Identification of a General Anesthetic Binding Site in the Diacylglycerol-binding Domain of Protein Kinase Cδ*

Protein kinase C (PKC) is an important signal transduction protein that has been proposed to interact with general anesthetics at its cysteine-rich diacylglycerol/phorbol ester-binding domain C1, a tandem repeat of C1A and C1B subdomains. To test this hypothesis, we expressed, purified, and characterized the high affinity phorbol-binding subdomain, C1B, of mouse protein kinase Cδ, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintoxin-D, bound to PKCδ C1B in buffer at a molar ratio of 1:2, its fluorescence emission maximum, λmax, shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted λmax of the PKCδ C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to ∼415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCδ C1B was photolabeled with three photo-activable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCδ C1B at a stoichiometry of 1:1 in the labeled fraction. The photolabeled PKCδ C1B was subjected to tryptic digest, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKCδ C1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within ∼10 Å of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.

The molecular mechanisms of general anesthesia remain poorly understood (1)(2)(3)(4). Anesthetics are relatively nonspecific drugs that interact with many transmembrane ion channels and soluble proteins, often causing unwanted side effects.
Gaining a detailed understanding of the structural motifs governing anesthetic-protein interactions is a critical step in elucidating the molecular mechanisms underlying general anesthesia and in developing more selective anesthetic agents. However, little progress can be made until such sites are unequivocally identified. The physiological sites of anesthetic action remain difficult to define, and some potential targets such as the transmembrane domains of the ligand-gated ion channel superfamily are so poorly characterized compared with soluble proteins that atomic level principles governing their anesthetic binding cannot yet be defined. Consequently, several crystallizable soluble proteins such as myoglobin (5), hemoglobin (6), adenylate kinase (7), luciferase (8), human serum albumin (9), and insulin (10) have been used as surrogate targets to obtain structural information on protein-anesthetic interactions.
An important signal transduction protein, protein kinase C (PKC), 1 has been proposed as the target of anesthetics such as alcohols, halothane, and enflurane (11,12); for a review, see Rebecchi and Pentyala (13). The protein kinase C superfamily plays a central role in signal transduction, regulating divergent cellular functions by phosphorylation of target proteins such as ion channels (14,15). It can be separated into two major categories, the conventional (␣, ␤I ␤II, and ␥) and the novel (␦, ⑀, , and ) kinases, each having four domains, termed C1 though C4, that play distinct roles in the function of the kinase. C1 and C2 are the regulatory domains, C3 is the ATP-binding domain, and C4 is the catalytic domain. The two phorbol-binding sites are in the C1 domain, and consist of a tandem repeat of highly conserved cysteine-rich zinc finger subdomains C1A and C1B (residues 159 -208 and 231-280, respectively, in the ␦ isoform). These domains differ in their binding affinities for phorbol ester and sn-diacylglycerol (16 -19). Homology between different members of the superfamily is high within domains but the novel kinases differ in having their C2 domain N-terminal to their C1 domain (Fig. 1) and in associating with membranes in a calcium-independent manner.
Stimulated PKC activity is modulated by anesthetics, and it has been proposed that discrete binding sites for general anesthetics could be present and possibly located near or within the phorbol ester-binding sites (11,20). The interaction between PKC activators and anesthetics, however, is complex and incompletely understood. The isolated catalytic domain, C4, is unaffected by anesthetics, indicating that the primary anesthetic interaction occurs within the regulatory domains (11, 18, * This work was supported in part by United States Public Health Service Grants GM 58448 and GM 069726, and the Department of Anesthesia and Critical Care, Massachusetts General Hospital. 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. § Contributed equally to the results of this work.  20,21). In intact PKC␣, volatile anesthetics and long-and short-chain alcohols modulate PKC activation in vitro and alter activator binding affinities. The enhancement of PKC␣ activity induced by octanol in the TPA/phospholipid system was attributed to a positive allosteric interaction between it and the high affinity phorbol ester-binding site (20).
To study the interaction between anesthetics and high affinity phorbol ester binding, we expressed the C1B subdomain of mouse PKC␦ in Escherichia coli and purified it by affinity chromatography. We chose PKC␦ C1B, which is highly homologous with other major isoforms, because it is the best characterized, both structurally and functionally, of the isolated subdomains (16,(22)(23)(24). We found that butanol and octanol allosterically enhanced the binding of a fluorescent phorbol ester to PKC␦ C1B, and that diazirine derivatives of butanol and octanol photoincorporated into a single residue identified as Tyr-6 of C1B, corresponding to Tyr-236 in intact PKC␦.
Bacterial Expression and Purification of the PKC␦ C1B Subdomain-The PKC␦ C1B subdomain fused with glutathione S-transferase was expressed in BL21 gold E. coli as described earlier (28). Briefly, cell pellets were treated with 1% Triton X-100 and lysozyme (1 mg/ml), followed by sonication and centrifugation. The clarified supernatant was applied to a glutathione-Sepharose column. The bound protein was thoroughly washed, released by thrombin cleavage, eluted with phosphate-buffered saline, and concentrated by ammonium sulfate precipitation. It was further purified by fast performance liquid chromatography (Bio-Rad Biologic Work station) using a Superdex TM 75 column (Amersham Biosciences), a mobile phase of 50 mM Tris, 100 mM NaCl, pH 7.0, and a flow rate of 0.5 ml/min. CD Measurements-Circular dichroism spectra were recorded from 260 to 190 nm at 25°C on an Aviv 62 DS instrument (Aviv Associates, Lakewood, NJ) in a quartz cell of 0.1-cm path length. Data were collected at 0.5-nm intervals and an accumulation time of 20 s. CD spectra were fitted using a CDNN program and the neural network approach of Bohm et al. (29).
Fluorescence Measurements-Fluorescence measurements were performed on a Jobin Yvon Spex (model Fluoromax-2, ISA Instruments S.A., Inc.) fluorimeter equipped with temperature and stirring control systems. A 1.5-ml cuvette (Hellma) with a Teflon stopper was used for fluorescence measurements. PKC␦ C1B and SAPD were mixed in a buffer (50 mM Tris, pH 7.2) and stirred gently for 20 min with a small Teflon-coated magnetic stir bar at room temperature. SAPD was excited directly at 355 nm and emission spectra were recorded from 375 to 575 nm. Effects of alcohols on the SAPD binding were studied in a 1-ml mixture containing 4 M protein and 2 M SAPD. Alcohols were titrated by adding successive 2-l aliquots of Me 2 SO containing either 0.5 M octanol or 10 M butanol. Spectra were recorded after 30 min incubation with slow stirring. PKC␦ C1B was denatured by heating the solution at 90°C for 20 min, and its fluorescence was monitored after cooling and transferring to the cuvette.
The wavelength maxima of the emission spectra were determined by fitting the symmetrical top of the peaks to a Gaussian function with Igor Pro 4 (WaveMetrics, Inc., Lake Oswego, OR). Butanol, octanol, and TPA titration curves were fitted using the following equation (30), where, min and max are the minimum and maximum values of the effect, EC 50 is the equilibrium ligand concentration at half-maximum effect, n H is the Hill coefficient, and x is the concentration of the alcohol or TPA. For fluorescence quenching experiments, phorbol esters (TPA or 4␣-TPA, 1 mM in Me 2 SO) were titrated by additions of successive 1-l aliquots into 1 ml of PKC␦ C1B (1 M) in buffer (50 mM Tris, pH 7.2). The sample was excited at 290 nm and intrinsic fluorescence quenching was monitored by recording fluorescence emission from 300 to 500 nm. The average fluorescence intensity between 348 and 350 nm was normalized to that without additions (F/F max ) and used in the analysis of binding curves and plots to calculate the Stern-Volmer constant, K SV (31).
Determination of Stoichiometry of Photolabeled PKC␦ C1B by Electrospray Ionization Mass Spectrometry-The stoichiometry of photoincorporation of 3-azioctanol into PKC␦ C1B was determined using microcapillary liquid chromatography coupled to an ion trap mass spectrometer with electrospray ionization. Samples of PKC␦ C1B (95 pmol/sample) were photolabeled at 365 nm for 20 min after 10 min preincubation with 1 mM 3-azioctanol, 7-azioctanol, or 3-azibutanol using a model UVL-56 20W Black-Ray handheld lamp (Upland, CA). A 1-l aliquot of each reaction mixture was diluted 1:20 with 0.1% acetic acid to yield a 1 pmol/l solution. A 1-l aliquot of each of these diluted samples was then analyzed by microcapillary liquid chromatography coupled to an ion trap mass spectrometer with electrospray ionization (LCQ, Finnigan MAT, San Jose, CA). The column (75 ϫ 360 m) was packed with POROS 10R2 (Perceptive Biosystems, Framingham, MA) to 15 cm and butt-connected to a fused silica nanospray tip (5 m). The reaction mixture was eluted from the column with a gradient of 0 -80% acetonitrile in 15 min at a flow rate of 0.2 l/min. Mass spectra were acquired from m/z 300 to 2000 with a maximum ejection time of 400 ms. Molecular weights for both the modified and unmodified forms of PKC␦ C1B were calculated from the resulting charge envelopes. Essentially, the charge state of an ion was determined by subtracting two adjacent ions from each other and the reciprocal of this difference multiplied by the ion of lower mass/charge minus one. The result is the charge state of the ion with the higher m/z of the two adjacent peaks. To calculate the unprotonated molecular weight, the determined charge state is multi-FIG. 1. Photolabels and protein used in the present study. A, diazirine analogs of butanol and octanol. B, schematic representation of the domains of protein kinase C␦: the C1 regulatory domain binds to lipids, diacylglycerol, and phorbol esters; the C2 regulatory domain binds to anionic lipids, but does not bind to Ca 2ϩ ; C3 is the ATP-binding domain, and C4, the catalytic domain. P denotes the pseudo substrate-binding domain. C1B (residues 231-280) binds to phorbol esters with higher affinity than C1A (residues 159 -208). The 50 residues of subdomain C1B used in this study are shown in bold and additional residues used in the construct are shown in regular type.
plied by the mass/charge of the ion and then the charge state is subtracted from the product.
Proteolytic Digestion of Azioctanol-labeled PKC␦ C1B-To the photolabeled PKC␦ C1B (95 pmol, 100 l), 100 mM ammonium bicarbonate was added to a final volume of 300 l. The sample was then digested overnight at room temperature with trypsin at a ratio of 1:40, protease: protein. The digested samples were stored at Ϫ20°C.
Online Reverse-phase Chromatography and Sequencing of Azialcohol-labeled PKC␦ C1B by Electrospray Ionization Mass Spectrometry-Each digested sample was analyzed by automated LC/MS/MS on the ion trap mass spectrometer using online LC. A 0.5-l aliquot from the 30 l of digested protein was loaded onto the column, prepared as described above. Peptides were eluted directly into the mass spectrometer with a two-step gradient of 5-65% acetonitrile in 0.1 M acetic acid for 25 min, followed by 65-90% acetonitrile in 0.1 M acetic acid for 5 min at a flow rate of 0.2 l/min. MS/MS spectra were acquired under automatic acquisition of the most intense ion from each MS scan.

Protein Purification and Characterization-SDS-PAGE
(18%) analysis of affinity purified and thrombin-cleaved PKC␦ C1B showed a single band around 7 kDa (data not shown) similar to that previously reported (23). A number of criteria suggest that the purified PKC␦ C1B protein is correctly folded. First, fast performance liquid chromatography on a gel filtration column confirmed that the PKC␦ C1B subdomain migrated as a single symmetrical peak with the expected Stokes radius. Second, analysis of the CD spectrum ( Fig. 2) suggests that the secondary structure of PKC␦ C1B has about two times more ␤-sheet content than ␣-helix content and a high content of random coil (ϳ50%) (32). This estimate is consistent with the published crystal structure (23). Third, phorbol ester binding had the required specificity (see below).
Phorbol Ester Binding-In 50 mM Tris buffer, pH 7.4, SAPD showed an emission maximum at 437 nm that was unaffected by addition of Me 2 SO, anesthetics, or the combination of both in the concentrations used in the present studies. Addition of a 10-fold excess of PKC␦ C1B to SAPD caused a broad blueshifted emission spectrum that could be deconvoluted into two components with maxima at 404 and 437 nm representing bound and free SAPD, respectively (Fig. 3). When the spectrum of the same solution was recorded at 90°C for 20 min, the spectrum narrowed to a single component centered on 437 nm, indicating release of SAPD from PKC␦ C1B. Consistent with this interpretation, when PKC␦ C1B, preheated at 90°C for 20 min, was added to SAPD, no wavelength shift was observed.
PKC␦ C1B contains a single tryptophan (Trp-252) whose fluorescence was quenched in a concentration-dependent manner when TPA was added (Fig. 4, inset). Quenching of the intrinsic fluorescence of PKC␦ C1B depended in a sigmoidal manner on the concentration of the phorbol ester TPA (Fig. 4, trace a). By contrast, the inactive isomer, 4␣-TPA (33), quenched significantly less efficiently and with linear dependence on concentration (Fig. 4, trace b). Assuming that the 4␣-TPA data points represent nonspecific quenching, they were subtracted from the TPA quenching data to yield corrected data points that fitted well to the mass action equation (Fig. 4, trace c) with an EC 50 of 0.31 Ϯ 0.072 M (total concentration). Controls showed that addition of TPA (0.1-1.2 M) to 1 M Ltryptophan in buffer did not cause any quenching of the tryptophan fluorescence, nor did addition of Me 2 SO in the concentration range used cause significant changes in the fluorescence emission spectra of PKC␦ C1B.
When the TPA fluorescence quenching data were plotted according to the Stern-Volmer equation, 4␣-TPA quenching was linear with a slope (K sv ) of 0.54 ϫ 10 6 M Ϫ1 . On the other hand, TPA quenching was linear up to 0.5 M TPA with a much higher K sv of 2.9 ϫ 10 6 M Ϫ1 . At higher concentrations, however, the plot curved upwards suggesting additional quenching mechanisms.
Consistent with the TPA study, SAPD also quenched intrinsic PKC␦ C1B fluorescence. With increasing concentration of SAPD, intrinsic fluorescence decreased and bound SAPD fluorescence increased in parallel (data not shown).
Phorbol Ester-Anesthetic Interactions-As shown above (Fig.  3), the emission spectrum of SAPD in the presence of 10 -50fold excess protein consists of overlapping free and bound contributions with max at 437 and 404 nm, respectively. The addition of butanol (100 -200 mM) or octanol (1-2 mM) caused no significant change in the composite spectrum under these conditions. However, when the protein to SAPD molar ratio was reduced from 10:1 to 2:1 the composite peak was more symmetrical and exhibited a maximum at 425 nm (Fig. 5). Titration of alcohols caused more SAPD to bind resulting in a blue shift that reached a plateau of ϳ415 nm at 80 mM butanol or 1 mM octanol (Fig. 6). These concentration-dependent emission shifts were fitted to logistic curves yielding EC 50 values of 34 Ϯ 16 mM for butanol and 410 Ϯ 150 M for octanol and Hill coefficients that did not differ significantly from one (2 Ϯ 1.6 for butanol and 2 Ϯ 1.4 for octanol). Control experiments in which identical additions of butanol, octanol, or Me 2 SO were made to SAPD in buffer did not cause any blue shift.
To test the stability of the diazirine group during the binding assay, the absorbance change of a 2 mM aqueous 3-azioctanol solution at 350 nm was recorded before and after a single emission scan. About 10% of the chromophore was photodecomposed. Because of the photosensitive nature of the diazirine group we did not titrate the azialcohols. However, 2 mM 3-azioctanol caused a substantial, near maximum blue shift to 417 nm under the above conditions.
Determination of the Mole Ratio of Alcohol Photoincorporation into PKC␦ C1B by LC/MS-The photolabeled samples were diluted (1 pmol/l) and analyzed using online microcapillary liquid chromatography coupled to an ion trap mass spectrometer with electrospray ionization. An ion chromatogram for the photoreaction mixture of PKC␦ C1B and 1 mM 3-azioctanol can be seen in Fig. 7A. Complete separation of unla-beled and labeled PKC␦ C1B was not achieved. However, in LC/MS experiments, peak heterogeneity does not prevent molecular weight determination of the eluted proteins because of their unique charge envelopes. Thus, it was unnecessary to refine chromatographic conditions to achieve resolution of the components. Fig. 7B shows the mass/charge envelope for samples eluting between 10.8 and 13.5 min obtained by summing the spectra obtained every second during this time period. Deconvolution of the mass/charge envelope yielded two major peaks with molecular masses that did not differ significantly from the expected ones of 7,363 Da for PKC␦ C1B and 7,491 Da for PKC␦ C1B modified by photoincorporation of azioctanol (Fig. 7C). The observed difference between the two major peaks was 128 Ϯ 1 Da, corresponding to photoincorporation of a single 3-azioctanol molecule in PKC␦ C1B. No higher molecular weight peak corresponding to the photoincorporation of two azioctanols (7,619 Da) was observed. Nonetheless, it is hard to rule this out because the probability of such labeling is likely to be low (26). A major unidentified contaminant occurred at 7458 Da, but only in this particular preparation.
Similarly, samples of PKC␦ C1B photolabeled by 3-azibutanol and 7-azioctanol (1 mM) were analyzed, and their deconvoluted charge spectra are shown in Fig. 8, A and B, respectively. The deconvoluted charge envelope of the 3-azibutanol sample appears quite complex at first sight. The largest peak is observed at 7,363 Da, corresponding to the unmodified protein.
The two satellites marked a and b in Fig. 8A are offset from the main peak by 16 and 32 Da and most likely correspond to oxidized methionines, of which PKC␦ C1B has two, Met-9 and Met-36. The second largest peak is observed at 7,435 Da and corresponds to the protein modified by photoincorporation of a single 3-azibutanol (ϩ72). Interestingly, both oxidized states are also photolabeled. A small peak in the vicinity of, but not centered on, 7,507 Da makes it difficult to rule out that a second azibutanol photoincorporated into PKC␦ C1B.
The charge envelope derived from spectra of 7-azioctanollabeled PKC␦ C1B eluting between 10.2 and 13.1 min was deconvoluted to yield molecular masses corresponding to the expected values of 7,363 and 7,491 Da. The observed molecular mass difference of 127 Ϯ 1 Da being consistent with photoin- corporation of 7-azioctanol at a molar ratio of 1:1 (Fig. 8B). No peak that would correspond to double photoincorporation was found at 7,619 Da (not shown). Once again, both peaks exhibited satellites consistent with two oxidized methionines. In addition, an unidentified contaminant was observed around 7,455-7,475 Da.
Identification of Photolabeled Amino Acid Residues by LC/ MS/MS-All peptides generated by trypsin digestion of photolabeled PKC␦ C1B were analyzed by LC/MS/MS. In this type of experiment, eluted peptides are ionized and randomly fragmented along the peptide backbone, resulting in several smaller observed ion fragments. Fragment ions containing the N terminus of the peptide are called "a," "b," and "c" ions; fragment ions containing the C terminus are called "x," "y," and "z" ions. Under our experimental conditions, b and y ions predominate. Conventionally, numbers following these letters denote the number of residues in the fragment.
Digested samples were eluted directly from the online high performance liquid chromatography column into the mass spectrometer as described above, and MS/MS spectra were acquired under automatic acquisition of the most intense ion from each MS scan. Fig. 9 shows the collision-activated dissociation MS/MS spectrum of the triply charged peptide ion with m/z 887.6 that presented in the tryptic digest of 3-azioctanol-labeled PKC␦ C1B. The 22-amino acid sequence of the peptide, VYNYMSPTFCDHCGSLLWGLVK, and the amino acid modified with 3-azioctanol were deduced from this spectrum. Photoincorporated azioctanol caused a mass shift of 128 Da for all fragment ions containing the modified amino acid. The b2 (m/z 391) and a2 (m/z 363; loss of 28 Da from carbon monoxide) ions and all subsequent observed b ions (b3 through b5) contained a mass shift of 128 Da, indicating that either Tyr-6 or Val-5 is photolabeled. This ambiguity was resolved in favor of Tyr-6 by the y ions. The y ions with 4, 5, 6, 9, and 13 through 20 amino acids were all unmodified, whereas a mass shift of 128 Da was observed for the doubly charged y21 ion (m/z 1280). Although the latter peak appears small in Fig. 9, this is an artifact of the different scales employed in the three panels. Furthermore, its position is secured by being a member of the complete doubly charged sequence from y17 to y21.
MS/MS spectrum of the triply charged peptide with m/z of 887.5, derived from a digest of 7-azioctanol-photolabeled PKC␦ C1B showed b2 (m/z 391) and a2 (m/z 363; loss of 28 Da from carbon monoxide) ions (data not shown). All subsequent observed b ions (b3 through b5) contained a mass shift of 128 Da, indicating that either Tyr-6 or Val-5 is labeled with the alcohol. The y ion spectrum was identical to that with 3-azioctanol confirming photoincorporation into Tyr-6. affinity copy of the two phorbol sites in PKC. Surprisingly, in the isolated C1B subdomain, which contains only 50 of the 674 amino acid residues from the whole protein (Fig. 1), we observed the major features of alcohol-PKC interactions that have been reported in the intact kinase (20). Whereas we would not expect all features of alcohol action on PKC to be rationalized by actions within a single subdomain, nonetheless this provides a further indication of the modular nature of this kinase. That the general anesthetic site is intact is also consistent with studies on individual human serum albumin subdomains, which bind general anesthetics in much the same way as does the intact protein (34).
Our work shows that there is a positive heterotropic interaction between alcohols and phorbol-binding sites because both butanol and octanol enhance the binding of the fluorescent phorbol ester, SAPD, to PKC␦ C1B (Figs. 5 and 6). Our finding for octanol is remarkably similar to that reported for membrane-bound intact PKC␣, where SAPD titrations indicate that octanol in the 100 M range increases the affinity of high affinity SAPD binding, an observation that has been confirmed in a membrane-free assay (35). However, in those studies, unlike in ours, butanol was not observed to enhance high affinity SAPD binding. Whether this reflects subtle differences in the structure of the two isoforms, or a limitation of our simple model, remains to be determined.
Having established that the effect of alcohols on PKC␦ C1B resembled those in intact PKC␣, we went on to more directly test the hypothesis that an alcohol-binding site is responsible for this action. To do so, we employed diazirine derivatives of butanol and octanol to photolabel PKC␦ C1B. Each of these alcohols, 3-azibutanol, 3-azioctanol, and 7-azioctanol, photoincorporated into PKC␦ C1B with a stoichiometry of 1:1 in the labeled fraction (Figs. 7 and 8). It is not possible from mass spectrometry alone to establish the percentage of PKC␦ C1B that was photolabeled, but, based on previous experience with adenylate kinase, it is unlikely to be high (26). Consequently, as discussed previously (26), it is difficult to rule out the existence of fractions photoincorporating two alcohols per PKC␦ C1B because of the predicted low probability of such species occurring. However, two observations make this unlikely. First, in adenylate kinase, 3-azibutanol photoincorporated with a stoichiometry up to at least 5, whereas in PKC␦ C1B, only 1:1 was observed unequivocally. Second, no second site of photoincorporation was observed in the 22 positively identified amino acid residues, whereas in adenylate kinase three additional sites were identified for 3-azibutanol that were not ob- served with either octanol derivative. The single alcohol that photoincorporated into PKC␦ C1B was found on the same residue for each of the three diazirine derivatives. This residue was identified by MS/MS as Tyr-6 of PKC␦ C1B, corresponding to Tyr-236 in intact PKC␦.
A secondary objective of our study was to delineate the alcohol-binding site by employing 3-and 7-azioctanol in parallel. The efficacy of this approach was based on a study using these two azioctanols on adenylate kinase where they photolabeled a histidine and an aspartate, respectively, whose side chains were separated by ϳ5 Å, corresponding to the distance between the 3-and 7-positions on an extended methylene chain (26). This suggested that the azioctanols bind to adenylate kinase in an oriented fashion, with the hydroxyl end closer to the histidine than to the aspartate. In the current study, by contrast, both azioctanols photolabeled the same residue, Tyr-6. There are several possible explanations: the alkyl end of octanol might assume a coiled conformation in the binding pocket, bringing the 3-and 7-position into close proximity; the alkyl chain might wrap around the tyrosine, and the chain may have no single preferred orientation relative to Tyr-6. Some precedent for the latter observation is provided by complexes of peroxisome proliferatoractivated receptors ␦ with fatty acids (36).
A major rationale for choosing to study the ␦ isoform of C1B is that it is the only isoform for which a high-resolution crystal structure has been reported (23). To aid in the discussion, this structure, which was solved in a complex with a phorbol ester, is reproduced in Fig. 11. The protein contains two zinc ions, which are coordinated by conserved cysteine and histidine residues. The phorbol ester binds in a groove between two loops at the top end of the structure (23) and the photolabeled Tyr-6 is located on the right of the structure as shown and about halfway between the N terminus and the beginning of the phorbol binding loop. It is part of the first ␤ strand and is exposed on the surface of the protein, thus being readily accessible to alcohols from the aqueous phase.
Inspection of the surface close to Tyr-6 reveals no deep cavities but some plausible binding grooves. Tyr-6, shown in red in Fig. 11B, forms an isolated hump on the surface of PKC␦ C1B, and there is a wide surface groove between it and Glu-32 and a narrower one between it and Met-9. Either groove is large enough to accommodate octanol. However, the failure of the two azioctanols to delineate the site means that it would be unwise to draw further conclusions. It is clear, however, by inspection that both putative surface grooves contain polar residues, which is in keeping with observations in other proteins reviewed in Ref. 3. Furthermore, it seems likely that butanol has a relatively tighter interaction with PKC␦ C1B than does octanol because the ratios of their EC 50 values for their allosteric effects on SAPD binding to their general anesthetic potency are ϳ3 and ϳ9, respectively.
A comparison of unliganded and phorbol-bound C1B structures has shown that phorbol binding results in a subtle widening of the binding site (23) with the two loops forming the groove moving farther apart. Thus the groove has a certain plasticity. It seems plausible that anesthetics can influence the shape of the phorbol-binding site, and therefore the affinity of the protein for phorbol, by interacting with nearby regions. We note that Tyr-6 is only three residues away from residue Met-9, and close to Ser-10 and Pro-11, all of which are in direct contact with the bound phorbol and form one of the two loops that constitute the phorbol-binding site (Fig. 11A). The closest contact between the Tyr-6 side chain and the Met-9 main chain amide is only 5.3 Å, which is almost close enough to be classified as a van der Waals contact. The tyrosine is also only 7.7 Å away from the Ser-10 main chain carbonyl atom. The close proximity of Tyr-6 to a phorbol-contacting loop can thus help to explain the observed allosteric interaction between the alcoholand phorbol-binding sites. Binding of an alcohol molecule could easily perturb the location of this loop, thereby affecting the affinity of C1B for phorbol esters.
A priori, a possible site for general anesthetics on PKC might be at the lipid-protein interface. Our work suggests this is unlikely because the residues where mutations affect lipid insertion following phorbol binding, Leu-20, Trp-22, and Leu-24 (24), are found on the opposite face of PKC␦ C1B from Tyr-6 (see Fig. 11B for location of Trp-22). However, we cannot rule out that lipid association of phorbol-bound PKC␦ C1B may possibly cause indirect effects on the structure of C1B, or its force field, that might be transmitted to the general anesthetic site.
The most detailed functional studies of alcohol and anesthetic action on PKC have been carried out on human PKC␣. How similar is it to the mouse ␦ isoform that we have studied here? Fig. 11B shows a surface representation of the structure of PKC␦ C1B, in which those residues that are not identical in human PKC␣ are colored green (see legend for details). In addition Tyr-6 of PKC␦ C1B, which is conservatively replaced by a histidine in PKC␣ C1B, is colored red. This diagram reveals that Tyr-6 is on a boundary dividing two surface regions. One of these is highly conserved and the other quite variable. Overall, with the current evidence there is no reason to suppose that alcohol binding should not occur in the ␣-isoform, although minor differences in the alcohol structure-activity relationships might be expected.
In conclusion, we have demonstrated an allosteric interaction between the anesthetic and phorbol sites on PKC␦ C1B, and have located the allosteric alcohol site as being in the proximity of Tyr-6. This new data supports the hypothesis that discrete alcohol-binding sites exist in the phorbol ester-binding regulatory domain, C1, of PKC as proposed by Slater et al. (20). Because the structure of PKC␦ C1B has been reported, our study also suggests the possibility that high-resolution structural studies will be able to explore the binding site and the mechanism with atomic resolution in this isoform. FIG. 11. Structure of the PKC␦ C1B domain and comparison to PKC␣. A, ribbon diagram of PKC␦ C1B, (23) with selected features shown in ball-and-stick representation. The side chain of Tyr-6, which was photolabeled in this study, is shown in orange. The side chain of Trp-22 (blue) and the bound phorbol ester (orange) are also shown. Two bound zinc ions are represented with dark blue spheres. B, surface representation of PKC␦ C1B, viewed from two opposite sides. The orientation on the left is similar to that shown in panel A. Tyr-6 is shown in red, and the bound phorbol ester is represented as a ball-andstick model. Residues colored in green are not conserved in PKC␣, with light green representing conservative substitutions (e.g. Val 3 Ile) and dark green representing more drastic substitutions (e.g. Met 3 Gly). Residues shown in white are identical in PKC␣ and PKC␦. Panels A and B were produced with RIBBONS (37) and GRASP (38), respectively.