilin-ligand complex.
a) Two different substrates, a phosphopeptide andp-nitrophenyl phosphate, are
bound to CN. b) Immunophilin-ligand complexes, FKBP12- FKS06 and CyPCsA, affect the enzymatic activity of CN on the two substrates (phosphopeptide
and p-nitrophenyl phosphate).
involves calmodulin-facilitated binding of FKBP-FK506 or
CyP-CsA in a single or in two interacting hydrophobic binding sites that are adjacent to the phosphatase active site
present on the A subunit of CN.
6. Additional Roles of the Immunophilins
A variety of data indicate that in addition to mediating the
signal inhibition of FK506, rapamycin, and CsA, immunophilins may also have a number of other roles in the cell. For
example, FKBP13 is a membrane-associated protein and the
predominant FKBP in mast cells.['. 5 5 , Although FKBP12
and FKBP13 share 43 YOamino acid sequence identity, and
show exact identity for all residues lining the ligand-binding
pocket, the two proteins are significantly different in the
regions immediately surrounding the binding pocket. In particular, residues in the highly charged basic loop of FKBP12
(Arg40 to Lys44) have been replaced with much different,
uncharged residues in FKBP13 (RDRNK -+ LPQNQ, using
the one-letter amino acid code). If this loop is important for
binding of the FKBP12-FK506 complex to CN, then these
differences imply a different biological role for FKBPl3.
Indeed, the FKBP13-FK506 complex does not bind CN,
although the structural basis for this has not yet been determined.[791Clues to the biological role of FKBP13 also can be
drawn from the protein sequence. Unlike all other known
FKBPs, FKBPI 3 is initially synthesized with a signal peptide
at its N-terminus that directs the protein into the endoplasmic reticulum (ER).[55*
801 In the ER the peptide is cleaved,
leaving the soluble form of the molecule. At its extreme
C-terminus FKBPl3 also contains a possible ER-retention
sequence,1811Arg-Thr-Glu-Leu-CO; (Fig. 12). This causes
the protein to be retained in the ER rather than secreted or
directed to other parts of the cell. These two signal peptides
indicate that FKBP13 is a resident protein of the ER, and
suggest that it may play a role in regulating protein traffic in
this organelle. Thus. FKBP13 may be similar in function to
the heat-shock proteins[821and protein disulfide isomerase,182,831 which stabilize nascent unfolded or improperly
folded polypeptide chains in the ER, preventing their aggregation and precipitation. It is not unreasonable that
FKBPl3, too, may stabilize unfolded peptides through interactions with its hydrophobic pocket. It may also act as a
protein foldase by facilitating the cis-trans isomerization of
peptidyl-prolyl amide bonds, an event shown to be rate limiting in the in vitro folding of a number of proteins.[s41
Similar roles have also been postulated for two members
of the cyclophilin family, CyP-BfS8',s8g1 and the protein
product of the ninaA gene.f58d,
8 5 ] Both these molecules
reside in the ER, although CyP-B is a soluble protein whereas ninaA is bound to the membrane through a transmembrane C-terminal domain. The ninaA protein, which is located specifically in photoreceptor cells in the eye of Drosophila
nzelanognster is necessary for proper function of the visual
pigment rhodopsin. Drosophila photoreceptor cells containing a mutant ninaA gene have dramatically reduced levels of
rhodopsin, resulting in impaired visual function (the
rhodopsin is trapped in the ER[861).When the mutant gene
is replaced with the wild type, rhodopsin function is restored
along with normal vision. The actions of ninaA have also
been shown to be substrate specific: Of the homologous
rhodopsins Rhl, Rh2 (70 YOamino acid identity to Rhl), and
Rh3 (35% amino acid identity to Rhl), only the levels of
Rhl and Rh2 are affected by ninaA mutations. These data
suggest that the ninaA cyclophilin is required for the proper
trafficking of rhodopsin from the ER to the plasma membrane of the eye.[58d. 86J
Another interesting member of the FKBP family that was
discovered using a synthetic rapamycin-based affinity matrixfS5]is FKBP25.r57b1
This protein consists of two clearly
defined domains. The C-terminal region consisting of 114
residues is homologous to FKBP12 (42 'YO sequence identity).
But the N-terminal region consisting of 101 residues does not
show significant homology to any known protein. Unlike
FKBP12 and FKBP13, which bind FK506 and rapamycin
with roughly equal affinity, FKBP25 shows almost 200-fold
selectivity for rapamycin over FK506 (K,(rapamycin) =
0.9 nM; Ki(FK506) = 160 nM). FKBP25 also possesses a possible nuclear localization seq~ence,[~'1
consisting of a Lys,
tetrapeptide and one of two Lys, dipeptides. The apparent
sequestering of FKBP25 in the nucleus suggests a different
role for this protein than for FKBP12 and FKBP13. Rapamycin selectivity, and the presence of an N-terminal domain also point to this conclusion. Identification of the role
of FKBP25 must wait, however, for a number of analyses.
These include confirmation of its presence in the nucleus,
determination of the function and structure of its N-terminal
domain, and assessment of its ability to form inhibitory complexes with FK506 and rapamycin.
The body of data accumulated in the various immunophilins, including those described above, clearly suggest multiple roles for this family of proteins. They are found in
virtually all organisms examined to date. They show tissue
8 5 3
specificity. They are localized in the cell in compartments
ranging from the cytoplasm to the nucleus. They exist in
soluble and membrane-bound forms. Finally, nearly all proteins tested to date show rotamase activity, yet the substrate
specificity varies from protein to protein. These data, while
providing only hints as to the exact nature of the biological
roles of this family, do indicate that there are many interesting aspects of immunophilin function that are still unexplored.
7. Summary and Outlook
In summary, a family of natural products was used to
identify a new family of protein receptors, named immunophilins. Investigations of these proteins resulted in the understanding that they function as complexes with their cognate
immunosuppressive ligands. This led to the use of immunophilin-ligand complexes as probes of cellular signal transduction pathways, allowing the discovery of a common
target, calcineurin. Identification of substrates of CN may
provide yet a further step in this pathway. In this manner, by
beginning with inhibitory natural products, a signal transduction pathway can be investigated molecule by molecule
through the black box of the cytoplasm.
It is not unusual for natural products from one organism
to bind to proteins in another. Examples of this include
many pharmacologically useful compounds including the
opiates and antimitotic agents. It is also not unprecedented
that ligands are able to enhance the interaction between two
proteins. The uniqueness of the immunophilin story resides
in the fact that the ligands FK506, CsA, and rapamycin
possess both these properties. Not only do they bind to
proteins in organisms other than those that produce them,
but in binding they enhance the interactions between these
proteins and others. Thus, all three of the known natural
immunophilin ligands have evolved with two separate but
related abilities. It seems difficult to believe that this dual
function arose fortuitously, especially if one considers the
complex relations between the three ligands described in the
introduction of this review.
This quandary leads to a number of basic, and still unanswered questions regarding the function of these fascinating
compounds and their biological receptors. To begin with,
why do the immunosuppressants FK506, CsA, and rapamycin exist? What purpose does it serve fungi to make compounds with their complicated and interrelated spectrum of
properties? Similarly, why do the immunophilins exist?
Surely they did not evolve to bind molecules found only in
microorganisms. If their purpose is to regulate signal transduction in cells, how are they activated? Are there endogenous molecules whose actions are mimicked by the various
immunosuppressants? If so, can we identify these molecules
and use their bound structures to design better signal transduction inhibitors? At a more detailed level there are still
many questions. What is the physiological role of CN, and
what are its natural substrates? Thus, how close are we to
defining all of the molecules in the black box of the signal
transduction pathway from cell surface receptors to the nucleus? (For a speculative proposal addressing this question
see [89].) What is the target of the later acting FKBPrapamycin complex? Is this molecule related to CN? Finally,
Angew. Clieni. Int. Ed. E q I . 31 (1992) 384 -400
what is the structural basis for the formation of the different
ternary immunophilin-ligand-target complexes? Can the
structures of these complexes provide us with general knowledge about the mechanisms of molecular recognition? The
resolution of these questions will depend, as have the studies
that led us to them, on a variety of experimental methods
and approaches. It is hoped that by applying the complementary tools of chemistry, biology, and structural analysis,
we will be in a position to pose even more exciting questions
in the future.
We would like to thank our many friends and colleagues who
have been involved in the structural studies o f the immunophilins. In particular, our continuing collaborations with Jon
Clardy and his group at Cornell Universit.y have yielded a
wealth of structural insights into the molecular basis of immunophilin function. Our success in determining the solution
structure of unbound FKBPl2 would also have been impossible
witlioul the patience, guidance, and ejyorts of Stephen Michnick, and the hard work and criiical analysis of Tom Wandless
and Professor Martin Karplus of the Harvard Chemistry Department. Thanks go out to Mark Albers and Tom Wandless
,for critiquing the manuscript, Robert Standaert Jor kindly
providing Figure 12, and Dr. Hengming Ke of the University of
North Carolina for providing coordinates of CyP (used to
prepare Fig. 7 ) prior to publication. Finally, we owe a great
debt to other members of the Schreiber group, including M . A . ,
Jun Liu, Pat Martin, R. S., und 7: W whose commitment and
enthusiasm have made the immunophilin project in this laboratory exciting and successful. M . K . R. would like to thank the
American Chemical Society Division of Organic Chemistry
and Merck, Sharp & Dohme,jorfinancial support in the form
of a graduate,fellowship. Immunophilins research in the S. L.
S. laboratory is supported by grants from the National Institutes of General Medical Sciences (GM-38627 and GM40660).
Received: October 25, 1991 [A859IE]
German version: Angew. Chem. 1992. 104, 413.
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