Fourier Transform Infrared Spectroscopy Study of the Secondary Structure of the Gastric H+,K+-ATPase and of Its Membrane-associated Proteolytic Peptides*

(Received for publication, April 22, 1996, and in revised form, September 9, 1996)

Vincent Raussens Dagger , Jean-Marie Ruysschaert and Erik Goormaghtigh §

From the Laboratoire de Chimie-Physique des Macromolécules aux Interfaces, CP 206/2, Université Libre de Bruxelles, Campus Plaine, B-1050 Brussels, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Membrane topology of the H+,K+-ATPase has been studied after proteolytic degradation of the protein by proteinase K. Proteinase K had access to either the cytoplasmic part of the protein or to both sides of the membrane. Fourier transform infrared attenuated total reflection spectroscopy indicated that membrane-associated domain of the protein represented about 55% of the native protein, meanwhile the cytoplasmic part represented only 27% of the protein. The secondary structure of the ATPase and of its membrane-associated domains was investigated by infrared spectroscopy. The secondary structure of the membrane-associated structures and of the entire protein was quite similar (alpha -helices, 35%; beta -sheets, 35%; turns, 20%; random, 15%). These data were in agreement with 10 alpha -helical transmembrane segments but suggested a participation of beta -sheet structures in the membrane-associated part of the protein. Polarized infrared spectroscopy indicated that the alpha -helices were oriented nearly perpendicular to the membrane plane. No preferential orientation could be attributed to the beta -sheets. Monitoring the amide hydrogen/deuterium exchange kinetics demonstrated that the membrane associated part of the ATPase molecule is characterized by a relatively high accessibility to the solvent, quite different from that observed for bacteriorhodopsin membrane segments.


INTRODUCTION

The gastric H+,K+-ATPase is an alpha ,beta heterodimer (1, 2) that belongs to the P-type ATPase family. These ATPases, which include the Na+,K+-ATPase, the Ca2+-ATPases and the yeast and Neurospora plasma membrane H+-ATPases, are thought to share a common mechanism and a common structure. Both N- and C-terminals have been found cytoplasmic (3), thus the catalytic subunit must have an even number of transmembrane segments. According to the hydropathy plots of their catalytic subunits, the P-type ATPases are thought to adopt a similar topological insertion in the membrane. The first four transmembrane segments located in the N-terminal third of the protein are clearly defined on the hydropathy plots. Their existence was demonstrated by proteolysis and in vitro expression experiments (4, 5). The other predicted transmembrane segments, located in the C-terminal third of the protein, are more ambiguous. Predictions and experiments suggested from four to six transmembrane segments in this region of the protein (4, 6, 7).

It must be pointed out that until now all the transmembrane segments of the ATPase are predicted to be alpha -helices, according to the hydropathy plots designed for localizing potential transmembrane alpha - helices on the sequence of membrane proteins. This idea was further strengthened by the recent knowledge of the three-dimensional structure of bacteriorhodopsin (8), of a photosynthetic complex (9), and of a bacterial cytochrome c oxidase (10). Bacterial porins (11) whose membrane segments adopt a beta -sheet structure were considered as an exception. However, recently a 9-Å resolution structure of the nicotinic acetylcholine receptor obtained by high resolution electron microscopy (12) indicated that three out of the four predicted transmembrane alpha -helices do not adopt the expected helical conformation. The secondary structure of the membrane part of the nicotinic receptor was analyzed by Fourier transform infrared spectroscopy after removal of the receptor's extramembrane moieties by enzymatic proteolysis (13). beta -Sheet structures were clearly present in the membrane part of the protein.

In the present paper, we investigated by a similar approach the structure of the membrane part of the H+,K+-ATPase inserted in tubulovesicles isolated from stomach parietal cells. After proteolysis of either the cytoplasmic part or of both the vesicle interior and the cytoplasmic parts of the protein, the analysis of the secondary structure of the remaining membrane-associated part of the ATPase by Fourier transform infrared spectroscopy showed an amount of alpha -helical structure compatible with 10 alpha -helical transmembrane segments. These segments were oriented perpendicularly with respect to the membrane plane according to their infrared linear dichroism. Our results also suggested the presence of a large amount of beta -sheet structures in the membrane-associated segments of the tubulovesicle proteins.


EXPERIMENTAL PROCEDURES

Materials

Proteinase K, ATP, and nigericin were purchased from Sigma. Deuterium oxide was from Janssen Chimica (Geel, Belgium). All other reagents were of the highest purity grade commercially available.

Preparation of the Tubulovesicles

Tubulovesicles were isolated from hog gastric fundus by differential centrifugation as described (14). They were further purified from the microsomal fractions by centrifugation on a sucrose discontinuous density gradient at 100,000 × g overnight. The material collected at the 8-30% sucrose interface is referred to as the tubulovesicles. A single band at 95 kDa corresponding to the alpha -subunit of the H+,K+-ATPase was visible after SDS-PAGE1 analysis of a 6-µg sample on a mini-protean II (Bio-Rad).

Gel Electrophoresis

Gel electrophoresis was carried out on a 7.5% gel according to Laemmli (15) or on a 16% Tricine gel according to Schagger and von Jagow (16). Typically, 6-9 µg of protein were applied per lane on a 7-cm gel in the case of SDS-PAGE and 40-50 µg on a 10-cm 16% gel (+2 cm of spacer gel) in the case of SDS-Tricine gel. Bands were revealed by Coomassie Blue coloration. The samples were not boiled before running. In the case of SDS-Tricine gel, all the steps (polymerization and running) were performed at 4-5 °C.

ATPase Activity

ATPase activity of the tubulovesicles was determined in a medium containing 40 mM HEPES, 2 mM ATP, 2 mM MgCl2 at pH 7.2 in the presence or in the absence of 20 mM KCl. The vesicles were incubated at 37 °C for 15 min in this medium. The reaction was stopped by addition of 7% SDS (final concentration 1.75%). Inorganic phosphate formation was assayed according to Stanton (17) excepted that the coloration was revealed with ascorbate. Sucrose was 8% (w/v) in order to keep iso-osmotic conditions and nigericin was 14 µM.

Protein Assay

Proteins were assayed using the BCA kit from Pierce or by a modified Lowry method (18). Bovine serum albumin was used as standard in both cases.

Lipid Assay

Lipids were assayed with the enzymatic test for the choline reaction group from Boehringer Mannheim, assuming that lipids carrying a choline function represent 50% of the total of the lipids present in the tubulovesicles (19).

Proteolytic Digestion

We used two conditions for the proteolytic degradation of the water-exposed domains of the H+,K+-ATPase.

In the first case, vesicles containing the H+,K+-ATPase were diluted to a concentration of 2.5 mg/ml in the conservation buffer (8% sucrose, 50 mM HEPES, pH 7.2 with Tris) in order to keep iso-osmotic conditions. A freshly prepared solution of proteinase K (Sigma) in the same conservation buffer was added at a protease/protein ratio of 1/4 (w/w).

In the second case, tubulovesicles were first diluted and washed by centrifugation in a 1 mM HEPES, pH 7.2 (with Tris) buffer and then resuspended in the same buffer at a final concentration of 2.5 mg/ml. A freshly prepared solution of proteinase K in the 1 mM HEPES buffer was added at a protease/protein ratio of 1/2 (w/w). In this case, the same amount of protease was added after 3 and 7 h.

In both cases, samples were incubated at 37 °C and aliquots were removed after 0 min (in this case the inhibitor phenylmethylsulfonyl fluoride is added just before the protease), 5 min, 30 min, 1 h, 4 h, 7 h, 24 h, 48 h, and 72 h. A fresh solution of phenylmethylsulfonyl fluoride (100 mM in EtOH) was added to each aliquot at a final concentration of 6 mM to stop the proteolysis. NaCl was added to the digested vesicles to 0.5 M final concentration. The sample was then shaken on ice for a few minutes, and the samples were centrifuged at 45,000 × g for 45 min. The resulting pellets were twice resuspended in 1 mM HEPES, pH 7.2, and recentrifuged at 45,000 × g for 45 min. The final pellets were resuspended in 100 µl of 1 mM HEPES, pH 7.2, and stored at -20 °C.

Deglycosylation of the beta -Subunit

The beta -subunit of the H+,K+-ATPase was deglycosylated using the N-glycosidase F (Boehringer Mannheim) in denaturing conditions. To 100 µl of tubulovesicles (1 mg/ml) was added 3 µl of 10% (w/v) SDS and 7 µl of 10% beta -mercaptoethanol (v/v). The sample was boiled for 1 min. Then, 21 µl of n-octyl-beta -D-glucopyranoside 10% (w/v), 154 µl of phosphate-buffered saline-EDTA buffer (137 mM NaCl, 2.7 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, and 20 mM EDTA) and 15 µl of N-glycosidase F (250 units/ml) were added, before incubation at 37 °C during 24 h.

Amino Acid Microsequence Analysis of the beta -Subunit

The protein was isolated on a 14% SDS-polyacrylamide gel electrophoresis as described by Laemmli (15). The protein band was electroblotted onto polyvinylidene difluoride membranes by the method of Matsudaira (20), and the blot was stained with Coomassie Blue. Alkylation of cysteine in proteins with acrylamide was carried out in the reaction cartridge of the sequencer as described by Brune (21). Amino acid microsequence analysis was performed by automated Edman degradation of 1-10 pmol of peptides on a Beckman LF3400 protein sequencer (Beckman Instruments, Inc.). The samples was sequenced using standard Beckman sequencer procedure 4. All sequencing reagents were from Beckman Instruments, Inc.

FTIR Spectroscopy

Attenuated total reflection infrared (ATR-FTIR) spectra were recorded on a Perkin-Elmer infrared spectrophotometer 1720X equipped with a liquid nitrogen-cooled mercury-cadmium telluride detector. The internal reflection element was a germanium ATR plate (50 × 20 × 2 mm) with an aperture angle of 45° yielding 25 internal reflections. 128 scans were averaged for each spectrum. Spectra were recorded at a nominal resolution of 4 cm-1. The spectrophotometer was continuously purged with air dried on a silica gel column (5 × 130 cm) at a flow rate of 7 liters/min. Every 4 scans, reference spectra of a clean germanium plate were automatically recorded by a sample shuttle accessory. At the end of the scans, the spectra were transferred from the memory of the spectrophotometer to a computer for subsequent treatments.

Preparation of the Samples

Thin films were obtained as described by Fringeli and Günthard (22) by slowly evaporating 40-100 µl of the cleaved and noncleaved tubulovesicles under a N2 stream on one side of a germanium plate. This results in the formation of oriented multilayers at the surface of the plate. The ATR plate was then sealed in a universal sample holder and rehydrated by flushing D2O-saturated N2 at room temperature.

Lipid/ATPase Ratio

The lipid/protein (w/w) ratio is related to the gamma (C=O)lipid/delta (N-H)protein absorption bands ratio in the FTIR spectra. The ATPase/lipid ratio after hydrolysis was estimated from the ratio of the protein absorption peak (amide I (1600-1700 cm-1)) with respect to the lipid (C=O) absorption peak (1700-1800 cm-1) as established by Goormaghtigh et al. (23).

Secondary Structure Determination

Fourier self-deconvolution was applied to narrow the different components of the amide I region, which is the most sensitive to the secondary structure of proteins. The self-deconvolution was carried out using a Lorentzian line shape for the deconvolution and a Gaussian line shape for the apodization. To quantify the area of the different components of amide I revealed by the self-deconvolution, a least squares iterative curve fitting was performed to fit Lorentzian line shapes to the spectrum between 1700 and 1600 cm-1. Prior to curve fitting, a straight base line passing through the ordinates at 1700 cm-1 and 1600 cm-1 was subtracted. The spectrum arising from the lipid part of the system was found to be completely flat between 1700 cm-1 and 1600 cm-1 and was therefore not subtracted. To avoid introducing artifacts due to the self-deconvolution procedure, the fitting was performed on the nondeconvoluted spectrum. The proportion of a particular structure was computed to be the sum of the area of all the fitted Lorentzian bands having their maximum in the frequency region where that structure occurs divided by the total area of amide I. In these conditions, the difference with the x-ray determination amounted to 8% (standard deviation) for a large set of proteins tested (23).

Orientation of the Secondary Structures

The determination of molecular orientations by infrared ATR spectroscopy was performed as described by Goormaghtigh and Ruysschaert (24). When orientation was to be evaluated, additional spectra were recorded with perpendicular (0°) and parallel (90°) polarized incident light. The dichroism spectrum was computed by subtracting the 0° polarized spectrum from the 90° polarized spectrum taking into account the difference in the relative power of the evanescent field for each polarization as described before (25). A larger absorbance at 90° (upward deviation on the dichroism spectrum) indicates a dipole oriented close to a normal to the ATR plate. Conversely, a larger absorbance at 0° (downward deviation on the dichroism spectrum) indicates a dipole oriented closer to the plane of the ATR plate. The dichroic ratio R is defined as the ratio of the amide I area recorded for the parallel polarization (A90°) and perpendicular polarization (A). If only the alpha -helix component is ordered while the other structures have no particular orientation in the membrane,
A<SUP>90°</SUP><SUB>&agr;</SUB>=A<SUP>90°</SUP>−A<SUP>90°</SUP><SUB>u</SUB> (Eq. 1)
A<SUP>0°</SUP><SUB>&agr;</SUB>=A<SUP>0°</SUP>−A<SUP>0°</SUP><SUB>u</SUB> (Eq. 2)
where the index u refers to the fraction of the polypeptide chain with no preferential orientation. In ATR, the dichroic ratio for an isotropic sample Riso is different from unity (see Equation 3).
R<SUP><UP>iso</UP></SUP>=A<SUP>90°</SUP><SUB>u</SUB>/A<SUP>0°</SUP><SUB>u</SUB> (Eq. 3)
If x is the fraction of alpha -helix component in the protein, we can write the following (see Ref. 24).
(1−x) · (A<SUP>90°</SUP>+2A<SUP>0°</SUP>)=A<SUP>90°</SUP><SUB>u</SUB>+2A<SUP>0°</SUP><SUB>u</SUB> (Eq. 4)
Combining the equations above yields the alpha -helix dichroic ratio,
R<SUP>&agr;</SUP>=A<SUP>90°</SUP><SUB>&agr;</SUB>/A<SUP>0°</SUP><SUB>&agr;</SUB>=<FR><NU>R−<FR><NU>R+2</NU><DE>2R<SUP><UP>iso</UP></SUP>+1</DE></FR>(1−x)</NU><DE>1−<FR><NU>1</NU><DE>R<SUP><UP>iso</UP></SUP></DE></FR> <FR><NU>R+2</NU><DE> 2R<SUP><UP>iso</UP></SUP>+1</DE></FR>(1−x)</DE></FR> (Eq. 5)
where Ralpha is the dichroic ratio of the alpha -helix component. In the beta -sheet structure, the dipole associated with amide I lies in the plane of the sheet, essentially parallel to the amide C=O axis. In the alpha -helical structure, the amide I dipole is oriented at about 27° with respect to the helix axis (for a review, see Ref. 26). The mean orientation of the helix axis with respect to the membrane plane was estimated as described before (for a review, see Ref. 24).

Hydrogen/Deuterium Exchange

Films containing 70-100 µg of protein were prepared on a germanium plate as described above. Nitrogen gas was saturated with D2O (by bubbling in a series of five vials containing D2O) at a flow rate of 51 ml/min (controlled by a Brooks flow meter). Bubbling was started at least 1 h before starting the experiment. At zero time, the tubing was connected to the cavity of the sealed chamber surrounding the film. For each kinetic time point, 12 spectra are recorded and averaged at a resolution of 4 cm-1. Two kinetics were recorded simultaneously as described before (25, 27). The areas of amides I, II, and II' were obtained by integration between 1702 and 1596, 1596 and 1502, and 1492 and 1412 cm-1, respectively. For each spectrum, the area of amide II and amide II' was divided by the area of amide I. This permitted us to take into account small but significant variations of the overall spectral intensity due in part to the presence of D2O, which, by virtue of its presence, induces the swelling of the sample layer and therefore increases the average distance between the protein sample and the germanium crystal surface. Since the ATR spectrum intensity depends on this distance (28), this resulted in a loss of a few percent of the band intensity for all measured bands. Undeuterated spectra were recorded before the kinetic experiment as explained above, and 100% deuterated samples values were extrapolated by assuming a value of zero for the amide II surface. The area of amide II and II' (reported to the area of amide I) was finally expressed between 0 and 100% for each kinetic time point.

For the analysis of the deconvoluted spectra during the time course of exchange, each spectrum was self-deconvoluted with a 28-cm-1 FWHH Lorentzian lineshape and apodized with a Gaussian lineshape with a FWHH of 14 cm-1, yielding to a resolution enhancement factor of 2 (29).


RESULTS

Orientation of the ATPase Molecules in the Vesicles

In a first approach, the integrity and the orientation of the vesicles was assessed from ATPase activity measurements. ATPase activity (mean values ± S.D. from four experiments) expressed in µmol·h-1·mg-1 protein at 37 °C is 36 ± 7 in isotonic conditions; 111 ± 30 after addition of 14 µM nigericin and 122 ± 20 in hypotonic conditions (see "Experimental Procedures"). With these figures of activity, it was possible to evaluate the proportion of sealed vesicles oriented with the cytoplasmic side outside (30). The increased activity upon nigericin addition indicated that at least 68% of the ATPase molecules were pumping into the interior of a sealed vesicle. This is a minimum estimate arrived at by assuming that in the absence of nigericin, 32% (= 36/111 × 100) of the ATPase molecules was fully active, and 68% was completely inhibited by the electrochemical gradient. In fact, it is more likely that more ATPase molecules pumped protons into sealed vesicles but that the ion leak through the membrane was largely responsible for the residual ATPase activity. The fact that rupture of the vesicles by hypotonic conditions did not reveal significantly more activity than collapsing the electrochemical gradient by nigericin indicated that the ATP binding site of the ATPase molecules was homogeneously oriented toward the outside of the vesicles. The 10% activity increase recorded in hypotonic conditions indicated that no more than 10% of the vesicles was oriented with the ATP binding site located inside. That the hypotonic conditions opened the vesicles is demonstrated below.

In a second approach, we took advantage of the asymmetric localization of the beta -subunit of the ATPase. While most of the alpha -subunit mass is located on the cytoplasmic side (7, 31), the largest part of the beta -subunit is located inside (~35 amino acids outside for ~228 amino acids inside) and spans the lipid bilayer only once. Accessibility of the beta -subunit to proteolysis was therefore a good test for defining the orientation of the ATPase molecule. Visualization of native beta -subunit on SDS-PAGE was difficult, since it migrates as a broad band between 60 and 90 kDa. When deglycosylated (see "Experimental Procedures"), the beta -subunit appeared as a well defined band around 31 kDa (32, 33). Proteolytic degradation of the beta -subunit was therefore followed by SDS-PAGE after deglycosylation of the beta -subunit by N-glycosidase F in denaturing conditions (see "Experimental Procedures"). After proteolysis in iso-osmotic conditions, the beta -subunit appeared at 30.5 kDa on the gel just below the intact beta -subunit (31 kDa), with the same intensity (data not shown). Sequencing of the band at 30.5 kDa showed that the N-terminal part of the proteolyzed beta -subunit in iso-osmotic conditions begins at the 31st amino acid of the beta -subunit (G31RTLSRWVWISLYYV). This result indicated the accessibility of the sole cytoplasmic side of the ATPase to proteases even though the cytoplasmic part of the subunit represents only about 35 amino acids. After proteolysis in hypotonic conditions, the band of the beta -subunit completely disappeared (data not shown), indicating accessibility of both sides (cytoplasmic and vesicle interior) of the ATPase to proteases.

ATPase Membrane Part Isolation and Characterization

In order to isolate the membrane-embedded domain of the gastric H+,K+-ATPase, gastric tubulovesicles were treated with proteinase K. In a first set of experiments, we treated sealed tubulovesicles with proteinase K in iso-osmotic conditions (see "Experimental Procedures"). With this procedure, we potentially removed the cytoplasmic domain of the H+,K+-ATPase, leaving intact the membranous and vesicle interior parts of the protein. In a second set of experiments, the vesicles were opened by an osmotic shock before proteinase K was added. In this case, the protease had access to both sides of the membrane and was able to hydrolyze the cytoplasmic and extracytoplasmic parts of the ATPase molecules.

Kinetics of the Proteolytic Degradation

The percentage of proteolysis as a function of digestion time was monitored by following the evolution of the vesicle lipid:protein ratio after removal of digestion products. Lipid:protein ratios were calculated from the FTIR spectra of the re-isolated tubulovesicles (Fig. 1A) (see "Experimental Procedures"). The relative amount of protein remaining associated to the vesicles was reported as a function of the incubation time for the two proteolysis conditions (Fig. 2). The proteolysis kinetic was analyzed as the sum of two components (see Fig. 2). The fast component could represent the easily accessible part of the protein, and the slow one could represent less accessible segments (presumably because of their association with the membrane). For the sealed vesicles, the fast component represented 27% of the protein. This component rose up to 45% for the opened vesicles. The additional 18% could account for the extracytoplasmic parts of the ATPase made accessible to the protease after opening the vesicles. The kinetics constants (k1 and k2) (see Fig. 2) are similar for both conditions of proteolysis. Since k1 = 12 h-1, the rapid components (45% of the protein in hypotonic digestion conditions) were virtually totally hydrolized (99.999%) after 4 h. On the other hand, k2 = 6.10-3 h-1, indicating that less than 3% of the slow component was hydrolyzed after the same period of time.


Fig. 1. A, comparison of the spectra between 1800 and 1400 cm-1 of tubulovesicles (0 min) and tubulovesicles digested by proteinase K in isotonic conditions during 5 min, 30 min, 1 h, 4 h, 7 h, and 24 h. In order to visualize the decrease of the amide I surface (around 1650 cm-1) as a function of digestion time, the spectra were scaled so that the area of the lipid nu (C=O) band between 1770 and 1710 cm-1 is equal for all experiments, i.e. the spectra represent a same amount of vesicles. For the clarity of the figure, spectra were offset. B, spectra obtained by subtraction of each of the spectra in A from the spectra of the native tubulovesicles (0 min spectrum) after rescaling of the spectra on the lipid nu (C=O) peak area as described above. These calculated spectra represent the spectra of the part of the protein removed from the vesicles by the proteolysis. Spectra were reported on the same scale as A spectra. Addition of A and B corresponding spectra yield the spectrum of the undigested sample (0 min, A). Vertical dotted lines are placed every 50 cm-1 from 1500 to 1700 cm-1.
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Fig. 2. Amount of protein associated with the tubulovesicles during the time course of proteolysis with proteinase K. The percentage of protein was estimated by calibration of the protein amide I-integrated absorption peak (1600-1700 cm-1) compared with the lipid nu (C=O)-integrated absorption peak (1700-1800 cm-1), as established by Goormaghtigh et al. (23). The values are reported for samples digested in isotonic conditions (filled circles) and for samples digested in hypotonic conditions (open circles). The experimental data were fitted as a sum of two exponential components (solid line) by nonlinear regression (algorithm of Marquardt-Levenberg) of a function f = a1e-k1t + a2e-k2t, where a1 and a2 are the proportions of each of the two populations and k1 and k2 the corresponding time constants.
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Membrane-associated Peptides

On SDS-PAGE (Fig. 3), after proteolysis in hypotonic conditions, we observed the disappearance of the 95-kDa alpha -subunit band of the H+,K+-ATPase. This disappearance was accompanied with the appearance of several bands with molecular weight below 11 kDa (see Fig. 3, lane 1) which were not present in the intact tubulovesicles (Fig. 3, lane 2). Four bands were resolved by SDS-PAGE and migrated at 10,800, 8900, 7800, and 6600 Da, respectively. This peptide pattern was already stabilized after 4 h of incubation for both conditions of proteolysis and was not modified for longer digestion times (up to 48 h). The peptide pattern was identical for both proteolysis conditions.


Fig. 3. SDS-PAGE tricine. Lane 1, tubulovesicles digested by proteinase K in hypotonic conditions during 48 h; lane 2, during 0 min; lane 3, molecular weight ladder (low range molecular weight standards supplied by Life Technologies, Inc.).
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Structure of the ATPases in the Tubulovesicles: Secondary Structure of the ATPases and of Its Membrane-associated Fraction

Attenuated total reflection IR spectroscopy (ATR-IR) was used to determine the structure of the ATPase and of its isolated membrane-associated part. Fig. 1A reports the shape of amide I in the course of the proteolysis in isotonic conditions (from 5 min to 24 h). Amide I bands were relatively broad with a maximum at 1655 cm-1 and a shoulder near 1635 cm-1 for all digestion times. Fig. 1B reports the spectra obtained by subtraction of the spectrum of the native ATPase (i.e. the 0-min spectrum in A) from every spectrum reported in A. The spectra in B are therefore the spectra of the cleaved parts of the ATPase molecules and are representative of the structure of the removed peptides as they were in the native protein. The maximum near 1655 cm-1 and the shoulder near 1640 cm-1 suggest the presence of both alpha -helix and beta  structure in the peptides accessible to the protease. Assigning the different amide I components revealed by Fourier self-deconvolution to different secondary structures is best substantiated by monitoring their shift upon hydrogen/deuterium exchange. Figs. 6 and 7 report, respectively, the spectra of the intact ATPase (Fig. 6) and of the membrane-associated ATPase fragments isolated after incubation in the presence of proteinase K for 48 h in hypotonic conditions (Fig. 7) for deuteration times from 0 to 512 min. In B of Figs. 6 and 7, spectra have been self-deconvoluted with a 28-cm-1 FWHH (full width at half-height) Lorentzian line shape and apodized with a 14-cm-1 FWHH, Gaussian line shape as described elsewhere (24). The lipid nu (C=O) band was located at 1744 cm-1 before and after protease treatment (Figs. 6 and 7). For the native ATPase (Fig. 6B), the amide I region presented two main features located at 1654 and 1636 cm-1. We assign them, respectively, to the alpha -helix and beta -sheet structure. The exchange of the amide hydrogen by deuterium (52% of exchange after 60 min, see below) did not alter the frequency of the two main features resolved here (the frequency shift upon deuteration reaches large values only when deuteration is close to completion). This confirmed the assignment of the 1654 and 1636 cm-1 bands to alpha -helix and beta -sheet stable secondary structures. This observation is important, since several structures (loops, various turns, extended chains; see Goormaghtigh et al. (26) for a more exhaustive compilation) may absorb in the same frequency range but are expected to exchange much faster because of weaker internal hydrogen bonds. The random coil-associated band was probably too broad to be resolved in the self-deconvolution conditions used here (25, 34) and was therefore hidden in the base line underlying the resolved features described above. Two similar peaks, located at 1653 and 1633 cm-1, were found in the membrane part of the ATPase (Fig. 7B) and were assigned, respectively, to alpha -helix and beta -sheet structures. The additional small 1695-cm-1 component resolved here was tentatively assigned to the high frequency component of the beta -sheet structure. These results suggest a significant amount of beta -sheet structure in the membrane-associated domain of the ATPase.


Fig. 6. A, infrared spectra between 1800 and 1400 cm-1 of the H+,K+-ATPase in the tubulovesicles. Spectra were recorded as a function of time of exposure to D2O-saturated N2 flow, which is indicated (in minutes) in the right margin of the figure. Integration of amide I, amide II, and amide II' was performed using the base lines indicated by the dotted lines drawn between the intersection of the spectra with the vertical line segments. B, same as A, but after deconvolution of the spectra in order to better visualize the evolution of the different components of the amide I band during the deuteration.
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Fig. 7. Same as Fig. 6, but the membrane-associated ATPase fragments isolated after digestion by proteinase K in hypotonic conditions during 48 h were analyzed.
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A quantitative analysis of the amide I band of deuterated spectra by Fourier self-deconvolution and least squares curve fitting (23) allowed us to quantify this secondary structures (Fig. 4). Results are reported in Table I.


Fig. 4. Decomposition of the amide I' peaks in Lorentzians, obtained as described (23) for the native H+,K+-ATPase (a), for the digested H+,K+-ATPase in isotonic conditions during 48 h (b), and for the digested H+,K+-ATPase in hypotonic conditions during 48 h (c).
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Table I.

Percentage of secondary structures (and corresponding number of amino acid residues) determined by Fourier self-deconvolution followed by a least squares curve fitting of the amide I peak for the native tubulovesicles and for the tubulovesicles digested by the proteinase K during 48 h in isotonic conditions and hypotonic conditions (see text)

The data presented are the mean value obtained for four independent experiments (n = 4). Standard deviation is 4%. aa, amino acids.
 alpha -Helix  beta -Sheet Turn Random coil

Native tubulovesicles (= 1324 aa) 32% 31% 16% 20%
424 aa 410 aa 212 aa 265 aa
Tubulovesicles digested in Isotonic 32% 28% 14% 26%
  conditions (= 967 aa) 309 aa 271 aa 135 aa 251 aa
Tubulovesicles digested in 34% 34% 18% 14%
  Hypotonic conditions (= 728 aa) 248 aa 248 aa 131 aa 102 aa

Orientation of the Secondary Structures of the ATPase in the Tubulovesicles

Spectra of native and digested tubulovesicles were recorded using polarized light at 90 and 0° (see Ref. 24 for the geometry of the experiment). The dichroic spectra were obtained by subtracting spectra recorded with 0° polarized light from spectra recorded with 90° polarized light, for native tubulovesicles (Fig. 5) and for the vesicles digested in the two conditions used (data not shown). In all cases, a positive deviation of the dichroic spectra was present in the 1656-1660-cm-1 spectral domain assigned to the alpha -helical structure. As the amide I nu (C=O) dipole lies approximately parallel to the helix axis, this deviation suggested a perpendicular orientation of the helix axes with respect to the membrane plane for the majority of the alpha -helices. In the beta -strand region of the spectra (1637-1613 cm-1), no deviation could be observed. Integration of the amide I area between 1706 and 1597 cm-1 yielded a dichroic ratio of 1.53 for the native ATPase, 1.63 for the ATPase digested in isotonic conditions, and 1.66 for the ATPase digested in hypotonic conditions with a value of Riso (see "Experimental Procedures") of 1.45. Taking into account the alpha -helical content reported in Table I, the corresponding Ralpha values are, respectively, 1.59, 1.77, and 1.82. Since it is not legitimate to assume an uniaxial distribution of the helix axis orientations in the entire protein, it was not possible to translate the value of Ralpha  = 1.59 into a mean angular orientation. On the other hand, the values of 1.77 and 1.82 correspond to a near-perpendicular orientation of the helix axes with respect to the membrane plane, taking into account a 27° angle between the transition dipole and the helix axis (see Refs. 24 and 27 for a thorough discussion on the method and on the 27° value). The latter values of Ralpha implied little orientational disorder in the helical component.


Fig. 5. Infrared polarized parallel (90°) and perpendicular (0°) absorption spectrum between 1800 and 1400 cm-1 of native ATPase. The spectra were recorded after 6 h deuteration of the samples. The dichroic spectrum (top of the figure) is the difference between the 90 and 0° spectra (see "Experimental Procedures"). The dichroic spectrum is enlarged four times as compared with the 90° spectrum and smoothed by apodization of its Fourier transform by a 6-cm-1 FWHH Gaussian line shape.
[View Larger Version of this Image (14K GIF file)]


Amide Hydrogen/Deuterium Exchange Kinetics

At constant pH and temperature, the rate of amide hydrogen exchange by deuterium is related to the stability of the secondary structures and to the solvent accessibility to the NH amide groups. Amide hydrogen exchange was followed by monitoring the amide II absorption peak (maximum at 1544 cm-1) decrease or amide II' absorption peak (maximum at 1450 cm-1) increase as a function of the time of exposure to D2O-saturated N2 flow (from 15 s to 8 h; for details, see "Experimental Procedures"). This is illustrated in Figs. 6 and 7, where a series of spectra recorded as a function of the deuteration time for the ATPase (Fig. 6) and for the ATPase digested in hypotonic conditions (Fig. 7) are reported. The evolution of the area of the amide II for each sample was then computed between 0 and 100% of deuteration as explained under "Experimental Procedures." The hydrogen/deuterium exchange was faster for the digested ATPase than for the native one (Fig. 8). After 1-h deuteration, 67% of the peptide N-H was exchanged in the protease-treated in hypotonic conditions sample and 61% in isotonic conditions of digestion, whereas for the intact ATPase the exchange was only 52% after 1 h.


Fig. 8. Percentage of deuteration reported as a function of the deuteration time for native ATPase (open circles), for the ATPase digested by proteinase K during 48 h in isotonic conditions (filled circles), and in hypotonic conditions (open triangle). The percentage was estimated after the amide II surfaces evolution as described under "Experimental Procedures."
[View Larger Version of this Image (19K GIF file)]



DISCUSSION

The aim of this work was to test experimentally the models proposed to describe the mode of insertion of the gastric H+,K+-ATPase into the membrane. The approach chosen was to use proteases to attempt to cleave the segments of ATPase protruding from the membrane while preserving the integrity of the membrane domain and to investigate the structure of this membrane domain by ATR-FTIR spectroscopy.

Critical assumptions in the present work are that 1) proteolytic cleavage is efficient outside the membrane but does not reach the membrane part of the protein, and 2) the structure of the membrane part of the protein is not affected by removal of the extramembrane loops. These two assumptions have been discussed previously in detail and experimentally verified in similar experimental conditions on a membrane protein of know structure: bacteriorhodopsin (25).

The choice of proteinase K has been suggested by the fact that it is one of the most active unspecific protease and because it can be efficiently separated from lipid vesicles after proteolysis as described before (25). The fact that proteinase K completely cleaves the parts of the protein protruding from the vesicle is crucial for the conclusions of this paper. Proteinase K has been shown to be one of the most effective proteases to remove the extramembrane part of proteins (e.g. Refs. 13, 25, and 35, 36, 37, 38, 39). Furthermore, as some specificity of proteinase K for selected amino acids or secondary structures could eventually hinder the proteolytic action, the association of different proteases is likely to overcome this barrier. Incubation of the ATPase with trypsin or subtilisin before the incubation with proteinase K did not improve the extent of digestion (data not shown). Therefore, we consider the possibility of extramembrane domain(s) resistant to the protease as unlikely. The second critical assumption is that the isolated transmembrane segments keep their native structure. From thermodynamic consideration, it seems likely that the structure of the membrane embedded segments is essentially dependent on the membrane environment and that changes in the extramembrane region would have little influence on it. The only well documented membrane protein behavior upon proteolysis is that of bacteriorhodopsin, which confirms this hypothesis. Experiments carried out previously under similar experimental conditions indicated that the strong amide I polarization which demonstrates a transmembrane orientation of the alpha -helices is identical in both the intact bacteriorhodopsin molecule and in its membrane domain isolated after proteinase K treatment (25). These data bring additional indication of the integrity of isolated membrane domain structures. These conclusions are in line with cleavage experiments performed by Draheim et al. (40) which showed that the secondary structure and the orientation of the isolated membrane domain of bacteriorhodopsin do not change after proteolysis. Moreover, the integrity and stability of membrane fragments of bacteriorhodopsin after proteolysis or after separated reconstitution is well documented (41, 42, 43). In the present work, the similar polarization of the alpha -helical component of amide I in the intact protein and in its membrane part indicated the stability of the orientation of this membrane protein structure upon removal of the connecting segments. A similar stability of the isolated transmembrane segments was also demonstrated on the Neurospora crassa plasma membrane H+-ATPase (25).

The amount of protein removed by the proteolytic treatment when either only the outside of the vesicle or both sides of the vesicle were accessible to the proteases can be discussed with respect to the current model of the ATPase. The comparison of the model with the experimental data supposes that the orientation of the vesicles is characterized. ATPase activity measurements indicated that more than 68% of the ATPase molecules was oriented with their cytoplasmic side facing the outside of sealed vesicles. Proteolytic cleavage confirmed that more than 95% of the ATPase molecules had this orientation. In isotonic conditions, proteinase K removed only the first 30 amino acids of the beta -subunit of the ATPase. These 30 amino acids correspond to the N-terminal and sole cytoplasmic part of the subunit. Whereas after opening of the vesicles in hypotonic conditions cytoplasmic and extracytoplasmic sides of the subunit became accessible to the protease. Our experimental results indicated that the sole membrane-associated domain of the ATPase represented about 55% of the protein (i.e. 728 amino acids, see Table I), i.e. more than expected from the hydropathy plots (about 15-20%) (7, 31). Only a few works addressed the question of the amount of protein present in the lipid bilayer. Sequencing of the isolated membrane-associated peptides could demonstrate the presence of some peptide sequences in the membrane, although the estimation of their length rely on the apparent mass deduced from SDS-PAGE. The presence of other peptides which either do not migrate as well resolved bands or do not stain is likely (44). An additional hypothesis to explain the discrepancy between prediction and experimental data is that only transmembrane alpha -helix secondary structures have been considered so far. It has also been shown for the Na+,K+-ATPase that models based on the hydrophobicity of the amino acid sequence underestimate there protein insertion into the bilayers, in comparison with models based on chemical labeling experiments (45). Accessibility increases upon opening the vesicles in hypotonic conditions precisely accounted for the extracytoplasmic side of the beta -subunit, suggesting little degradation of the alpha -subunit from the extracytoplasmic side of the membrane. Extracytoplasmic parts of the alpha -subunit of other P-type ATPases have been described as small: the Ca2+-ATPase extracytoplasmic domain is small according to crystal analysis by x-ray diffraction (46, 47, 48), and the extracytoplasmic loops of the H+-ATPase from N. crassa (49) and of the Na+,K+-ATPase alpha -subunit (50) are highly resistant to tryptic digestion.

Quantitative evaluation of the structure of the ATPase before incubation with protease (Table I) is in good agreement with secondary structure determined for other P-type ATPases (25, 51, 52). Analysis of the sole membrane-associated part of the protein indicated the presence of 34% beta -sheet (248 amino acid residues). Particularly convincing as to the presence of beta -sheet structures in the membrane-associated proteolytic peptides of the ATPase were the IR spectra reported in Fig. 7B, where one of the two main resolved features in amide I was located at 1633 cm-1, a frequency assigned to the beta -sheet structure and which is never overlapped by helical contribution (26, 53). Extended polypeptide chains have been found at similar frequencies in some protein (34), but this hypothesis is unlikely here since only beta -sheet structures can form the internal hydrogen bonding required in the membrane environment. Furthermore, if not buried in the membrane environment, such an extended polypeptide chain should be easily accessible to the proteases and available for fast H/D exchange, at odds with the experimental results. The absence of a distinct dichroism from the beta -sheet structure implies that these beta -sheets are either disordered or are oblique, in agreement with the lack of distinct infrared linear dichroism observed for porine (54). The presence of beta -sheet structures in the membrane-associated domain of the ATPase implies that the current models for these proteins need to be revised. In the transmembrane structure of the porin monomer, 16 segments of 11 ± 5 residues length form a beta -pleated sheet structure and are connected by turns (55), but segments as short as 5 residues in length are sufficient to span the hydrophobic core of the membrane (56). It is premature to describe how beta -sheet structures could be associated with the membrane in the case of the ATPase, but the presence of a rather large number of short stretches spanning the membrane would explain the relatively large number of amino acids involved in turn structures. The 248 amino acid residues involved in alpha -helices are compatible with the presence of about 10 transmembrane helices in the ATPase, in good agreement with the predictions based on the protein sequence. The analysis of the dichroic spectra reveals an orientation of the alpha -helices in agreement with a transmembrane orientation.

Little data exist on the amide hydrogen/deuterium exchange rate in membrane proteins (for a review, see Ref. 57). For bacteriorhodopsin, the exchange reaches a plateau when 30% of the amide protons has been exchanged (25, 58, 59). This value corresponds roughly to the fraction of the protein protruding from both sides of the membrane and suggests that the transmembrane helices are exchanged at an extremely slow rate. Similarly, the single transmembrane alpha -helical segment of glycophorin is virtually not exchangeable (38, 60). On the other hand, the human erythrocyte glucose transporter exchanges about 80% of its amide protons by deuterium within the first hour (61), even though structure predictions suggest the presence of 12 alpha -helices accounting for 50% of the protein in the membrane. It was concluded that an access to the aqueous medium exists in the transmembrane part of the protein. It must be mentioned that the 12-alpha -helix model has been recently challenged (62). Similarly, the channel-forming peptide of colicin E1, which has been shown to be predominantly alpha -helical, also has very fast exchange (80% exchange in 5 min (63)). The results obtained here for the H+,K+-ATPase show that for the membrane-associated part of the ATPase, 67% of exchange is observed after 1 h (corresponding to about 488 amino acids out of 728), indicating that the membrane region is much less tightly protected against hydrogen/deuterium exchange than the membrane region of bacteriorhodopsin. These data suggest that building a model of the transmembrane region of the ATPase based on the structure and on the packing of the transmembrane segments of bacteriorhodopsin is not legitimate, as already evident from secondary structure analysis.

In a recent paper, Corbalan-Garcia et al. (64) applied a proteolytic approach to the sarcoplasmic reticulum Ca2+-ATPase. Digestion by trypsin (30 min) and proteinase K (30 min) on the cytoplasmic side of the membrane resulted in the removal of 63% of the protein in agreement with the prediction model derived value of 66% (65). Fourier self-deconvolution and partial least squares analysis of amide I demonstrated the absence of beta -sheet structure in the proteinaceous material associated with the membrane after proteolytic digestion. Experiments carried out in our laboratory (not shown) confirm the Ca2+-ATPase results obtained by Corbalan-Garcia et al. (64) and suggest that gastric ATPase and N. crassa H+-ATPase (25) show a larger membrane-associated protease-resistant fraction than the Ca2+-ATPase. Whether a serious structural difference exists between H+,K+- and H+-ATPase on one hand and Ca2+-ATPase on the other hand is now to be confirmed by other means. The analysis of the primary structure of the H+,K+-ATPase by the algorithm of Schirmer and Cowan (66), designed to reveal porin-like beta -sheet transmembrane segments, indicated the presence of 12 such segments in the catalytic subunit of the H+,K+-ATPase, but only 7 in the Ca2+-ATPase (not shown).

In conclusion, our results showed the presence in the membrane-associated domain of the gastric H+,K+-ATPase of 248 amino acid residues involved in alpha -helical structures, in agreement with the presence of 10 transmembrane helices. Linear dichroism data confirmed their transmembrane orientation. In addition, 248 amino acid residues were also involved in beta -sheet structures associated with the membrane, 131 were involved in turns, and 102 in random coil structure. The hydrogen/deuterium exchange rate of the membrane part of the protein suggested an organization of these different secondary structures in such a way that the solvent had access to a large fraction of the protein, perhaps through a membrane hole or pore delimited by the protein.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Fonds pour la Promotion de la Recherche dans l'Industrie et dans l'Agriculture fellow (Belgium).
§   Senior Research Associate of the National Fund for Scientific Research (Belgium). To whom correspondence should be addressed: Free University of Brussels, Campus Plaine CP 206/2, B1050 Brussels, Belgium. Tel.: 32-2-6505386; Fax: 32-2-6505113; E-mail: egoor{at}ulb.ac.be.
1    The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ATR, attenuated total reflection; FTIR, Fourier transform infrared; FWHH, full width at half-height.

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