Structural characterization of the C2 domain of ... - Wiley Online Library

Eur. J. Biochem. 268, 1107±1117 (2001) q FEBS 2001

Structural characterization of the C2 domain of novel protein kinase C: Josefa GarcõÂa-GarcõÂa, Juan C. GoÂmez-FernaÂndez and Senena CorbalaÂn-GarcõÂa Departamento de BioquõÂmica y BiologõÂa Molecular `A', Facultad de Veterinaria, Universidad de Murcia, Spain

Infrared spectroscopy (IR) and differential scanning calorimetry (DSC) were used to study the biophysical properties of the PKC:-C2 domain, a C2 domain that possess special characteristics as it binds to acidic phospholipids in a Ca21-independent manner and no structural information about it is available to date. When the secondary structure was determined by IR spectroscopy in H2O and D2O buffers, b sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D2O showed a broadening in the amide I 0 band starting at 45 8C. Curve fitting analysis of the spectra demonstrated that two components appear upon thermal denaturation, one at 1623 cm21 which was assigned to aggregation and a second one at 1645 cm21, which was assigned to unordered or open loop structures. A lipid binding assay has demonstrated that PKC:-C2 domain has preferencial affinity for PIP2 although it exhibits maximal binding activity for phosphatidic acid

when 100 mol% of this negatively charged phospholipid was used. Thus, phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure and thermal stability. These experiments showed that the secondary structure did not change upon lipid binding and the thermal stability was very high with no significant changes occurring in the secondary structure after heating. DSC experiments demonstrated that when the C2-protein was scanned alone, it showed a Tm of 49 8C and a calorimetric denaturation enthalpy of 144.318 kJ´mol21. However, when phoshatidic acid vesicles were included in the mixture, the transition disappeared and further IR experiments demonstrated that the protein structure was not modified under these conditions.

C2 domains are modules present in a wide variety of proteins and participate in different types of interactions. The C2 domain was originally identified as the second of four conserved domains in the classical isoforms of mammalian PKCs [1]. Further studies revealed the presence of homologous C2 domains in other proteins, including the novel PKC family that was initially thought not to have a C2-domain and is Ca21-independent. This C2-domain is located at the N-terminus and does not bind Ca21 [2,3].

In general, most of the proteins containing C2-domains function in signal transduction or membrane traffic [4]. Pioneer studies with the C2A-domain of synaptotagmin I revealed that the C2 domain acts as a Ca21 binding motif [5±7]. This function has also been demonstrated in several other C2-domain-containing proteins such as classical PKCs [8±11], cPLA2 [12,13] and Nedd4 [14] all of which bind to phospholipids in a Ca21-dependent manner. Furthermore, it has recently been found that Ca21 forms a bridge between the C2 membrane-binding domain of PKC and PtdSer [15]. Moreover, there are many C2-domains that are involved in lipid binding and do not bind Ca21, as it is the case of the C2 domains from the novel PKCs, the functions of which are still not well characterized [16,17]. Another function attributed to C2-domains is that they are involved in protein±protein interactions: for example, the C2A domain of synaptotagmin I binds to syntaxin 1 in a Ca21-dependent manner [18,19], while the C2B domain of the same protein binds to clathrin AP-2, inositol polyphosphates, b-SNAP and Ca21 channels in a Ca21-independent way [20±23]. It has been described recently how the C2-domain of PKCd is involved in the interaction with GAP-43, a growth-associated protein [24]. A further example is given by RACKs, which are not PKC substrates but which instead, interact with C2 domains of PKCs to direct them to cellular compartments [25±27]. X-ray diffraction analysis of several C2 domains has revealed that the structure consists of a compact b sandwich composed of two four-stranded b sheets [6,15,28±32]. Basically, three loops at the top of the domain and four at the bottom connect the eight b strands and, interestingly, two distinct but easily interconverted topological folds have

Correspondence to S. CorbalaÂn-GarcõÂa, Departamento de BioquõÂmica y BiologõÂa Molecular `A', Facultad de Veterinaria, Universidad de Murcia, Apartado Postal 4021, E-30080, Murcia, Spain. Fax: 1 34 968364766, Tel.: 1 34 968364785, E-mail: [email protected] Abbreviations: CBR1, Ca21 binding region 1; CBR3, Ca21 binding region 3; GAP-43, GTPase-activating protein-43; Hepes, N-(2-hydroxyethiyl)piperazine-N 0 -(2-ethanesulfonic acid); IR, infrared spectroscopy; PtdIns3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; cPLA2, cytosolic phospholipase A2; PLC1, phospholipase C1; PtdSer, phosphatidylserine; PTEN, protein-tyrosin phosphatase; RACK, receptor for activated C kinase; -SNAP, soluble NSF attachment protein; IPTG, isopropyl thio-b-d-galactoside; POPA, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate. Enzymes: GTPase-activating protein-43 (GAP-43; EC 3.1.5.1); phosphatidylinositol 3-kinase (PtdIns3K; EC 2.7.1.137); protein kinase C (PKC; EC 2.7.1.37); cytosolic phospholipase A2 (cPLA2; EC 3.1.1.4); phospholipase C1 (PLC1; EC 3.1.4.3); protein-tyrosin phosphatase (PTEN; EC 3.1.3.48). (Received 18 October 2000, revised 15 December 2000, accepted 19 December 2000).

Keywords: protein kinase C; C2 domain; phosphatidic acid; Fourier transform infrared spectroscopy.

1108 J. GarcõÂa-GarcõÂa et al. (Eur. J. Biochem. 268)

been found: topology I becomes topology II when its Nand C-termini are fused and new termini are generated by cutting the loop between strands 1 and 2 [4,17]. The C2 domains of classical PKCs are classified as having a type I topology, while those of novel PKCs exhibit a type II topology [17]. The existence of two topologies is a still unsolved question and, in spite of some studies, it is not clear why C2 domains occur in two modes. As mentioned above, novel PKCs present a unique type of C2 domain and, differently to classical PKCs or phospholipases, they are able to bind to acidic phospholipids in a Ca21-independent manner. So far, very little is known about the lipid binding mechanism of these isoforms. In this study, IR was used to characterize the structure and thermal denaturation mechanism of the C2-domain of PKC in the absence and in the presence of phospholipid. Interestingly, the structural components of this C2-domain show several differences from the C2 domain of PKC, which we previously studied [33] and these differences might be attributed to different structural motifs. A phospholipid binding assay has enabled us to determine that the electronegative character of the phospholipids is the driving force to promote binding of the PKC:-C2 domain. Furthermore, it has been demonstrated that PKC:-C2 domain presents a slightly higher affinity for phosphatidic acid than for other anionic phospholipids. A differential scanning calorimetry study of the heat-induced denaturation of this PKC:-C2 domain revealed that the presence of phospholipid protects against thermal denaturation.

E X P E R I M E N TA L P R O C E D U R E S Construction of expression plasmids The DNA fragment corresponding to the C2 domain of PKC: (residues 6±134) was amplified using PCR. The PKC cDNA was a kind gift from Drs Nishizuka and Ono (Kobe University, Kobe, Japan). The resulting 384 bp PCR fragment was subcloned into the BamHI and HindIII sites of the bacterial expression vectors, pGEX-KG [34] and pET28a(1), in which the inserts are fused to GST and 6His tag, respectively. All constructs were confirmed by DNA sequencing. PKCa-C2 constructs were described previously [33]. Expression and purification of the His-PKC-C2 and GST-PKC-C2 domains The pET28a(1) plasmid containing the PKC:-C2 domain was transformed into BL21(DE3) Escherichia coli cells. The bacterial cultures (D600 0.6) were induced for 5 h at 30 8C with 0.5 mm of isopropyl thio-b-d-galactoside (IPTG) (Roche, Germany). The cells were lysed by sonication in lysis buffer (25 mm Hepes, pH 7.4, and 100 mm NaCl) containing protease inhibitors (10 mm benzamidine, 1 mm phenylmethanesulfonylfluoride and 10 mg´mL21 of trypsin inhibitor). The soluble fraction of the lysate was incubated with Ni/nitriloacetic acid Agarose (Qiagen, Hilden, Germany) for 2 h at 4 8C. The Ni beads were washed with lysis buffer containing 20 mm imidazole. The bound fractions were eluted with the same buffer containing 250 mm imidazole. 6His tag was removed after thrombin cleavage and, finally, the PKC:-C2 domain was

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desalted and concentrated using an Ultrafree-5 centrifugal filter unit (Millipore Inc., Bedford, MA). The pGEX-KG plasmid containing PKC:-C2 domains was transformed into HB101 Escherichia coli cells. The bacterial cultures (D600 0.6) were induced with 0.2 mm IPTG for 5 h at 30 8C. The cells were lysed by sonication in phosphate buffer saline (NaCl/Pi) containing protease inhibitors (10 mm benzamidine, 1 mm phenylmethanesulfonylfluoride and 10 mg´mL21 of trypsin inhibitor). The soluble fraction of the lysate was incubated with glutathione-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) for 30 min at 4 8C, which were then washed with NaCl/Pi three times. Protein concentration was determined using either the method described by Lowry et al. [35] or by densitometry after analyzing the samples on a 15% SDS/ PAGE gel and by Coomassie Blue (Sigma, St Louis, MO, USA) staining. The PKCa-C2 domain was purified as described by Garcia-Garcia et al. [33]. Preparation of lipid vesicles. Lipid vesicles were generated by mixing chloroform solutions of 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (phosphatidylcholine) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) and 1,2-dioleoyl-sn-glycero-3-phosphate (phosphatidic acid) (Lipid Products, Nutfield, Surrey, UK) or 1-palmitoyl-2oleoyl-sn-glycero-3-phosphate (POPA) (Avanti Polar Lipids, Inc., Alabaster, AL) in the desired proportions and then dried from the organic solvent under a nitrogen stream and further dried under vacuum for 60 min. 1,2-Dipalmitoyl-l-3-phosphatidyl-N-methyl-[3H]choline (Dupont, Boston, MA, USA; specific activity 56 Ci´mmol21) was included in the lipid mixture as a tracer, at approximately 3000±6000 cpm´mg21 of phospholipid. Dried phospholipids were resuspended in buffer containing 25 mm Hepes pH 7.4, 0.1 m NaCl and 0.2 mm EGTA by vigorous vortexing and subjected to direct probe sonication (30 cycles of 30 s). The samples were then centrifuged at 13 000 g during 20 min to discard remaining multilamellar vesicles. Phospholipid binding measurements Standard assay. The procedure described by Davletov and SuÈdhof [5], was used with minor modifications. 20 mg of PKC:-C2 domain bound to glutathione±sepharose beads were used. Beads were prewashed with the respective test solutions and resuspended in 0.1 mL of buffer containing 50 mm Hepes pH 7.2, 0.1 m NaCl 0.5 mm EGTA and 20 mg of the corresponding lipids. The mixture was incubated at room temperature for 15 min with vigorous shaking, then briefly centrifuged in a tabletop centrifuge. The beads were washed three times with 1 mL of the incubation buffer without liposomes. Liposome binding was then quantified by liquid scintillation counting of the beads. IR spectroscopy Lyophilized PKC:-C2 domains were dissolved in H2O or D2O at approximately 20 and 8 mg´mL21, respectively. The proteins were incubated overnight at 4 8C to maximize H-D exchange when D2O was used. To study infrared amide bands of the proteins in the presence of lipids, small unilamellar vesicles in D2O or H2O buffer containing 25 mm Hepes, 20 mm NaCl and 0.2 mm EGTA, pH 7.4,

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PKC:-C2 domain secondary structure (Eur. J. Biochem. 268) 1109

were mixed in the desired proportions with the protein solution. Infrared spectra were recorded using a Bruker Vector 22 Fourier transform infrared spectrometer equipped with a MCT detector. Samples were examined in a thermostated Specac 20710 cell (Specac, Kent, UK) equipped with CaF2 windows and 6 mm spacers for samples in H2O medium or 50-mm spacers for samples in D2O medium. The spectra were recorded after equilibrating the samples at 25 8C for 20 min in the infrared cell. A total of 128 scans were accomplished for each spectrum with a nominal resolution of 4 cm21 and then Fourier transformed using a triangular apodization function. A sample shuttle accessory was used to obtain the average background and sample spectra. The sample chamber of the spectrometer was continuously purged with dry air to prevent atmospheric water vapor obscuring the bands of interest. Samples were scanned between 25 8C and 75 8C at 5-8C intervals with a 5-min delay between each scan using a circulation water bath interfaced to the spectrometer computer. Spectral subtraction was performed interactively using the spectracalc program (Galactic Industries Corp., Salem, NH, USA). The spectra were subjected to deconvolution and secondderivation using the same software. Fourier selfdeconvolution was carried out using a bessel apodization function, a Lorentzian shape with a resolution enhancement parameter, K, of 2.6 and a full width at half-height of 20 cm21. Both deconvolution and derivation gave the number and position, as well as an estimation of the bandwidth and intensity of the bands making up the amide I region. Thereafter, curve-fitting was performed and the heights, widths and positions of each band were optimized successively [36,37]. The fractional areas of the bands in the amide I region were calculated from the final fitted band areas. With the aim of compairing the results obtained for the PKCa-C2 domain in previous work [33] and those obtained in the present work, we performed a control spectrum of the PKC-C2 domain with a nominal resolution of 2 cm21 and both deconvolution and second derivative were performed. The number of components and band possitions assigned were the same than those obtained in the present work. Differential scanning calorimetry A high sensitivity MicroCal MC-2 scanning calorimeter (Northampton, MA) was used in these experiments. Scan rates were 60 8C´h21. The protein concentration was 100 mm. A buffer profile was subtracted from the sample

Fig. 1. IR, Fourier and second-derivative spectra. (A) IR spectra of the PKC:-C2 domain in the amide I region at 25 8C in H2O (continuous line) and D2O (dotted line) buffer containing 25 mm Hepes pH 7.4 and 0.2 mm EGTA. Protein concentration was 20 mg´mL21 and 8 mg´mL21 in H2O and D2O, respectively. Resolution-enhanced amide I and I 0 band contours of PKC:-C2 domain. (B) Fourier self-deconvolved spectra in H2O and D2O using a Lorentzian line-shape function with 20 cm21 full width at half-height and a enhancement factors of 2.3 (a) and 2.6 (b). (C) Second-derivative spectra as calculated by 11 and 17 points Savitzky/Golay algorithm in H2O and D2O, respectively.


Fig. 2. Amide I band decomposition of the PKC:-C2 domain in H2O (A) and D2O (B) at 25 8C. The position of the individual bands was obtained from the resolution-enhanced spectra represented in Fig. 1. The parameters corresponding to the component bands are reflected in Table 1. The dashed line represents the curve-fitted spectra. The increment of absorbance units (K) was 0.02 (A) and 0.1 (B).

scans. Baselines were created by the cubic spline and subtracted. The calorimetric parameters Tm (midpoint temperature of the transition) and Hcal (calorimetric enthalpy) were extracted from the data by the origin software provided by MicroCal.

R E S U LT S Structure of the PKC:-C2 domain To study the structure of the PKC:-C2 domain, a recombinant fusion protein was generated and purified as described in the Experimental procedures section. Protein

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structure was studied by analysing the amide I region of the infrared spectrum both in H2O and in D2O. Figure 1A depicts the Amide I and Amide I 0 spectra of this protein, dispersed in H2O and D2O buffers, respectively, after solvent subtraction. It can be observed that the maxima are located at 1637 cm21 and 1639 cm21 in the D2O and H2O spectra, respectively. Deconvolution of the spectrum obtained in H2O-buffer showed four components (Fig. 1B). Similar results were found after obtaining the second derivative spectrum (Fig. 1C). Five components were found when the D2O spectrum was submitted to deconvolution and second derivation (Fig. 1B,C). Quantitation was carried out by curve fitting to the original spectra (Fig. 2A,B). The corresponding parameters, i.e. band position, percentage area, and bandwidth of each spectral component are displayed in Table 1. In the H2O spectrum, the main component was centered at 1639 cm21 and amounted to 51%. This wavelenght can be attributed to b sheet structure [37], the major structural component of C2 domains [4,17]. The component at 1658 cm21, which amounted to 18%, is usually assigned to a helix and/or unordered structures [38] but could also be assigned to large loops with dihedral angles similar to those of a helix [39±42]. The component at 1671 cm21, amounting to 22%, is assigned to b turns [43,44]. Finally the band at 1689 cm21, which can be assigned to the high frecuency component of antiparallel b sheet in the case of proteins in H2O solution [38] amounted to 9% of the total area. When the protein was studied in D2O, five components were basically detected both by deconvolution and derivation (Fig. 1B,C). The band centered at 1636 cm21 corresponds to a b sheet structure and was the main component, representing 51% of the total area of the amide I 0 band. The component located at 1651 cm21 is usually assigned to a helix but, as in the case of the 1658 cm21 component in the H2O buffer, it may also correspond to large loops or turns with dihedral angles similar to those of a helix [37,44,45]. It is clear that the 1658 cm21 component detected in the H2O buffer (Table 1) did not include unordered structures as they would have shifted to a lower frequency of about 1643 cm21 in D2O buffer, but such a component was not detected [46]. The components located at 1662 (15%) and 1686 cm21 (4%) arise from -turns and the 1674 cm21 band (9%) is usually assigned to antiparallel b sheet structure [36,47±49]. Quantitation of the secondary structure and assignments of the PKC:-C2 domain in D2O are also summarized in Table 1 and will serve as a basis for the interpretation of temperature-dependent structural changes of the protein in D2O buffer. Thus, there was very good agreement between the quantitation made in H2O and in D2O solutions, with 60% b sheet being identified both in H2O (adding the 1639 and 1689 cm21 components) and D2O (adding the 1636 and 1674 cm21 components); 18% a helix (or loops) in H2O and 21% in D2O and 22% b turns (adding the 1686 and 1662 cm21 components) in H2O and 19% in D2O. Study of the thermal stability of the PKC:-C2 domain by IR Protein thermal denaturation can be followed using infrared spectroscopy by studying the temperature-induced changes

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Table 1. Peak positions and assignement of the Amide I 0 and Amide I 0 bands of PKC:-C2 domain. H2O

D2O

Position (cm21)

Area (%)

Width (cm21)

1689 1671 1658

9 22 18

19 27 23

1639

51

31

Assignment Antiparallel b sheet b turns a helix, unordered structure or large loops b sheet

Position (cm21)

Area (%)

Width (cm21)

1686 1674 1662 1651

4 9 15 21

14 19 20 21

1636

51

27

produced in the amide I 0 band. Protein thermal denaturation profiles are sensitive tools which reveal small conformational differences that are not always apparent from the individual infrared spectra. The band decomposition procedure used in this work enabled us to analyse the thermally induced changes in protein structure in detail and the thermal behaviour of the individual structural elements. In general, large changes (10 cm21) in band position seem to indicate variations in the secondary structure, whereas smaller shifts (6 cm21) reflect local changes in a given conformation [37]. Figure 3A shows the pattern of PKC:-C2 domain upon heating, with the spectra basically revealing a typical broadening of the overall amide I 0 contour [33,50,51]. Figure 3B depicts a plot of half-bandwidth vs. temperature, which permitted the Tm transition temperature to be calculated (45 8C approximately). In order to study the thermal denaturation characteristics of this domain we have decomposed the different spectra into their constituents by curve-fitting. The number of components and positions were determined by previous deconvolution and derivation similarly to the spectra obtained at 25 8C. Table 2 show the results obtained at 45, 60 and 75 8C. The most significant changes compared with the spectrum recorded at 25 8C (Fig. 2C) are the appearance of two new components at 1645 and 1622 cm21 at 45 8C, which gradually increased with increasing temperatures up to 60±80 8C. The former can be attributed to an unordered structure [38] and the second to certain structures that arise upon thermal denaturation, such as extended chains not forming b sheet (b strands),

Fig. 3. (A) IR spectra of the PKC:-C2 domain in D2O buffer containing 0.2 mm EGTA in the amide I 0 region (1700±1600 cm21) as a function of temperature from 25 to 75 8C (increment of absorbance units (K) was 0.1) and (B) half-bandwidth of the amide I 0 region of the IR spectra in cm21 as a function of temperature, for the PKC:-C2 domain.

Assignment b-turns Antiparallel b sheet b turns a helix, large loops or turns with dihedral angles similar to a helix b sheet

irreversible aggregation or denaturated conformation [37,52±55]. An analysis of the peak positions, and the percentile areas of each component obtained by curve-fitting also provided an idea of the secondary structural changes that occur during the thermal process (Table 2). For example, the area of the component appearing at 1636 cm21, which has been attributed to b sheet, abruptly decreased at 50 8C from 49 to 19% (Fig. 4A). Furthermore, this decrease was correlated by an increasing in the percentage of the area of the 1622 cm21 band from 0 to 25% at 50 8C, suggesting that the emergence of the last component was a consequence of the process that induced the disappearance of the 1636 cm21 component. Figure 4B shows the changes which occurred, with another new component appearing at 1644 cm21 (assigned to unordered structure) and representing a maximum of 10±12% at the highest temperatures. This band seems to arise from the 1651 and 1636 cm21 components that may be partially denaturated during the heating. A further significant change concerned the component at 1686 cm21, which shifted to 1681 cm21 at the same time that the percentage of its area increased from 4% at 25 8C to 10% at 75 8C (Fig. 4C). These data suggest that there is only a small reorganization in the initial conformation of the b turns, for example, the component at 1651 cm21 slightly shifted to 1653 cm21 at 40 8C. Furthermore, the percentage of the area decreased from 21% at 25 8C to 14% at 40 8C and stayed constant during denaturation (Table 2), indicating that this component, which is attributed to a helix or large loops, is very stable during the heating process.


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Fig. 5. Phospholipid-dependent binding of PKC:-C2 domain. The binding capacity of the PKC:-C2 domain was measured as described in the materials and methods section. Small unillamelar vesicles containing 30 (white bars), 60 (hatched bars) and 100 (black bars) mol% of every negatively charged phospholipid were used. Binding was monitored by using 3H-labelled PC. Every experiment was performed in triplicate and the results are the averages for three independent experiments. We used as a 100% binding activity control, the GST-PKCa-C2 domain incubated with SUV containing 25 mol% PtdSer in the absence (horizontal bar) and in the presence of 100 m CaCl2 (gray bar).

Fig. 4. Thermal profiles of the amide I 0 components. (A) Percentage of band area detected at 1636 cm21 (b sheet) (X) and at 1623 cm21 (aggregation) (W) vs temperature. (B) Percentage of band area detected at 1645 cm21 (unordered) vs temperature. (C) Band position starting at 1680 cm21 at 25 8C (X) and percentage of band area of the same component (W) vs temperature.

Lipid binding specificity of the PKC:-C2 domain To test the specifity of phospholipid binding by the PKC:C2 domain, the binding of liposomes containing different phospholipid compositions was investigated (Fig. 5). Thus, Table 2. Peak positions of the Amide I 0 bands of PKC:-C2 domain at different temperatures. 25 8C

45 8C

60 8C

75 8C

Position (cm21)

Area (%)

Position (cm21)

Area (%)

Position (cm21)

Area (%)

Position (cm21)

Area (%)

1686 1674 1662 1651

4 9 15 21

1636

51

1681 1671 1662 1654 1646 1636 1622

8 7 11 17 4 44 10

1681 1671 1662 1653 1645 1636 1623

8 9 11 14 13 19 26

1681 1671 1662 1653 1645 1636 1622

10 9 12 14 10 21 24

the PKC:-C2 domain was fused to glutathione S-transferase, which enabled us to purify the proteins by high affinity chromatography. Afterwards, binding assays were performed using small unilamellar vesicles containing increasing mol% concentrations (30, 60 and 100 mol%) of different negatively charged phospholipids such as: PIP2, PI, PA, PG and PtdSer. These experiments revealed that at low mol% of negatively charged phospholipid, the PKC:-C2 domain has preferential affinity for PIP2 and after for PA PG . PtdSer . PI. Strikingly, when 100 mol% of negatively charged phospholipid were used, the domain showed maximal binding affinity when the vesicles contained PA and after for PI PG . PtdSer PIP2. In contrast, liposomes containing PC alone showed no significant binding to the domain, sugesting that in the absence of DAG, the negative charge of the phosphatidic acid and PIP2 mainly, is the principal factor in determining the selectivity of the PKCa-C2 domain, although we can not discard that other negative groups can also participate in this interaction. The figure also shows a 100% binding activity control, for that we used GST-PKC-C2 domain incubated with SUV containing 25 mol% PtdSer in the absence and in the presence of 100 mm CaCl2. These results demonstrate that the binding extension observed for PKC:-C2 domain is significantly important if we compare to that exhibit by the PKC-C2 domain. Effect of lipid binding on the structure of PKC:-C2 domain and its thermal denaturation pattern As shown above, phosphatidic acid vesicles were used as a membrane model as we could get the maximal binding activity under the specified conditions. IR was used to determine the effect of phosphatidic acid vesicles binding on the secondary structure of the PKC:-C2 domain. The spectrum was obtained and curve fitting was carried out for the PKC:-C2 domain bound to vesicles containing

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Fig. 6. IR spectrum of the PKC:-C2 domain in the presence of small unilamelar vesicles containing 100% phosphatidic acid, corresponding to the 1800±1600 cm21 region. (A) Spectrum at 25 8C. The parameters corresponding to the component bands are shown in Table 3. The dashed line represents the curve-fitted spectrum. (B) Deconvolved IR spectra obtained at different temperatures. Deconvolution was carried out using a gamma factor of 2 and a smoothing factor of 0.46. The increment of absorbance units (K) was 0.05 in both spectra.

100 mol% of phosphatidic acid in D2O at 25 8C in the 1780±1600 cm21 region (Fig. 6A). The curve fitting data point to a predominance of b sheet, as represented by the 1635 and 1675 cm21 bands, which amount to 59% of the total area. In summary, the secondary structure calculated is very similar to that obtained in the absence of lipids (compare Table 1 and 3), which suggests that no differences or changes in the secondary structure occur when lipid binding takes place. It is interesting to note that these results are different from those observed for the PKC:-C2 domain, which showed an increase in the component representing unordered and open loop structures upon lipid binding [33]. The effect of heating on the secondary structure of PKC:-C2 in the presence of phosphatidic acid was also followed by IR. Figure 6B depicts the heating profile,

which showed few changes in the temperature range that was studied. To better characterize these changes, the spectrum obtained at 75 8C was analyzed by curve fitting (Table 3), which revealed that the component at 1635 cm21 decreased to 48% of the total area (from 53% at 25 8C). Nevertheless, if we consider the contribution of the 1674 cm21 band, the total percentage of the b sheet components was 58%, which implies that no significant changes took place with this heating process, as it amounted to 60% at 25 8C. The other components were very similar to those obtained at 25 8C (Table 3), suggesting that in the presence of phosphatidic acid, the heating process carried out here produced only slight modifications in the secondary structure under our experimental conditions.

Differential scanning calorimetry DSC measurements were performed to ascertain whether the heating process induced a transition peak in the PKC:-C2 domain using this technique (Fig. 7). The PKC:-C2 domain showed a calorimetric denaturation enthalpy of 144.348 kJ´mol21 and a Tm of 49 8C (line A). Denaturation of the PKC:-C2 domain in the presence of phosphatidic acid vesicles (line B), on the other hand, showed the solid to fluid phase transition of the phospholipid, while the transition of the protein could not be clearly observed and a small transition appeared starting at 30 8C. It seems then, from these calorimetric experiments, that the structure of the protein was not substantially modified in the presence of lipid. Table 3. Peak positions of the Amide I 0 band of PKC:-C2 domain bound to phosphatidic acid at 25 and 75 8C. 25 8C

Fig. 7. DSC thermograms for PKC:-C2 domain. In the absence (A) and in the presence of small unilamelar vesicles (B) containing 100% phosphatidic acid. The increment of heat capacity (Cp) was 0.008 J´8C21.

75 8C

Position (cm21)

Area (%)

Width (cm21)

Position (cm21)

Area (%)

Width (cm21)

1686 1674 1662 1651 1635

5 6 13 21 53

21 19 23 26 32

1686 1673 1662 1650 1635

8 10 15 19 48

30 21 21 24 36


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Table 4. Peak positions of the Amide I band of PKC:-C2 domain after DSC. Position (cm21)

Area (%)

Width (cm21)

1688 1670 1656 1637

10 23 15 51

27 28 25 35

To further prove this, the sample was recovered and analyzed by IR after scanning in the calorimetric instrument. After the calorimetric experiment, running from 5 to 85 8C in the presence of POPA, the IR spectrum was obtained (in H2O solution) and quantified by curve-fitting (Table 4). A main component was found at 1637 cm21, which, together with the 1688 cm21 component, can be attributed to b sheet (61% b sheet). Taking into account that these two bands contribute to the b sheet signal, there were no differences between the native (60%) and the heated protein (61%) in this secondary structure. No significant changes appeared in the 1656 and 1670 cm21 components either. All these data suggest that, upon lipid binding, the PKC:-C2 domain is fairly stable and no thermal denaturation takes place in this temperature range.

DISCUSSION Among the PKC isozymes, PKC: has been reported to exhibit full oncogenic potential [56±59]. PKC: has also been implicated in the regulation of other biological processes, including neuronal differentiation [60], antiviral resistance [61], hormone secretion [62] and the regulation of transporters [63,64]. Furthermore, it has been found that PKC: has unique properties in terms of its membrane association, oncogenic potential and substrate specificity [63,65]. The C2 domain is present in conventional PKCs and, in a modified form, in novel PKCs [16,66,67]. To date, very little is known about the physiological role of the C2 domain in the different PKC isoforms. As no structural data are available from PKC:, we have attempted to study in this work the biophysical properties of PKC:-C2 domain, and compare them with the findings of previous studies of the PKCa-C2 domain [33]. The results of these studies show important diferences concerning to the secondary structure and thermal denaturation stability between PKCa and PKC:-C2 domains (see Table 5).

Infrared spectroscopy is recognized as a valuable tool for examining protein conformation in aqueous solutions. The reliability and wide applicability of this technique have resulted in its greatly expanded use in studies of protein secondary structure [40]. In addition, this work also demonstrates that the results obtained in the thermal denaturation studies using IR are compatible with those obtained by DSC, and a combination of the two techniques provide complementary information of the denaturation process. As mentioned above, the 3D structure of the PKC:-C2 domain has not been determined. Nevertheless, the 60% of b sheet calculated for PKC correlates well with the percentages calculated by X-ray diffraction for C2 domains of the same topology, such as PLCd (53%), cPLA2 (60%) and PKCd-C2 (53%) domains [28,29,32]. It is interesting to note that in spite of sharing a core b sheet with many other C2 domains, including the PKCa-C2 domain, IR detected significant differences between this last domain and the PKC:-C2 [33]. For example, the component at 1644 cm21 assigned to open loops and unordered structure in the PKCa-C2 cannot be found in the PKC:-C2 domain. Moreover, the band at 1651 cm21 which is attributed to a helix or large loops, is significantly greater in the PKC:-C2 in D2O solution (21%) than in PKCa-C2 (12%). These results suggest that the PKC:-C2 domain possesses a more ordered secondary structure than other C2 domains, which may be due to the connecting loops as the core b sheet is similar in all C2 domains. Recent studies have shown the crystal structure of the PKCd-C2 domain, which belongs to the group of novel PKCs. However, the low sequence homology between PKCa and PKCd-C2 domains suggests that further structural variations remain to be found in this group [32]. For example, there is an extended sequence in the CBR1-like loop of PKCa that could adopt a large loop conformation similar to that described in the CBR1 of PLC1, PTEN or PtdIns3K [28,68,69]. Another possibility is that this part of the domain adopts a helical conformation similar to that of cPLA2-CBR1, which is involved in membrane interaction [29]. Thus, one of those structures may be the origin of the extra helical component found in the IR spectrum of PKC:-C2 domain at 1651 and 1658 cm21, in D2O and H2O, respectively, as both a helix and large loops appear together at those frequencies [38±41]. With respect to thermal stability, it has been shown that in PKCa-C2 domain, the unordered structures and those components characteristic of thermal denaturation (1646, 1625 and 1680 cm21) increase substantially at 75 8C, a finding which correlates well with the decrease observed in the percentage of the a helical and b sheet components

Table 5. Important differences found between PKCa and PKC:-C2 domains.

Random or open loops structure a Helix or large loops Structural changes upon lipid binding Random component upon thermal denaturation a Helix component upon thermal denaturation

PKCa-C2 domain

PKC-C2 domain

12% of the total area 12% of the total area Yes Up to 34% of the total area Disappeared

0% of the total area 21% of the total area No Up to 10±12% of the total area Persisted until 14% of the total area

q FEBS 2001


[33]. On the other hand, the PKC:-C2 domain retains the 1651 cm21 component and part of the b sheet structure after heating. These results demonstrate that the PKC:-C2 domain is more stable than the PKCa-C2 domain in the absence of Ca21. These results support the idea that one of Ca21 functions is to stabilize the domain before lipid binding and thus, in the cases where the function of the C2 domain is independent of calcium this role is played by the protein itself. Furthermore, many studies of the C2 domains of topology II, such as cPLA2, have demonstrated that CBR1 and CBR3 contain hydrophobic residues that are very important for membrane binding [13,70]. The homologous residues of PKC:-C2 domain, which contribute to CBR1 and CBR3, could be potential candidates for the absorbance seen at this particular wavelength (1651 cm21). The C2 domains of novel PKCs constitute a special case as they have been classified as topology II although their membrane binding capacity depends on acidic phospholipids in a Ca21-independent way [71]. It has been demonstrated recently that PKC:, due to the lack of Ca21 binding, interacts with low specificity with PtdSer and DAG, which implies the presence of other physiological activators for this form [72]. For example, it is well established that PLD activation induces an increase of phosphatidic acid in biological membranes [73] and this could be a way of activating PKC:. As demonstrated in this work, the PKC:-C2 domain has an important affinity for phosphatidic acid vesicles although the secondary structure of the domain did not change upon lipid binding, in contrast with PKCa-C2 domain that underwent significant changes [33]. These results correlate well with the need for the PKCa to penetrate the membrane to be fully activated, the result of which could be the structural reorganization seen by IR. Experiments on phospholipid monolayer penetration, on the other hand, have revealed that PKC: penetrates the membrane to a lesser extent than PKCa. This might result in no secondary structural reorganization upon lipid binding, although some reorganization at the tertiary level cannot be discarded [72]. In summary, the regulation of PKC activity involves lipid cofactors, Ca21, protein phosphorylation and protein± protein interactions. Understanding the complexity of these regulation systems will allow the isozyme-specific signaling in different cellular systems to be elucidated and will help in the design of isozyme-specific pharmacological tools [74]. Meanwhile, the systematic study of the differences between C2 domains in PKCs may provide clues about how these domains adapt to fulfill different functions.

ACKNOWLEDGEMENTS We are very grateful to Dr Ono and Dr Nishizuka (Kobe University, Japan) for the kind gift of the cDNA codifying PKC. This work was supported by Grant PB98-0389 from DireccioÂn General de EnsenÄanza Superior e InvestigacioÂn CientõÂfica (Spain).

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