Solution structure of the fifth and sixth transmembrane segments of the mitochondrial oxoglutarate carrier

Abstract

The structures of the fifth and sixth transmembrane segments of the bovine mitochondrial oxoglutarate carrier (OGC) and of the hydrophilic loop that connects them were studied by CD and NMR spectroscopies. Peptides F215-R246, W279-K305 and P257-L278 were synthesized and structurally characterized. CD data showed that at high concentrations of TFE and SDS all peptides assume α-helical structures. ¹H-NMR spectra of the three peptides in TFE/water were fully assigned and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides were generated by distance geometry calculations. A well-defined α–helix was found in the region L220-V243 of peptide F215-R246 (TMS-V), in the region P284-M303 of peptide W279-K305 (TMS-VI) and in the region N261-F275 of peptide P257-L278 (hydrophilic loop). The helix L220-V243 exhibited a sharp kink at P239, while a little bend around P291 was observed in the helical region P284-M303. Fluorescence studies performed on peptide W279-K305, alone and together with other transmembrane segments of OGC, showed that the W279 fluorescence was quenched upon addition of peptide F215-R246, but not of peptides K21-K46, R78-R108 and P117-A149 suggesting a specific interaction between TMS-V and TMS-VI of OGC.

• Mitochondria
• oxoglutarate carrier
• structure
• NMR

Abbreviations
CD	=	circular dichroism
CSI	=	chemical shift index
DIPEA	=	di-iso-propylethylamine
DMF	=	N,N-dimethylformammide
DSS	=	4,4-dimethyl-4-silapentane-1-sulfonate
EDT	=	1,2-ethanedithiol
Fmoc	=	9-fluorenylmethyloxycarbonyl
HPLC	=	high performance liquid chromatography
NMR	=	nuclear magnetic resonance
NOE	=	nuclear Overhauser effect
OGC	=	oxoglutarate carrier
PyBop	=	benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophoshate
RMSD	=	root-mean-square deviation
SDS	=	sodium dodecyl sulphate
TCEP	=	tris (2-carboxyethyl)phosphine
TFA	=	trifluoroacetic acid
TFE	=	trifluoroethanol
TM	=	transmembrane
TMS	=	transmembrane segment

Introduction

The oxoglutarate carrier (OGC) is a nuclear-coded intrinsic membrane protein localized in the inner membranes of mitochondria. It catalyzes the electroneutral exchange of 2-oxoglutarate for malate by a rapid-equilibrium random sequential mechanism and plays a central role in several metabolic processes (Krämer & Palmieri [1992]). This transporter belongs to the mitochondrial carrier protein superfamily, which includes the ADP/ATP carrier and more than twenty other functionally-characterized members (for a review see Palmieri [2004]). Family members have three tandemly repeated domains about 100 amino acids in length. Each domain contains the signature sequence motif PX(D/E)XX(K/R)X(K/R) and two hydrophobic stretches linked by an extensive hydrophilic region (Walker & Runswick [1993]; Palmieri [1994]). Peptide specific antibodies and enzymatic cleavage revealed that OGC is folded in the inner mitochondrial membrane into six transmembrane segments with the N- and C-terminal regions protruding toward the cytosol and the three long hydrophilic loops between the odd and the even transmembrane segments on the matrix side of the membrane (Bisaccia et al. [1994]). Cross-linking studies showed that the functional unit is a homodimer (Bisaccia et al. [1996]). Cys-scanning mutagenesis identified two residues in TMS-IV of OGC that are essential for the carrier activity (Stipani et al. [2001]) and site-directed spin labelling identified TMS-IV as an amphipatic α-helix with the hydrophilic face containing the two residues essential for the carrier activity (Morozzo della Rocca et al. [2003]).

For the various members of the mitochondrial carrier family, direct methods of structure determination, i.e., crystallographic studies and spectroscopic analyses in solution, are difficult because of their hydrophobicity and because they undergo rapid structural changes required for their catalytic cycle. Not long ago, the X-ray crystallographic structure of the ADP/ATP carrier in complex with carboxyatractyloside, which binds the carrier very tightly and blocks the protein in a fixed inhibited conformation, was solved (Pebay-Peyroula et al. [2003]). The crystal unit cell contains one monomer per asymmetric unit. The transmembrane domain consists of six TM α-helices. The folding of the three repeated domains is very similar, each of the odd-numbered helices exhibiting a sharp kink, which is due to a proline residue located in the sequence motif characteristic of the mitochondrial carriers. Recently the structures of the first and second transmembrane segments of OGC were determined by NMR spectroscopy (Castiglione-Morelli et al. [2004]). The data obtained showed well-defined α-helices from K24 to V39 and from A86 to F106 in good agreement with the crystal structure of the first two TMSs of the ADP/ATP carrier-carboxyatractyloside complex.

In this work, we determined the structure of the last two TMSs of the bovine mitochondrial OGC and of the hydrophilic loop joining these TMSs. The peptides F215-R246 and W279-K305 were chosen on the basis of the hydrophobicity profile (Runswick et al. [1990]) and the transmembrane topology (Bisaccia et al. [1994]) of the bovine OGC, and the peptide P257-L278 was chosen for its homology with the α-helix region found in the loop between TMS-V and TMS-VI of the ADP/ATP carrier (Pebay-Peyroula et al. [2003]). The peptides were synthesized and characterized by CD and NMR spectroscopies. Within each peptide the NMR analyses identified a well-defined-helix structure. In addition, fluorescence quenching experiments of the tryptophan located in the first position of peptide W279-K305 suggested that TMS-V and TMS-VI of OGC interact with each other.

Materials and methods

Peptide synthesis and purification

Peptides FSDNILCHFCASMISGLVTTAASMPVDIVKTR (F215-R246), WKGFTPYYARLGPHTVLTFIFLEQMNK (W279-K305) and PEYKNGLDVLFKVVRYEGFFSL (P257-L278) were synthesized using standard Fmoc chemistry on a Pioneer synthesizer. Coupling reagents (0.5 M PyBop/DMF and 1.0 M DIPEA/DMF) were used with a fourfold excess amino acid. TFA/H₂O/thioanisole/phenol/EDT (88%, 3%, 3%, 4%, 2%) mixture was used for the deprotection and cleavage of the peptides (King et al. [1990]). Crude peptides were precipitated in cold diethyl ether and then lyophilized. Purification of the peptides was carried out on a reverse-phase C₁₈ column (25×300 nm, 5 µm particles) using a gradient of acetonitrile/water in 0.1% TFA. Sample identity was confirmed with electrospray mass spectroscopy.

Circular dichroism

Circular dichroism spectra were recorded on a Jasco 600A automatic circular dichrograph equipped with a thermoelectric temperature controller, using a 0.1-cm cylindrical quartz cell with a peptide concentration of 0.1 mg ml⁻¹ at 25°C. Measurements on peptide F215-R246 were performed in the presence of 5-fold cysteine residue molar excess of TCEP, a mild reducing agent. Spectra were obtained with 0.1 nm steps from 190 to 250 nm, 1 nm bandwidth, a time constant of 0.5 sec and 20 mdeg of sensitivity. After the baseline spectra of the solvents were subtracted, spectra were smoothed using the Fourier transform routine of the J-600A. Data were expressed in terms of the molar ellipticity per residues [θ]_R in units of deg×cm²×dmol⁻¹.

NMR experiments

Samples for NMR spectroscopy were approximately 1.5 mM in H₂O/TFE-d₃ 20:80 (v/v). The ¹H-NMR spectra were acquired on a Varian Unity Inova-500 spectrometer. One-dimensional spectra were acquired in Fourier mode with quadrature detection, and the water signal was suppressed by a 2.5 s pre-saturation pulse or with the WATERGATE pulse sequence (Piotto et al. [1992]). Two-dimensional DQF-COSY (Piantini et al. [1982]), TOCSY (Bax & Davis [1985]) and NOESY (Jeener et al. [1979]) spectra were collected in the phase-sensitive mode using the States method (States et al. [1982]). Typical data were 2048 complex data points, 8 or 32 transients and 256 increments. A mixing time of 80 ms was used for the TOCSY experiments and of 100–200 ms for NOESY. The methyl resonance of DSS was used as reference for chemical shift calibration. Sequential resonance assignments were made by the approach described by Wüthrich ([1986]). When necessary, to resolve ambiguities arising from chemical shift degeneracy, spectra were recorded at different temperatures (25 and 30°C). For isolated resonances, measurements of ³J_αH-NH coupling constants were obtained from 1D experiments acquired with 128 K points and application of strong resolution enhancement. For overlapping lines, coupling constants were estimated from DQF-COSY spectra.

Structure calculation

Experimental NOE intensities were converted into proton-proton distance constraints classified into three ranges: 1.8–2.7 Å, 1.8–3.3 Å and 1.8–5.0 Å corresponding, respectively, to strong, medium and weak NOEs (Wüthrich [1986]). Pseudoatoms were introduced when no stereospecific assignment was determined, and interproton distances were corrected accordingly (Wüthrich et al. [1983]). The structures were calculated with the DYANA 1.5 program (Güntert et al. [1997]) using a standard simulated annealing protocol. From an initial ensemble of 100 structures the best 50, in terms of target function values and residual distance restraint violations, were chosen to represent the conformations of the peptides. The resulting structures were analyzed with the MOLMOL graphics program (Koradi et al. [1996]), which was also used to produce all the molecular plots.

Fluorescence spectroscopy

Intrinsic fluorescence studies of the peptide W279-K305 were performed at 25°C on a Fluorog-3 spectrometer (Jobin Yvon/SPEX instruments) equipped with a thermoelectric temperature controller. All data collection was performed with DATAMAX software. The excitation wavelength was set at 295 nm, the maximal absorption for tryptophan. Fluorescence emission spectra were recorded from 305–500 nm in TFE/water 80:20 (v/v) in 1-cm quartz cuvette. The intrinsic fluorescence of peptides added to W279-K305 was negligible in comparison with that of W279-K305. To determine the dissociation constant of the interaction, aliquots of F215-R246 in concentration ranging from 5 to 55 µM were added to a solution 11 µM of W279-K305 in 80% TFE, and fluorescence at 355 nm was measured. The experimental results were plotted using GRAFIT software (Erithacus Software Limited) according to Pearce and Hawrot ([1990]).

Results

Circular dichroism measurements of F215-R246 and W279-K305

Figure 1a shows the CD spectra of peptide F215-R246 in TFE/water at different percentages (v/v). At 100% TFE the spectrum was characterized by a negative 222 nm band due to the peptide n-π* transition , a negative 208 nm and a positive 192 nm bands due to π-π* transition exciton splitting of the peptide (Sreerama & Woody [2000]). This strongly suggests the existence of a significant population of α-helical conformers. A decrease in molar ellipticity was observed as water percentage increases until it was difficult to record a CD spectrum at 20% TFE, where peptide is scarcely soluble. Further support to the presence of folded structures was obtained by the behaviour of peptide in SDS (Figure 1b). In SDS, from 0.1% to 5%, CD spectra exhibited the typical bimodal curve of the α-helical conformation.

Figure 1. CD spectra of peptide F215-R246 in: (a) TFE at 100% (x), 95% (-), 80% (Δ), 50% (□), 20% (---); (b) SDS 5% (Δ), 2% (---), 1% (x), 0.1% (□); and of peptide W279-K305 in (c): TFE 100% (x), 95% (-), 80% (Δ), 50% (□), 20% (---); (d) SDS 5% (Δ), 2% (---), 1% (x), 0.1% (□). The data were expressed in terms of [Θ]_R, the molar ellipticity per residue.

Peptide W279-K305 was already folded at 20% TFE (Figure 1c). To determine the effect of different TFE percentages on the helical content of this peptide, TFE was increased from 20% to 100%. This increase did not yield any significant change in the CD spectra. The spectrum recorded at 0.1% SDS was hardly detectable because of the peptide poor solubility. On increasing the SDS percentage, the spectra show a negative band at 208 nm, a shoulder at 222 nm and a positive molar ellipticity around 190 nm that are indicative of a predominant presence of helical structure and of a small contribution of β-turn conformations (Figure 1d). Taken all together, CD data indicate that the peptides assume predominantly α-helical conformation in both TFE and SDS.

NMR results

For the NMR analysis the membrane-mimicking agent TFE was used as a co-solvent, because of its small size compared to lipids, as well as its lipid-like characteristics (Raussens et al. [2002]). Furthermore TFE has been shown to promote helical structure in peptides only in regions with predicted helical propensity (Buck [1998]). Proton resonance assignments in 80% TFE/20% water (v/v) mixture were obtained from TOCSY experiments at 25°C and 30°C, and NOESY experiments were used for sequential resonance assignments. Secondary structure was determined from a qualitative interpretation of inter-residue interactions observed in NOESY spectra, ³J_αH-NH coupling constants, and αH chemical shifts. The chemical shifts and the line widths of the NH resonances in 1D-spectra and NOESY experiments were virtually independent from concentrations 1.5×10⁻³ M to 1.5×10⁻⁴ M, indicating that there is no intermolecular association under our conditions. For structure determination we used the NMR constraints collected from the spectra of the concentrated samples because of their better quality.

Peptide F215-R246

An almost continuous stretch of strong sequential NH_i-NH_i + 1 NOEs was observed from residue L220 to M238 and from V240 to V243 (Figure 2a). Many αH_i-βH_i + 3 and αH_i-NH_i + 3 connectivities were detected but peak overlaps prevented the observation of αH_i-NH_i + 3 connectivities in the region A225-V232 and of both αH_i-βH_i + 3 and αH_i-NH_i + 3 (marked with open bars in figure) in the region V240-V243. A secondary structure analysis based on chemical shift of αH protons (Wishart et al. [1991]) showed upfield shifts relative to random coil values (Merutka et al. [1995]) in the regions L220-A236 and V240-V243, indicating the formation of a helix there. ³J _αH-NH coupling constants <6.0 Hz were also observed.

Figure 2. Summary of sequential and medium-range NOEs observed for peptide F215-R246 (a) and peptide W279-K305 (b) in 80% TFE at 25°C. The thickness of lines is related to the intensity of NOEs. Open bars indicate overlapping peaks. Asterisks under the amino acid one-letter codes indicate apparent ³J_αH-NH coupling constants of non-Gly residues <6.0 Hz. Chemical Shift Index (CSI) of αH protons are shown below the bar diagrams for each peptide. Negative values indicate a helical conformation.

A total of 190 NOEs were obtained: 94 intraresidue, 81 short-range and 15 medium-range. They were translated into distance constraints for the structure calculations and the DYANA program (Güntert et al. [1997]) was used to obtain an ensemble of NMR derived structures for F215-R246. These structures have low target function values (average target function: 0.16±0.68 Ų) and satisfy well the NOE distance constraints (sum of NOE violations: 0.5±0.3 Å, maximum NOE violation: 0.12±0.08 Å).

Figure 3a shows a superposition of the 50 best structures of peptide F215-R246, best fitting backbone atoms from residue L220 to residue S237. This region is well-defined (RMSD: 0.90±0.32 Å on backbone atoms and 1.69±0.41 Å on heavy atoms) and adopts an α-helical structure. The helix presents a sharp kink due to the presence of P239. This was supported by the observation of three NOEs between sidechain protons of residues A235 and P239, namely the αH of P239 and βHs of A235; the αH of A235 and γH and δH protons of P239. A second short helical region follows from residue V240 to V243, which is better visible in Figure 3b . In 40 out of fifty selected structures, this is a 3–10 helix, as also suggested from the hydrogen bond pattern. For the V240-V243 region the RMSD is 0.28±0.32 Å on backbone atoms and 1.35±0.48 Å on heavy atoms. The structures are more flexible at the first five N-terminal residues and the last three residues at the C-terminus.

Figure 3. Left: Superposition of the fifty best structures of peptides F215-R246 (a) and W279-K305 (c), best-fitted on backbone atoms of residues L220-S237 and P284-M303, respectively. Structures are oriented with the N-terminus on the top of the figure. For peptide W279-K305 the side chains of Pro residues are arrowed, and are also shown in magenta colour online. For (a) the RMSD values are 0.90±0.32 Å on backbone atoms and 1.69±0.41 Å on heavy atoms and for (c) 0.81±0.32 Å on backbone atoms and 1.97±0.37 Å on heavy atoms, respectively. Right: Ribbon representations of the mean structure of the bundles of peptides F215-R246 (b) and W279-K305 (d). This Figure is reproduced in colour in Molecular Membrane Biology online.

Peptide W279-K305

For this peptide we observed a continuous stretch of strong sequential NH_i-NH_i + 1 in the region Y285-M303 (Figure 2b). The observation of many medium-range αH_i-NH_i + 3 NOEs together with quite a few αH_i-βH_i + 3 and αH_i-NH_i + 4 NOEs supports the presence of a long helical segment spanning residues P284-M303. The presence of P291 does not seem to interfere with the helix also because it might be included in a turn, as suggested by the presence of two αH_i-NH_i + 2 and NH_i-NH_i + 2 NOEs between G290 and H292. The conclusion of a long helical region is strengthened by an almost continuous region of negative CSI values from P284 to M303 and also by the ³J_αH-NH coupling constants <6.0 Hz in most of the region.

One-hundred-and-eighty NOEs were obtained from NOESY spectra (87 intraresidue, 63 short-range and 30 medium-range) and used as input in DYANA calculations. The 50 best conformers of W279-K305, in terms of target function values (average target function: 0.14±0.13 Ų), satisfy well the NOE distance constraints (sum of NOE violations: 0.7±0.4 Å, maximum NOE violation: 0.11±0.07 Å). Figure 3c superimposes the backbone atoms of the 50 best structures of peptide W279-K305, best-fitted on residues P284-M303 (RMSD: 0.81±0.32 Å on backbone atoms and 1.97±0.37 Å on heavy atoms). The peptide presents a well-defined helical structure from residue P284 to M303 and also the side chains of P284 but especially P291, arrowed in Figure 3c, are quite well-defined (see also Figure 3d). The RMSD of backbone atoms of residues P284-M303 increases only to 0.94±0.34 Å if the side chains of proline residues are included in the fitting. The first five residues and the last two residues do not converge to a unique structure.

Fluorescence measurements

In order to determine whether the fifth and sixth TMSs interact each other, we monitored the changes in tryptophan fluorescence of peptide W279-K305 upon addition of peptide F215-R246.

Figure 4a reports the fluorescence emission spectrum of W279-K305 in TFE/water 80%, alone or in the presence of equimolar amounts of peptides K21-K46, R78-R108, P117-A149, F215-R246, containing the predicted first, second, third and fifth TMSs of the OGC protein, respectively. (Bisaccia et al. [1994]). The maximal emission wavelength was at 355 nm. The addition of K21-K46, R78-R108 and P117-A149 did not change the spectrum of W279-K305 significantly. In contrast, in the presence of F215-R246, a blue shift of the emission maximum toward 353 nm and an appreciable quenching of fluorescence were observed. In Figure 4b the fluorescence emission spectra of W279-K305 in the presence of increasing concentrations of F215-R246 are reported. The quenching of fluorescence was saturable with respect to F215-R246, and a dissociation constant of 4.6 µM (±0.8 standard error) was calculated.

Figure 4. Quenching of intrinsic tryptophan fluorescence upon binding of TMSs to W279-K305. The spectra were recorded on a Fluorolog-3 spectrofluorimeter at an excitation wavelength of 295 nm. The fluorescence emission was recorded from 305 nm to 500 nm. (a) the spectra are W279-K305 alone (^_) and then in presence of peptides: K21-K46 (KSVKFLFGGLAGMGATVFVQPLDLVK) (Δ), R78-R108 (RGIYTGLSAGLLRQATYTTTRLGIYTVLFER) (x), P117-A149 (PGFLLKAVIGMTAGATGAFVGTPAEVALIRMTA) (□), F215-R246 (---); (b) W279-K305 alone (^_) and then in presence of 5 (---), 11 (□), 22 (Δ), 33 (•), 44 (x) and 55 (○) µM of F215-R246. The inset shows the fluorescence quenching of W279-K305 upon addition of different concentrations of F215-R246.

Peptide P257-L278

NMR results

Strong sequential NH_i-NH_i + 1 NOEs were observed from residue N261 to F275 (Figure 5). Moreover, αH_i-βH_i + 3 and αH_i-NH_i + 3 connectivities were detected in the region N261-E273. Secondary structure analysis based on chemical shift of αH protons (Wishart et al. [1991]) identified N261-E273 as a helical region. This was confirmed by the ³J_αH-NH coupling constants <6.0 Hz for the residues involved.

Figure 5. Summary of sequential and medium-range NOEs observed for peptide P257-L278 in 80% TFE at 25°C. The thickness of lines is related to the intensity of NOEs. Open bars indicate overlapping peaks. Asterisks under the amino acid one-letter codes indicate apparent ³J_αH-NH coupling constants of non-Gly residues <6.0 Hz. Chemical Shift Index (CSI) of αH protons are shown below the bar diagrams. Negative values indicate a helical conformation.

A total of 130 NOEs were obtained: 56 intraresidue, 61 short-range and 13 medium-range. They were translated into distance constraints for the structure calculations. The best fifty structures for P257-L278 have an average target function of 0.02±0.05 Ų and satisfy well the NOE distance constraints (sum of NOE violations: 0.11±0.10 Å, maximum NOE violation: 0.06±0.06 Å).

Figure 6 shows a superposition of backbone atoms of P257-L278, best fitted from residue G262 to residue F275. This region adopts a well-defined α-helical structure (RMSD: 0.66±0.21 Å on backbone atoms and 1.93±0.36 Å on heavy atoms). Also in this case the structures show ill-defined N- and C-termini.

Figure 6. (a) Superposition of the fifty best structures of peptide P257-L278 best-fitted on backbone atoms of residues G262-F275. Structures are oriented with the N-terminus on the top of the figure. The RMSD values are 0.66±0.21 Å on backbone atoms and 1.93±0.36 Å on heavy atoms. (b) Ribbon representation of the mean structure of the bundle. This Figure is reproduced in colour in Molecular Membrane Biology online.

Discussion

The results reported above shed light on the structure of the mitochondrial bovine OGC and in particular of its third repeated domain which consists of TMS-V, TMS-VI and the hydrophilic loop that connects them. Although the presence of helical structures in the hydrophobic regions of this transporter had already been foreseen (Runswick et al. [1990]), as in other members of the mitochondrial carrier family (Walker & Runswick [1993]; Palmieri [1994]), their extension and characterization have not yet been adequately explored. In this work we found a well-defined α-helix in peptide F215-R246 from L220 to V243 and in peptide W279-K305 from P284 to M303. Both helices have proline residues, which in water-soluble proteins are usually considered as helix breakers because of their side chain constraints and sterics (Chou & Fasman [1974]). However, a substantial number of proline residues are found in TM helices (Bywater et al. [2001]). Indeed, a proline residue is present in all the odd-numbered TMSs of the mitochondrial carriers, located in the characteristic signature motif PX(D/E)XX(K/R)X(K/R).

Our NMR data on peptide F215-R246, which contains the fifth TMS of the OGC protein (Bisaccia et al. [1994]), show that the helix from L220 to V243 presents a sharp kink due to the presence of P239. Interestingly, proline-induced kinks have been found in all the odd-numbered transmembrane helices of the ADP/ATP carrier by X-ray crystallography and have been proposed to be important in its transport mechanism and, precisely, in the reversible transition of the carrier from the closed to the open state on the matrix side (Pebay-Peyroula et al. [2003]).

The helical wheel projection of peptide F215-R246 (Figure 7) shows that the region preceding P239 is an amphipathic helix with the hydrophilic residues probably located towards the carrier crevice or translocation path of OGC and the hydrophobic ones towards the lipids and/or the hydrophobic faces of other TMSs of the protein. It is noteworthy that C221 and C224, which were found not to be essential for the transport activity (Palmieri et al. [1996]), reside on the hydrophobic face of this helix.

Figure 7. Helical wheel of peptides F215-R246, W279-K305 and P257-L278 obtained with the software ANTHEPROT (Deléage et al. [1988]). Hydrophobic residues are shown in blue, the hydrophilic residues in red, cysteine residues in green and the others in grey.

Unlike the ADP/ATP carrier, the uncoupling protein and the phosphate carrier, proline residues are present in the sixth TMSs of the oxoglutarate-, the dicarboxylate-, the tricarboxylate- and the glutamate carriers (for a review see Palmieri [2004] and references therein). This may be regarded as an evolutionary variation of the structure found in the ADP/ATP carrier.

The NMR data on peptide W279-K305 containing the sixth TMS of OGC show the presence of an α-helical structure in the region P284-M303. P284 is at the N-terminal end of the helix, an ideal location for this residue (Richardson & Richardson [1988]). P291 is at the end of the second helical turn, and does not interfere with the helical structure, only creating a bend in the helix axis, as found in other TM helices (von Heijne [1991]). This result shows that, in spite of the presence of two prolines, the sixth transmembrane segment of OGC has a helical structure. Similarly, in helix 6 of rhodopsin the presence of a proline residue (P267) does not significantly perturb the helical structure (Chopra et al. [2000]). Indeed, it has been reported that proline residues in the middle of α-helices may stabilize the helical structures by non conventional C-H…O hydrogen bonds, involving the ring CH groups (Bhattacharyya & Chakrabarti [2003]). Only a few charged and/or polar residues are present in the helical region P284-M303 of peptide W279-K305 (Figure 7). They reside on the same face and include R288, which was found to be essential for the carrier activity (Palmieri et al. [1996]) and H292. It should be noted that the other two even transmembrane helices of the OGC protein, TMS-II and TMS-IV, also contain positively charged residues (Palmieri et al. [1996]; Stipani et al. [2001]). It is likely that these charged residues form a cationic cluster that may line the water-accessible crevice or channel between helices of the OGC (see Stipani et al. [2001]) and may be involved in binding and/or translocation of oxoglutarate through the OGC protein. The only negatively charged residue E301 is located on the hydrophobic face of helix 284-303 presumably at the border of the membrane (see Bisaccia et al. [1994]; Stipani et al. [2001]).

Our observation that the fluorescence of W279 located at the N-terminus of peptide W279-K305 (TMS-VI of OGC) is quenched in a saturable manner by peptide F215-R246 (Figure 4) indicates that there is an interaction between the fifth and the sixth TMSs of OGC. It is interesting that in the crystal structure of the ATP/ATP carrier-carboxyatractyloside complex (Pebay-Peyroula et al. [2003]) an interaction has been found between TMS-V and TMS-VI (as well as between TMS-I and TMS-II and between TMS-III and TMS-IV) at the level of the N-terminus of the latter TMS and the C-terminus of the former. No change in fluorescence was observed when TMS-I, TMS-II or TMS-III, instead of TMS-V, were added to peptide W279-K305.

Another significant result of this paper is the demonstration that α-helices can also be present in the hydrophilic loops of OGC. In fact, NMR data on peptide P257-L278, corresponding almost to the complete matrix loop between TMS-V and TMS-VI, show an α-helix in the region N261-F275. Its helical wheel projection shows that positively and negatively charged residues are concentrated on one face of the helix with the exception of E273 that is located on the hydrophobic face (Figure 7). This suggests a precise orientation of this helix relative to the membrane.

It has been shown that the solution structures of transmembrane helices and loops of rhodopsin and bacteriorhodopsin closely resemble the high resolution crystallographic structures of these proteins (Chopra et al. [2000]; Katragadda et al. [2000]; Katragadda et al. [2001]). In the absence of a crystal structure of the OGC protein, we used the only available X-ray structure of a mitochondrial carrier, i.e., that of the ADP/ATP carrier (PDB accession code: 1okc).

The helical structures from peptides F215-R246, W279-K305 and P257-L278, reported here, were compared with the structures of the corresponding amino acid sequences of the ADP/ATP carrier.

Backbone atoms of residues L220-M238 of peptide F215-R246 were superimposed on the corresponding amino acid sequence of helix H5 (residues 210–228 of 1okc) with an RMSD of 1.72 Å. If the fit includes the whole helical region L220-V243 found in our peptide (with residues 210–233 of 1okc) the RMSD increases to 3.18 Å because the region after P239 is not well-defined by our data and this causes a reduction in the quality of the fitting. Backbone atoms of residues P284-E301 of peptide W279-K305 were superimposed with the corresponding residues of helix H6 (1okc: residues 273–290) with an RMSD of 1.76 Å. Finally, the fitting of backbone atoms of residues G262-E273 of peptide P257-L278 with residues 253–264 of 1okc, corresponding to helix h56 of the ADP/ATP carrier, gives an RMSD of 1.66 Å (Figure 8).

Figure 8. Superposition of backbone atoms of the mean structures of peptides F215-R246 (only residues L220-M238 are shown, in deep pink), W279-K305 (only P284-E301, in sky blue) and P257-L278 (only G262-E273, in yellow) on the corresponding amino acid sequences of helices H5 (residues 209–238), H6 (residues 273–290) and h56 (residues 253–264) in the crystal structure of the ADP/ATP carrier (Pebay-Peyroula et al. [2003]) (PDB accession code: 1okc, in dark blue). Only the region of the ADP/ATP carrier, which is relevant for the comparison with the peptides studied in this paper, is shown for clarity.

The experimental data presented in this paper indicate quite a good agreement between the crystal structure of the helices H5, H6 and h56 of the ADP/ATP carrier and the solution structure of our peptides, in spite of the low sequence homology between the OGC and the ADP/ATP carrier. In particular, our results suggest that the structures of their third repeated domain, corresponding to TMS-V, TMS-VI and the matrix loop between them, may be very similar. They further suggest that the solution structures reported here may exhibit the same secondary structure they adopt in the intact protein.

This paper was first published online on prEview on 21 April 2005.

This work was supported by grants from MIUR-PRIN, MIUR-FIRB, CEGBA, Universities’ Local Funds (ex-60%), by the European Social Fund and by the European Community's sixth Framework Programme for Research, Priority 1 “Life sciences, genomics and biotechnology for health”, contract number LSHM-CT-2004-503116.