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Originally published In Press as doi:10.1074/jbc.M705061200 on October 17, 2007

J. Biol. Chem., Vol. 282, Issue 50, 36496-36504, December 14, 2007
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A Novel Calmodulin-Ca2+ Target Recognition Activates the Bcl-2 Regulator FKBP38*Formula

Frank Edlich1, Mitcheell Maestre-Martínez, Franziska Jarczowski, Matthias Weiwad, Marie-Christine Moutty, Miroslav Malesevic, Günther Jahreis, Gunter Fischer2, and Christian Lücke

From the Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany

Received for publication, June 20, 2007 , and in revised form, October 16, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The FK506-binding protein 38 (FKBP38) affects neuronal apoptosis control by suppressing the anti-apoptotic function of Bcl-2. The direct interaction between FKBP38 and Bcl-2, however, requires a prior activation of FKBP38 by the Ca2+ sensor calmodulin (CaM). Here we demonstrate for the first time that the formation of a complex between FKBP38 and CaM-Ca2+ involves two separate interaction sites, thus revealing a novel scenario of target protein regulation by CaM-Ca2+. The C-terminal FKBP38 residues Ser290–Asn313 bind to the target protein-binding cleft of the Ca2+-coordinated C-terminal CaM domain, thereby enabling the N-terminal CaM domain to interact with the catalytic domain of FKBP38 in a Ca2+-independent manner. Only the latter interaction between the catalytic FKBP38 domain and the N-terminal CaM domain activates FKBP38 and, as a consequence, also regulates Bcl-2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FKBP38 is a member of the peptidyl prolyl cis/trans-isomerase (PPIase)3 family that belongs to the FK506-binding proteins (FKBPs). Its active form was found to inhibit Bcl-2-controlled neuronal cell death (1). The PPIase activity of FKBP38 is induced by the formation of a FKBP38/CaM-Ca2+ complex, which apparently affects the conformation of FKBP38. Thereby the active site of FKBP38 becomes accessible for substrate proteins, such as Bcl-2. Active site-directed ligands of FKBP38, on the other hand, can prevent the interaction between active FKBP38 and Bcl-2. As a consequence, active site-directed inhibition of FKBP38/CaM-Ca2+ (i) significantly increases cell survival rates of neuroblastoma cells after apoptosis induction by different apoptotic stimuli and (ii) shows substantial neuroprotective and neuroregenerative effects in the rat ischemia model of endothelin-1-induced cerebral middle artery occlusion (2). Furthermore, FKBP38 plays a role in cell size regulation (3) and participates in the development of murine neuronal tissues by regulating sonic hedgehog signaling (4). FKBP38 also interacts with the HIF1 prolyl-4-hydroxylase PHD2 and Hsp90 (5, 6); the latter interaction, however, occurs only in the presence of CaM-Ca2+.

FKBP38 contains two domains: (i) a PPIase domain in the N-terminal region and (ii) a C-terminal tetratricopeptide repeat (TPR) domain. The PPIase domain folds to a FKBP12-like structure consisting of five anti-parallel β-strands and an {alpha}-helix (7). In contrast to the prototypic FKBP12, the rim of the FKBP38 active site is more negatively charged, the bulge in β-strand D is shortened, and the flexible loop between β-strands E and F is slightly extended (7). The TPR domain in the closely related proteins FKBP51 and FKBP52 comprises three TPR motifs (8, 9). Compared with FKBP51 and FKBP52, whose sequences end with an {alpha}-helix following the TPR motifs, FKBP38 additionally contains a membrane anchor that localizes the protein to the membranes of the endoplasmic reticulum and mitochondria (1). Although several FKBP family members share the same domain composition, FKBP38 is the only CaM-Ca2+-regulated PPIase known to date.

The small Ca2+-sensing protein CaM consists of two globular domains connected by a linker that is rather flexible in solution (10). Each domain contains two helix-loop-helix elements, referred to as EF-hand motifs, that can bind Ca2+ among other positively charged ions (11). Upon Ca2+ binding, each CaM domain undergoes structural changes that lead to a concerted exposure of hydrophobic groups in a methionine-rich cleft (12, 13). Each EF-hand in the C-terminal domain binds one calcium ion with a KD value of about 0.2 µM, whereas the EF-hand motifs of the N-terminal domain show 10-fold lower Ca2+ affinities (14). The conformational change between the Ca2+-free and -complexed form of CaM mediates the signal of high cellular Ca2+ concentration for a plethora of target proteins, which participate in physiological processes such as control of muscle contraction, fertilization, cell proliferation, vesicular fusion, and apoptosis. In response to increased cellular Ca2+ concentrations, for example, CaM usually interacts with short amphiphatic helices of ~20 residues that are exposed in the target protein structures, as is the case for CaM kinases such as the CaM kinase II and myosine light chain kinases (15). In many target enzymes, like CaM kinase II and calcineurin A, the interaction with CaM displaces autoinhibitory elements from their active sites (16, 17). In contrast to proteins that interact only with CaM-Ca2+, the Ca2+ release channel ryanodine receptor 1 binds CaM in both the presence and the absence of Ca2+ (13, 18). Neuromodulin and neurogranin, on the other hand, have a higher affinity for Ca2+-free CaM (18–20). Finally, CaM can also interact with nonhelical sites in target proteins. For instance, the edemia factor from Bacillus anthracis not only binds to the Ca2+-coordinated C-terminal domain but also forms a complex with the surface of the Ca2+-free N-terminal CaM domain, thereby restructuring the active site of the edemia factor (21–23).

In the present study we show that two distinct binding sites exist between FKBP38 and CaM-Ca2+: (i) a helical motif, located in the FKBP38 sequence between the TPR motifs and the membrane anchor, forms a complex with the C-terminal CaM domain only in the presence of Ca2+, and (ii) the catalytic domain of FKBP38 binds to the N-terminal CaM domain in a Ca2+-independent manner. Hence, these results reveal a hitherto unknown scenario of Ca2+-independent enzyme activation by CaM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human FKBP38 lacking the membrane anchor (FKBP381–336), the PPIase domain of FKBP38 (FKBP3835–153), human FKBP12, human CaM, the N-terminal domain of human CaM (CaM1–75), the C-terminal domain of human CaM (CaM76–148), and human Bcl-2 were all expressed by using a pET28a vector in Escherichia coli RosettaTM cells, with the C-terminal CaM domain His6-tagged. The maltose-binding protein (MBP)-Bcl-2 fusion protein was purchased from Sigma. An affinity-purified section 4 polyclonal antibody from rabbit against the purified FKBP38 domain (FKBP381–165) was employed. Additional antibodies used were monoclonal hamster anti-Bcl-2 (BD PharMingen, San Diego, CA) and polyclonal rabbit anti-CaM (Santa Cruz Biotechnology, Santa Cruz, CA). Peptide substrates (succinyl-ALPF-p-nitroanilide and succinyl-AFPF-p-nitroanilide) were obtained from Bachem (Heidelberg, Germany).

The FKBP38290–313 peptide representing the CaM-binding site of FKBP38 was produced by solid phase peptide synthesis using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy. The peptide array was prepared using the standard SPOT synthesis protocol (35), with the peptides synthesized stepwise by an Abimed Asp 222 synthesizer on a cellulose membrane derivatized with two β-Ala residues as linker.

Western Blot Analysis of CaM Interaction with the Peptide Array
Before Western blot screening, the dry peptide array membranes were rinsed for 10 min in methanol and for 3x20 min in TBS buffer (30 mM Tris/HCl, pH 7.6, 170 mM NaCl, 6.4 mM KCl). CaM solution (100 nM) in TBS buffer was allowed to react with peptide array membranes in the presence of either 2 mM CaCl2 or 2 mM EGTA for 4 h at 4 °C under gentle shaking. The membrane was subsequently washed three times with TBS buffer before bound protein was blotted onto nitrocellulose membranes and analyzed using rabbit CaM antibodies.

PPIase Activity Measurements
PPIase activity was determined using a protease-coupled assay, with the oligopeptide succinyl-AFPF-4-nitroanilide as substrate (1). Typically, the PPIase activity of FKBP38 was measured in a reaction mixture containing 1 µM of either FKBP381–336 or FKBP3835–153, with 1.5 or 15 µM recombinant human CaM, respectively, and 1 mM CaCl2. Insensitivity of CaM to proteolytic digestion by {alpha}-chymotrypsin was verified in the time range of the kinetic experiments.

Protein-Protein Interaction Assays
Co-immunoprecipitation—The cells were grown in flasks, incubated for 16 h with 50 µM etoposide, and finally harvested. The cell lysis and co-immunoprecipitation experiments were performed according to manufacturer protocols of the immunoprecipitation starter kit (GE Healthcare, Uppsala, Sweden). Prior to incubation, 0.5 µM EGTA was added to the samples. In addition, 1 µM CaCl2 was added to those samples incubated in the presence of Ca2+.

CaM Binding Assay—CaM-Sepharose (GE Healthcare) was pre-equilibrated in 25 mM Tris/HCl buffer (pH 7.5, 200 mM NaCl, 1 mM dithiothreitol) in the presence of either 2 mM CaCl2 or 2 mM EGTA. Subsequently, 30 µg of recombinant FKBP381–336 was incubated with CaM-Sepharose. The Sepharose was washed, and the bound proteins were analyzed by 12.5% SDS-PAGE and Western blotting using polyclonal rabbit anti-FKBP38 antibodies. Competing CaM variants were applied in 5-fold excess.

Bcl-2 Binding Assay—40 µl of 6 µM MBP-Bcl-2 fusion protein was subjected to 40 µl of amylose-resin (New England Biolabs, Beverly, MA) and incubated for 30 min. Thereafter, the beads were washed twice with 25 mM Tris/HCl buffer (pH 7.5, 200 mM NaCl, 1 mM dithiothreitol) and subsequently incubated for 1 h with 40 µl of reaction mixture containing 200 µM of a CaM variant and either 50 µM FKBP381–336 or 100 µM FKBP3835–153. After three washing steps with 25 mM Tris/HCl buffer (pH 7.5, 200 mM NaCl, 1 mM dithiothreitol), the samples were boiled in Laemmli buffer and subjected to SDS-PAGE. Binding of FKBP381–336 was analyzed using polyclonal rabbit anti-FKBP38 antibodies. According to a competition model using Dynafit software, the presence of 1 mM EGTA causes the content of CaM-Ca2+ complex in the sample to drop below 0.6% of the CaM concentration under any chosen condition and is thus referred to as Ca2+-free.

Isothermal Titration Calorimetry
ITC experiments were performed at 25 °C using a MicroCal VP-ITC microcalorimeter. FKBP38 and CaM variants were dialyzed in 10 mM MES buffer (pH 6.8, 100 mM NaCl, 0.05% NaN3) with either 1 mM EGTA or 2 mM CaCl2. The FKBP381–336 solutions ranged in concentration from 35 to 70 µM; FKBP3835–153 was applied in a concentration range from 0.1 to 1.0 mM; the concentrations of the various CaM variants ranged from 0.2 to 2.0 mM in different experiments. The 24-mer peptide (0.25 mM) was applied in 10 mM MES buffer with 2 mM CaCl2.

Usually the FKBP38 solution was placed in the calorimeter cell, and the CaM solution was loaded into the syringe injector. The titrations were carried out in 5–10-µl aliquots, with a 240-s delay between each injection. Calorimetric titration data were fitted using the Origin 5.0 program supplied with the Microcal VP-ITC instrument, to obtain the stoichiometry (N), the association constant (K), the binding enthalpy ({Delta}H), and the binding entropy ({Delta}S). The reported binding constants are an average of duplicate measurements. As a control, the protein or peptide solutions were titrated into buffer.


Figure 1
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  		FIGURE 1.
CaM interacts with the FKBP38 residues Ser290–Asn313 only in the presence of Ca2+ ions. A, an array of 13-mer peptides spanning the FKBP38 sequence was synthesized with forward shifts by one amino acid. CaM interaction with the peptide array in the presence of 1 mM EGTA (upper panel) and 2 mM CaCl2 (lower panel) was analyzed by Western blot. The CaM-Ca2+ interaction pattern is marked by boxes. The respective binding motifs i and ii, comprising the peptides l21–23 and m14–17, correspond to segments Ile285–Ala298 and Glu303–Arg316 in the C-terminal FKBP38 domain, located between the TPR motifs and the membrane anchor. B, sequence of the FKBP38 segment Pro280–Cys321, featuring the Ca2+-dependent CaM-binding motifs identified in the FKBP38 protein sequence (boxes). Probability values (ranging from 0 for low and 9 for high) for an interaction with CaM according to the CaM Target Data Base (calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html) are shown below the FKBP38 sequence. C, Western blot analysis of endogenous proteins from SH-SY5Y cell lysate interacting with a biotin-labeled FKBP38290–313 peptide-bound streptavidin matrix in the presence of either 0.5 µM EGTA or 1 µM Ca2+, using CaM antibodies. The bound protein was dissected from SDS-PAGE, digested with trypsin, and analyzed by MALDI-TOF. D, Western blot analysis of FKBP38 binding to CaM-Sepharose in the presence of 2 mM Ca2+ ions. The flow through is displayed on the left, whereas eluted proteins are shown on the right. The FKBP38290–313 peptide competes with FKBP38 for CaM binding. E, PPIase activity assay, showing FKBP381–336/CaM-Ca2+ inhibition by the FKBP38290–313 peptide. The solid line is fitted according to the dose-dependent decrease in PPIase activity.

 
Fluorescence and NMR Spectroscopy
Steady-state fluorescence spectra were recorded with a PerkinElmer Life Sciences FluoroMax2 fluorescence spectrometer, using a 1 x 1-cm cuvette, an excitation wavelength of 280 nm, and excitation and emission slit widths of 5 and 3 nm, respectively. The protein samples were applied in 10 mM MES buffer with either 2 mM CaCl2 or 1 mM EDTA. The binding constant (KD) was calculated from the fluorescence intensity by using the equation,

Formula 1(Eq. 1)
where P0 = total protein concentration, {alpha} = (Fmax - F)/(Fmax - F0), Fmax = fluorescence intensity at saturation, F0 = initial fluorescence intensity, n = number of independent binding sites, C0 = total CaM concentration at each addition, and KD = dissociation constant.

All of the NMR spectra were acquired at 25 °C, using a Bruker DRX 500 spectrometer as previously described (7). The NMR sample contained 0.5 mM 15N-labeled CaM in 10 mM MES buffer (pH 6.8, 100 mM KCl, 6 mM CaCl2, 0.05% NaN3). FKBP38290–313 was added to the sample in 3-fold excess (1.5 mM). The backbone amide peaks were picked with the program Felix 2000 (Accelrys Inc., San Diego, CA) and assigned via 15N-edited three-dimensional spectra with the help of a previously reported assignment of CaM-Ca2+ (36). The backbone amide assignments are listed in supplemental Table S1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The FKBP38 Segment Ser290–Asn313 Interacts with CaM in a Ca2+-dependent Manner—The interaction between FKBP38 and CaM is dependent on the Ca2+ concentration both in vivo and in vitro (1). A peptide scan was performed to identify the CaM-binding site of FKBP38; CaM bound to two clusters of peptides in the presence of Ca2+, whereas in the absence of Ca2+, no binding of CaM to the array of FKBP38 peptides was detected (Fig. 1A). The two clusters of peptides, which interact with CaM-Ca2+ indicate minimal binding motifs corresponding to the FKBP38 segments Ile285–Ala298 and Glu303–Arg316 (Fig. 1B). Both segments are located in the C-terminal FKBP38 domain between the TPR motifs and the membrane anchor. The sequences of both segments consist largely of positively charged and hydrophobic amino acids and may therefore constitute an amphipathic {alpha}-helix. Interestingly, the putative CaM-binding site predicted by the CaM Target Data Base covers both segments nearly completely (Fig. 1B).


Figure 2
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  		FIGURE 2.
The PPIase domain of FKBP38 interacts with CaM in a Ca2+-independent manner. A, PPIase assay measuring the activity of 1 µM FKBP3835–153 at various CaM concentrations in the presence of 1 mM EGTA (•) or 2 mM CaCl2 ({circ}). In the absence of FKBP3835–153 ({blacktriangledown}) no PPIase activity was detected. B, fluorescence measurements at an excitation wavelength of 278 nm with 1 µM FKBP3835–153 (dashed line), 1 µM CaM (dotted line) and a 1:1 mixture of both proteins (dashed-dotted line) in the presence of 1 mM EGTA. In addition, the calculated spectrum (solid line) represents the sum of the individual protein spectra, as it should appear when the components do not interact. C, titration curve resulting from fluorescence measurements at 340 nm (excitation at 278 nm) of a sample containing 10 µM FKBP3835–153 and various concentrations of CaM in the absence of Ca2+ ions. The solid line represents the fit according to equation 1. D, Western blot analysis (using polyclonal anti-FKBP38 antibodies) of the FKBP381–336 and FKBP3835–153 interactions with a MBP-Bcl-2 fusion protein immobilized on amylose resin. The FKBP38 variants were applied at final concentrations of 20 µM, whereas CaM was used with a final concentration of 200 µM. The incubation was performed in presence of either 1 mM EGTA or 2 mM CaCl2. The input of the FKBP3835–153 sample is displayed in the right lane.

 
Because the matrix-bound peptides (i) exhibit the best accessibility and (ii) adopt the correct secondary structure generally in the central positions, we focused on the middle section of the identified motif. As a consequence, the peptide biotinyl-SKLVKKHAAQRSTETALYRKMLGN-NH2, which corresponds to FKBP38 residues Ser290–Asn313 (FKBP38290–313), was immobilized on streptavidin beads and subsequently tested for binding to endogenous CaM from SH-SY5Y cell lysate. CaM bound to the loaded affinity matrix only in the presence of Ca2+, as confirmed by MALDI-TOF analysis and Western blot (Fig. 1C). Moreover, the FKBP38290–313 peptide competed with FKBP381–336 for binding to CaM-Sepharose (Fig. 1D). To quantify the interference of the peptide with the activity of FKBP381–336/CaM-Ca2+, FKBP38290–313 was applied in a PPI-ase activity assay. The peptide competed with FKBP381–336 for CaM-Ca2+ binding, thus dissociating the FKBP381–336/CaM-Ca2+ complex into the PPIase-inactive constituents with an IC50 value of 1.4 ± 0.13 µM (Fig. 1E). Taken together, these results indicate a Ca2+-dependent complex formation between CaM and FKBP38290–313.

The Catalytic Domain of FKBP38 Features a Second CaM-binding Site—Given the interaction between FKBP38290–313 and CaM-Ca2+, we furthermore examined whether CaM interacts with an FKBP38 construct that lacks the C-terminal CaM-binding site. To this end, the PPIase activity of the catalytic FKBP38 domain (FKBP3835–153) was examined in the presence of CaM. Surprisingly, FKBP3835–153 showed PPIase activity when 10 µM CaM was added, although residues Ser290–Asn313, which form the CaM-binding site in FKBP38, were not present (Fig. 2A). Hence, CaM interacts with a second CaM-binding site located in the catalytic domain of FKBP38. Moreover, the presence of CaM induced the activity of FKBP3835–153 to the same extent in both the presence and the absence of Ca2+, as demonstrated by similar kcat/KM values, which amends the previously reported Ca2+-dependent activation of FKBP38 (1). The fact that this second binding site was not detected in the peptide array suggests a nonlinear binding motif.


Figure 3
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  		FIGURE 3.
The N-terminal domain of CaM interacts with the catalytic domain of FKBP38. A, co-immunoprecipitation of endogenous FKBP38 and Bcl-2 applying hamster anti-Bcl-2 antibody. The precipitates were analyzed by Western blot using anti-FKBP38 and mouse anti-Bcl-2 antibodies. B, co-immunoprecipitation of endogenous FKBP38 and Bcl-2 applying anti-FKBP38 antibody. Antibody/protein complexes were analyzed by Western blot using anti-Bcl-2 and anti-FKBP38 antibodies. C, FKBP381–336 (10 µM, input) was applied in the absence and presence of 20 µM CaM variants and 2 mM Ca2+ to MBP-Bcl-2 immobilized on amylose resin. Bound protein was eluted by 200 mM maltose and analyzed by Western blot using anti-FKBP38 antibodies. D, fluorescence measurements at 278 nm of 1 µM FKBP3835–153 (dashed line), 1 µM N-terminal CaM domain (solid line), and a 1:1 mixture (dotted line) in the presence of 1 mM EGTA. In addition, the calculated spectrum (dashed-dotted line) represents the sum of the individual protein spectra, as it should appear when the components do not interact. E, activity measurements of 1 µM FKBP3835–153 using the PPIase assay, in the presence of 2 mM CaCl2 and various concentrations of either the N-terminal (•) or the C-terminal ({circ}) domain of CaM. The dose-response curves are displayed for the presence of the N-terminal (dashed line) and the C-terminal (dotted line) CaM domain.

 
These results reveal a Ca2+-independent interaction between CaM and the catalytic domain of FKBP38, which apparently induces the active conformation of FKBP38. Remarkably, the dose response curves do not adopt hyperbolic character, as one might expect. This effect is due to the adsorption of FKBP38 and CaM variants at the cuvette surface and was successfully suppressed in the fluorescence assays described below.

The fluorescence measurements were performed to investigate the CaM interaction with FKBP3835–153 in the absence of Ca2+. Fig. 2B shows the fluorescence spectra of both proteins in the absence of Ca2+ ions. The spectrum of the FKBP3835–153/CaM complex is red-shifted by 4 nm, and its amplitude is reduced by 8.5% compared with the combined spectrum of the isolated proteins. A titration experiment shows the changes in the protein fluorescence of FKBP3835–153 in dependence of the CaM concentration, revealing a KD value of 6.63 ± 0.95 µM for this interaction (Fig. 2C). These data again indicate a Ca2+-independent, specific binding of FKBP3835–153 to CaM. In contrast, the combined fluorescence spectrum of separately measured FKBP12 and CaM is nearly identical to the spectrum of FKBP12 in the presence of CaM (supplemental Fig. S1).

To further investigate the consequences of this interaction between the catalytic FKBP38 domain and CaM on the binding of Bcl-2, we analyzed the binding of either FKBP381–336 or FKBP3835–153 to a Bcl-2 loaded affinity matrix. Fig. 2D shows that FKBP3835–153 bound to Bcl-2 both in presence and absence of Ca2+ whenever CaM was present, whereas no binding of FKBP3835–153 to Bcl-2 was detected in the absence of CaM. FKBP381–336 and FKBP3835–153 competed for Bcl-2 binding in the presence of Ca2+-free CaM, but when Ca2+ was present the FKBP381–336/CaM-Ca2+ complex prevented the binding of FKBP3835–153 to the Bcl-2 matrix. Taken together, these results (i) demonstrate the existence of a Ca2+-independent CaM-binding site in the catalytic domain of FKBP38 and (ii) indicate a higher affinity between the two proteins when both binding sites of FKBP38 are present.

The N-terminal Domain of CaM Interacts with the Catalytic Domain of FKBP38—To analyze the respective counterparts in CaM that interact with the two binding sites in FKBP38, a tryptic digest was performed to separate both CaM domains and analyze their interactions with FKBP38. The digest resulted in two fragments with a cleavage site between the CaM residues Lys75 and Met76, according to Edman digest analysis (data not shown). Therefore, two CaM fragments were cloned and expressed in E. coli. The fragment of the N-terminal CaM domain comprised residues Ala1–Lys75 (CaM1–75), whereas the fragment corresponding to the C-terminal CaM domain consisted of the sequence Met76–Lys148 (CaM76–148).

First, we used an affinity matrix loaded with the peptide that corresponds to FKBP38290–313 to bind endogenous CaM from SH-SY5Y cell lysate. Endogenous CaM was sequestered to the matrix and thus removed from the cell lysate (supplemental Fig. S2). Next, co-immunoprecipitation experiments were performed to analyze the interactions of endogenous FKBP38 and Bcl-2 in the presence of different CaM variants. When Ca2+ was present, FKBP38 interacted with Bcl-2 in the lysate of SH-SY5Y cells, but in CaM-depleted cell lysate no such interaction was observed (Fig. 3, A and B). The addition of full-length CaM to the CaM-depleted cell lysate restored the ability of endogenous FKBP38 to interact with Bcl-2. Upon the addition of the separate CaM domains, however, FKBP38/Bcl-2 interactions were restored only in the presence of CaM1–75, whereas no interactions between FKBP38 and Bcl-2 occurred in the presence of CaM76–148. These results indicate that it is the N-terminal domain of CaM that interacts Ca2+-independently with the catalytic FKBP38 domain.

Thus, we subsequently also investigated the interaction of FKBP381–336 with a Bcl-2 affinity matrix in the presence of different CaM variants. FKBP381–336 interacted with Bcl-2 in the presence of full-length CaM or CaM1–75, but no FKBP381–336 bound to the Bcl-2 affinity matrix when only CaM76–148 was present (Fig. 3C). Hence, the N-terminal CaM domain mediates the interactions with the catalytic FKBP38 domain, as is further evidenced by a decrease of the protein fluorescence amplitude by 17.8 ± 0.1% and a 1-nm red shift observed in the spectrum of FKBP3835–153 in the presence of CaM1–75 (Fig. 3D). Fluorescence measurements of the FKBP3835–153/CaM1–75 complex in the presence and absence of Ca2+ demonstrated that (i) the interaction between these two protein variants occurs both with and without Ca2+ and (ii) the spectral changes in the catalytic FKBP38 domain are identical under both conditions (supplemental Fig. S3).

Further, we tested the influence of the two separate CaM domains on the PPIase activity of the catalytic FKBP38 domain (Fig. 3E). Consistent with the previous data, only CaM1–75 caused an increase in the PPIase activity of FKBP3835–153, thus demonstrating that this interaction between the N-terminal domains is required and sufficient for the activation of FKBP38. In contrast, the addition of the C-terminal CaM domain did not alter the catalytic activity of FKBP3835–153.

Isothermal titration calorimetry (ITC) measurements with FKBP3835–153 and CaM1–75 yielded nearly identical results in both the presence and the absence of Ca2+. In both cases, the data revealed 1:1 stoichiometries with KD values of 6.9 ± 0.92 and 7.0 ± 0.55 µM, respectively, according to a one-binding-site model (Table 1 and supplemental Fig. S4).


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  		TABLE 1
Parameters for FKBP38 and CaM-Ca2+ interaction Parameters for this interaction between FKBP38 and CaM-Ca2+ at two different binding sites, measured in 10 mM MES (pH 6.8), 100 mM KCl at 20 °C in the presence of either 1 mM EGTA or 2 mM CaCl2. In each respective binding model, F1 and F2 represent the N-terminal and C-terminal binding sites of FKBP38, respectively, whereas C1 and C2 stand for the corresponding CaM-binding sites.

 
FKBP38290–313 Binds to the C-terminal Domain of CaM—Given the interaction between the catalytic FKBP38 domain and the N-terminal CaM domain, we were interested in identifying the part of CaM that binds to the Ca2+-dependent binding site of FKBP38. Therefore, we analyzed the influence of different CaM variants on the interaction between FKBP381–336 and CaM-Sepharose, because FKBP38 binds in a Ca2+-dependent manner to CaM-Sepharose. Fig. 4A shows that in addition to full-length CaM also CaM76–148 competed with CaM-Sepharose for FKBP381–336 binding, whereas the presence of CaM1–75 did not significantly reduce the amount of matrix-bound FKBP381–336. Hence, the C-terminal CaM domain interacts with FKBP38290–313.

Furthermore, PPIase activity measurements were performed to test whether CaM76–148 interferes with the activation of FKBP38 by CaM-Ca2+. Indeed, CaM76–148 lowered the activity of the FKBP381–336/CaM-Ca2+ complex, thus demonstrating a competition with full-length CaM for binding to FKBP381–336 (Fig. 4B). Thereby, CaM76–148/Ca2+ bound to FKBP381–336 with a Ki value of 2.01 ± 0.71 µM. Both CaM domains were subsequently used in ITC experiments to determine binding to FKBP38290–313. Only CaM76–148 interacted with the peptide that corresponds to FKBP38290–313, revealing a 1:1 complex with a KD value of 2.2 ± 0.24 µM according to a one-binding site model fit (Table 1 and supplemental Fig. S5).

The interaction between FKBP38290–313 and CaM76–148 on the one hand and the interaction between the catalytic FKBP38 domain and the N-terminal CaM domain on the other hand were also detected when ITC measurements were performed with FKBP381–336 and CaM in the presence of Ca2+ (supplemental Fig. S6). These measurements revealed two binding sites, each with a 1:1 molar ratio between both binding partners according to a two-binding-site model (Table 1). Thereby, one binding site interacted with a KD of 2.2 ± 0.22 µM, whereas the other binding site showed a KD value of 4.9 ± 0.97 µM. Both binding constants are similar to those derived from measurements with the single domains, although the interaction between the isolated N-terminal domains shows slightly lower affinity than the second binding site of the full-length proteins (Table 1).


Figure 4
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  		FIGURE 4.
The C-terminal domain of CaM interacts with FKBP38290–313. A, Western blot analysis of FKBP381–336 (15 µg) binding to CaM-Sepharose in presence of 2 mM CaCl2. Various CaM constructs were applied at a final concentration of 50 µM to compete with the Sepharose for FKBP38 binding. Bound proteins were dissolved in SDS-PAGE sample buffer after three washing steps and subjected to SDS-PAGE. Subsequently, Western blotting was performed using anti-FKBP38 antibodies. B, activity measurements of 1 µM FKBP381–336 using the PPIase assay, in the presence of 2 mM CaCl2 and various concentrations of CaM76–148. The solid line is fitted according to the dose-dependent decrease in PPIase activity, revealing a Ki value of 2.0 ± 0.71 µM. C, section of two overlaid [1H,15N]-HSQC spectra collected at pH 6.8 and 25 °C (1H resonance frequency of 500.13 MHz), representing free CaM-Ca2+ (blue) and CaM-Ca2+ complexed with FKBP38290–313 (red). Major signal shifts caused by peptide binding are found for specific residues that belong to the C-terminal CaM domain (i.e. residue numbers 82 and higher). Side-chain resonances are marked by sc. D, graphic representation of the C-terminal CaM domain (yellow, Protein Data Bank code 1CDL) and the peptide C{alpha}-traces of 16 superposed CaM complexes with various target proteins (Protein Data Bank codes 1CDL, 1CFF, 1IWQ, 1L7Z, 1MXE, 1NIW, 1QTX, 1SY9, 1YR5, 2BBM, 2BCX, 2BE6, 2F2O, 2F2P, 2F3Y, and 2FOT). CaM residues affected significantly by the presence of FKBP38290–313 are distributed throughout the target protein-binding cleft, as indicated by red coloring. E, same representation as in the previous panel, rotated by 90° to better visualize the binding cleft of the C-terminal CaM domain. The two calcium atoms (magenta) are partially visible in the upper part of the domain, at the opposite end relative to the binding cleft.

 
Finally, NMR spectra were collected to locate the site where FKBP38290–313 binds to CaM. [1H/15N]-HSQC spectra of 15N-labeled CaM-Ca2+ (Fig. 4C), collected both in absence and presence of FKBP38290–313, revealed significant chemical shift perturbations for several residues in the C-terminal CaM domain, comprising segments Val91–Phe92, Glu104–Val108, Ala128–Asn129, and Met145–Lys148, which are located within or near the cleft where peptide ligands generally bind to CaM (Fig. 4, D and E).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As shown previously, FKBP38 is the only enzyme catalyzing protein folding that is regulated by the Ca2+-sensing protein CaM (1). However, the interaction between both proteins remained puzzling. Using separate CaM domains, it was now possible to demonstrate that the FKBP38 regulation is mediated by two distinct interaction sites in both proteins, revealing a novel scenario of CaM target activation.

One interaction involves a short sequence in the C-terminal FKBP38 domain that comprises residues Ser290–Asn313 between the TPR motifs and the membrane anchor. It binds to the C-terminal CaM domain in a Ca2+-dependent manner with a 1:1 stoichiometry. Both the FKBP38290–313 peptide and the isolated C-terminal CaM domain proved sufficient to (i) prevent interactions between FKBP381–336 and a CaM-loaded affinity matrix and (ii) inhibit PPIase activity of the FKBP381–336/CaM-Ca2+ complex. Heteronuclear two-dimensional [1H, 15N]-HSQC experiments showed an interaction between the FKBP38290–313 peptide and the target protein-binding cleft in the C-terminal domain of CaM-Ca2+, suggesting binding site recognition of FKBP38290–313 by CaM76–148 that is comparable with the previously described CaM complexes (15, 24). Hence, the interaction between the C-terminal domains of FKBP38 and CaM-Ca2+ is in good agreement with previous observations of the Ca2+-dependent interaction between FKBP38 and CaM (1).

Moreover, our study discovered a second interaction in the FKBP38/CaM-Ca2+ complex that occurs between the N-terminal domains of both proteins. PPIase measurements revealed enzymatic activity of the catalytic FKBP38 domain in the presence of either Ca2+-free CaM or CaM-Ca2+. Furthermore, co-immunoprecipitation experiments using CaM-depleted lysate from SH-SY5Y cells confirmed that interactions between FKBP38 and Bcl-2 are induced only in the presence of either full-length CaM or CaM1–75, whereas CaM76–148 did not cause Bcl-2/FKBP38 interactions. These results clearly demonstrate that only CaM1–75 is responsible for the activation of FKBP38, independent of Ca2+. This interaction between the catalytic FKBP38 domain and the N-terminal CaM domain is therefore crucial for the participation of FKBP38 in the regulation of programmed cell death via its interaction with Bcl-2. These results suggest a novel scenario where the two CaM domains bind to separate interaction sites in FKBP38, one of which requires Ca2+, whereas the other does not.

Many CaM-regulated proteins, such as myosine light chain kinases or CaM-dependent kinases, interact with CaM only in presence of Ca2+ (25), whereas Ca2+-free CaM has only a low affinity for these particular target proteins (13). In the CaM-Ca2+ complex of the edemia factor from B. anthracis, only the C-terminal CaM domain coordinates Ca2+, whereas no calcium ions are found in the N-terminal CaM domain (22, 23). Furthermore, CaM binds with its Ca2+-free N-terminal domain to the Ca2+-ATPase in the membrane of erythrocytes (26). Finally, several CaM-binding partners are regulated by CaM in a Ca2+-independent fashion (18). For instance, the cGMP-dependent protein kinase is regulated by CaM, but the presence or absence of Ca2+ does not influence the regulation of this enzyme (27). Such a Ca2+-independent regulation by CaM was also identified for the adenylyl cyclase from Bordetella pertussis (28). The interaction of FKBP38 and CaM-Ca2+ therefore shares certain properties previously described for other CaM-binding partners but features a novel combination of both a Ca2+-dependent and a Ca2+-independent interaction to regulate enzymatic activity.


Figure 5
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  		FIGURE 5.
Proposed mechanism for FKBP38 activation by CaM-Ca2+ via two independent binding sites assuming a 1:1 complex stoichiometry. FKBP38 (white), containing a PPIase domain (PPIase), a TPR domain (TPR), a CaM target site (zigzag line), as well as a C-terminal membrane anchor (MA), and CaM (gray), consisting of two domains (N and C), do not interact in the absence of Ca2+ (I). After an increase of the cellular Ca2+ concentration, the C-terminal CaM domain coordinates two Ca2+ (II), whereas the N-terminal CaM domain can be found either in the Ca2+-free (depicted here) or Ca2+-coordinated form, depending on the cellular Ca2+ level. The Ca2+ binding causes structural changes in the C-terminal CaM domain. Subsequently, the FKBP38/CaM-Ca2+ complex is formed because of the binding of FKBP38290–313 to the target protein-binding cleft of the C-terminal CaM domain (III). In this complex the N-terminal CaM domain can now interact with the catalytic domain of FKBP38 in a Ca2+-independent manner (IV), thereby activating the catalytic domain of FKBP38. In the case of a 1:2 complex stoichiometry, the PPIase domain would rather be approached by a second CaM-Ca2+ molecule.

 
The interaction between FKBP3835–153 and CaM was also detected by protein fluorescence. Moreover, ITC measurements revealed a 1:1 complex for the interaction between the catalytic FKBP38 domain and the N-terminal CaM domain, with a KD value of 7.0 ± 0.55 µM. The binding between the C-terminal interaction sites of both proteins, however, increases the affinity significantly to a KD of 1.4 µM (1). These results apparently favor a 1:1 stoichiometry between FKBP38 and CaM-Ca2+, because FKBP38 is activated in cells in a Ca2+-dependent manner with higher affinity than the binding constants of the single interaction sites (Fig. 5). However, a 1:2 stoichiometry could theoretically also be possible, if allosteric effects induced by the C-terminal interaction assist the interaction of the N-terminal binding sites or other as yet unknown factors facilitate the binding of two CaM molecules to FKBP38.

The interaction between FKBP38290–313 and CaM76–148 is entropically driven, as also found for other CaM-Ca2+-binding sites, because of the dehydration of the binding interface caused by the dominant role of hydrophobic interactions (29). The higher affinity of the C-terminal interaction is essential for the regulation of FKBP38 under physiological conditions, because the availability of free CaM-Ca2+ limits the activation of target proteins in the cell (30, 31). Considering the high cellular abundance of CaM, reaching 0.5% of the total protein content in the brain (32), this appears surprising, but the cellular concentration of free CaM-Ca2+ does not exceed 1 pM (13). Thus, the CaM-Ca2+ concentration is far below the concentration of cellular CaM-Ca2+ target proteins, implying a coupling of CaM-dependent enzyme activities by the competition for the limiting CaM-Ca2+ species (33, 34). This competition with other CaM target proteins apparently results in a Ca2+-dependent interaction between FKBP38 and CaM, as indicated by the increased affinity of the full-length proteins.

Interestingly, in neuronal cells large amounts of CaM are sequestered to the endoplasmic reticulum and mitochondria by proteins such as neuromodulin and neurogranin, which exhibit a higher CaM affinity in the absence of Ca2+, thus forming a CaM reservoir (19, 20). This CaM pool is released in the presence of Ca2+, resulting in particularly high concentrations of free CaM-Ca2+ at the endoplasmic reticulum and mitochondria membranes of neuronal cells, where FKBP38 is located in the cell (1).

Therefore we propose that a cellular increase in the Ca2+ concentration initially induces the formation of a complex between the C-terminal domains of FKBP38 and CaM, subsequently leading to an activation of FKBP38 as a result of the interaction between the N-terminal CaM domain and the catalytic FKBP38 domain. Hence, a high cytosolic Ca2+ concentration is presumably favorable for the activation of FKBP38 by CaM and thus also for the regulation of Bcl-2 by FKBP38/CaM-Ca2+ in neuronal apoptosis.


   FOOTNOTES
 
* This work was supported by SFB 604 of the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6 and Table S1. Back

1 To whom correspondence may be addressed. Tel.: 345-55-22839; Fax: 345-55-11972; E-mail: edlich{at}enzyme-halle.mpg.de. 2 To whom correspondence may be addressed. Tel.: 345-55-22800; Fax: 345-55-11972; E-mail: fischer{at}enzyme-halle.mpg.de.

3 The abbreviations used are: PPIase, peptidyl prolyl cis/trans-isomerase; FKBP, FK506-binding protein; CaM, calmodulin; TPR, tetratricopeptide repeat; MBP, maltose-binding protein; MES, 4-morpholineethanesulfonic acid; ITC, isothermal titration calorimetry; HSQC, heteronuclear single quantum coherence. Back


   ACKNOWLEDGMENTS
 
We thank Angelika Schierhorn and Peter Rücknagel for MALDI-TOF analysis and Edman digest. We are grateful to Melanie Kirchner and Beate Rappsilber for excellent technical assistance.



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 INTRODUCTION
 MATERIALS AND METHODS
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 DISCUSSION
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