Structure of the C-terminal Fragment 300-320 of the Rat Angiotensin II AT1A Receptor and Its Relevance with Respect to G-Protein Coupling*

(Received for publication, October 28, 1996)

Lorella Franzoni , Giuseppe Nicastro Dagger , Thelma A. Pertinhez §, Marco Tatò Dagger , Clovis R. Nakaie , Antonio C. M. Paiva , Shirley Schreier § and Alberto Spisni par

From the Institute of Biological Chemistry, University of Parma, 43100 Parma, Italy, Dagger  Pharmacia & Upjohn, Nerviano, 20014 Milano, Italy, § Institute of Chemistry, Department of Biochemistry, University of São Paulo, CP 26077, 05599-970, São Paulo, Brasil, and the  Department of Biophysics, Federal University of São Paulo, São Paulo CP 04044-020, Brasil

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Angiotensin II AT1A receptor is coupled to G-protein, and the molecular mechanism of signal transduction is still unclear. The solution conformation of a synthetic peptide corresponding to residues 300-320 of the rat AT1A receptor, located in the C-terminal cytoplasmic tail and indicated by mutagenesis work to be critical for the G-protein coupling, has been investigated by circular dichroism (CD), nuclear magnetic resonance (NMR) and restrained molecular dynamics calculations. The CD data indicate that, in acidic water, at concentration below 0.8 mM, the peptide exists in a predominantly coil structure while at higher concentration it can form helical aggregates; addition of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. Using NMR-derived constraints, an ensemble of conformers for the peptide has been determined by restrained molecular dynamics calculations. Analysis of the converged three-dimensional structures indicates that a significant population of them adopts an amphipathic alpha -helical conformation that, depending upon experimental conditions, presents a variable extension in the stretch Leu6-Tyr20. An equilibrium with nonhelical structured conformers is also observed. We suggest that the capability of the peptide to modulate its secondary structure as a function of the medium dielectric constant, as well as its ability to form helical aggregates by means of intermolecular hydrophobic interactions, can play a significant role for G-protein activation.


INTRODUCTION

Mutational studies on angiotensin II AT1A1 receptor have indicated that residues or sequences of the cytoplasmic tail and loops, close to the membrane, are critical for G-protein coupling (1-4). As far as the molecular mechanism, although the current working hypothesis is yet very speculative, it is certainly accepted that contacts between the receptor cytoplasmic portions and G-protein must take place and, therefore, that the secondary structure of those sequences is of great importance. The modulation of the coupling due to endocytosis or to a shift, for all the cytosolic components, between some sort of an aggregate state and an "open" one are among the mechanisms proposed (5). It is interesting to note, however, that from a conformational point of view, both mechanisms might require an equilibrium between a helical and an extended conformation.

As far as the receptor C-terminal (CT) portion, the 21-mer near the membrane surface, corresponding to the sequence 300-320 in the rat AT1A receptor, has been predicted to have a high probability to assume an amphipathic helical structure. Moreover, recently, it has been shown that the synthetic peptide representing the fragment 306-320 of the rat AT1A receptor is able to activate purified Gi1, Gi2, and G0 proteins (3).

These observations have prompted us to investigate the solution conformation of the synthetic fragment LFYGFLGKKFKKYFLQLLKYI-NH2, fCT300-320, to verify if indeed it may assume a helical structure and if its secondary structure can be modulated by physical or chemical, physiologically relevant perturbations.

The investigation of the solution conformation of fCT300-320 has been carried out by means of circular dichroism (CD) and two-dimensional 1H-NMR spectroscopy together with restrained molecular dynamics calculations. The peptide was studied in water, as well as in acidic H2O, 2,2,2 trifluoroethanol (TFE) mixtures, at 28 and 5 °C.

The results obtained show that, in acidic water, fCT300-320 exists in a predominantly coil structure at concentration lower than 0.8 mM, whereas at the NMR concentration, it tends to form soluble helical aggregates; in the presence of TFE, the peptide does not aggregate and its structure is characterized by a well-defined amphipathic alpha -helix involving the stretch Leu6-Tyr20, with an increased flexibility of the helix in the N-terminal segment. Most importantly, the data clearly demonstrate the existence of an equilibrium between helical conformations and extended ones: changes in the dielectric constant of the medium, as well as temperature variations, can modulate the extent of the helix, and hydrophobic intermolecular interactions can favor the formation of helical aggregates. All of this evidence indeed supports the various models and functional evidence requiring a coil to helix transition of the AT1A receptor CT region associated to helix-helix interaction as a possible molecular mechanism for G-protein activation.

In this work, on the basis of both the obtained results and the current knowledge, we outline a possible general molecular model accounting for the various functions in which the CT tail of the AT1A receptor may be involved.


MATERIALS AND METHODS

Peptide Synthesis

The 21-amino acid residues peptide fCT300-320, corresponding to the fragment 300-320 of the cytoplasmic CT domain of the angiotensin II rat AT1A receptor, was synthesized manually in a 0.2 mM scale under the tert-butyloxycarbonyl protocol. It was purified by high performance liquid chromatography, the amino acid composition was analyzed, and the primary sequence was determined. Fig. 4 shows the sequence with both the numbering for the peptide residues and the one from the primary structure of rat AT1A (6).


Fig. 4. Summary of sequential and medium-range NOEs for fCT300-320 (1.6 mM) in 70% H2O, 30% TFE-d3, pH approx  4, at 28 and 5 °C. The relative intensities of the NOEs, classified into strong, medium, and weak, are indicated by the thickness of the lines. The stars indicate potential NOE connectivities that could not be obtained due to resonance overlap. Data were obtained from 600-MHz Watergate-NOESY experiments with a mixing time of 150 ms.
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Circular Dichroism Spectroscopy

CD spectra were recorded at 28 and 5 °C on a Jobin Yvon CD6 spectropolarimeter using a thermostatic water bath to control the cell temperature. The instrument was routinely calibrated with an aqueous solution of recrystallized d-10-camphorsulfonic acid. Ellipticity is reported as mean residue molar ellipticity, [theta ], (deg cm2 dmol-1). The peptide's concentration was determined spectrophotometrically from the tyrosine absorbance, using an epsilon 274 value of 1412 M-1 cm-1 and amino acid analysis. The concentration-dependence study, in the range of 0.2-1.2 mM, was performed by dissolving the peptide in water at pH 4.0 (adjusted using small amounts of NaOH or HCl solutions). The H2O/TFE cross-titration experiments were carried out mixing the appropriate aliquots of two 0.2 mM stock solutions, one in water at pH 4.0 and the other in TFE.

The helical population was estimated by using the mean residue ellipticity at 222 nm [theta ]222 according to the equation of Chen et al. (7), [theta ]lambda  = (fH - ik/N)[theta ]Hinfinity , where [theta ]lambda is the observed mean residue ellipticity at wavelength lambda , fH is the fraction of helix in the molecule, i is the number of helical segments, k is a wavelength-dependent constant (2.57 at 222 nm), N is the total number of residues, and [theta ]Hinfinity is the maximum mean residue ellipticity for a helix of infinite length. The expected value of the mean residue ellipticity for the peptide in a 100% helical conformation was calculated to be -34666 deg cm2 dmol-1.

NMR Spectroscopy

The peptide for the NMR sample was dissolved in 0.7 ml of 70% H2O, 30% TFE-d3 (v/v), pH approx  4, to yield a peptide concentration of 1.6 mM. TFE has been widely used as a solubilizing and structure-inducing cosolvent for peptides and protein fragments; in fact, a strong correlation between TFE-enhanced alpha -helicity and the intrinsic propensity of the sequence to adopt this structure has been extensively demonstrated (8). In this case though fCT300-320 is soluble in water, at the NMR concentration, it tends to form soluble helical aggregates that do not allow a proper analysis of the NMR spectra. Therefore, an H2O/TFE mixture has been used to overcome this problem. All two-dimensional 1H-NMR experiments were recorded with a pulse field gradient (z axis; 1H, {13C, 15N}) triple resonance probe on a Varian Unity 600 spectrometer, at temperatures of 28 and 5 °C. The proton chemical shifts were referenced to the H2O signal located at 4.774 ppm at 28 °C and 5.05 ppm at 5 °C, downfield from the external 4,4-dimethyl-4-silapentane-1-sulfonate (0.00 ppm). To assign the peptide resonance peaks, standard methods were used to perform DQF-COSY (9, 10), double quantum-two-dimensional (11), clean TOCSY (12), and NOESY (13, 14) experiments. The TOCSY spectra were acquired using a Waltz-16 spin-lock sequence at a field strength of 10 kHz and spin-lock evolution time of 80 ms; NOESY experiments with several mixing times, (tau m), in the range between 150 and 300 ms were carried out. All two-dimensional experiments were acquired in the phase-sensitive mode using the method of States et al. (15), and quadrature detection was achieved using hypercomplex data acquisition. The spectral width in both dimensions was typically 10 ppm, and 512 tau 1 experiments with 32 transients of 2048 complex points for each free induction decay were recorded. In both DQF-COSY and TOCSY spectra, water suppression was achieved by low power continuous wave irradiation during the relaxation delay, while in the NOESY experiments, the Watergate technique (16) was employed.

Data were processed on a Silicon Graphics Personal Iris workstation using the FELIX NMR 2.3 data processing package (BIOSYM Technologies, San Diego, CA). Prior to Fourier transformation, the time domain data were zero-filled in both dimensions to yield 4 K × 2 K matrices and apodized by a 30°-shifted squared sinebell window function to improve resolution; for quantitative measurements of NOESY data, a 90°-shifted squared sinebell window function was applied to avoid distortion of peak intensities. When necessary, a fifth-order polynomial base-line correction algorithm was applied after transformation and phasing.

To obtain distance constraints, cross-peak intensities were estimated from the 150 ms NOESY spectra recorded at 28 and 5 °C. To relate these NOE data with interproton distances, a calibration was made using the distance of 1.8 Å for the well defined geminal beta -protons, and the NOE intensities were classified as strong, medium, and weak, corresponding to upper bound constraints of 3.0, 4.0, and 5.0 Å, respectively. Lower bounds were taken to be the sum of the van der Waals radii (1.8 Å) for the interacting protons in all cases. Upper distance restraints involving nonstereospecific assigned methylenes, aromatic, and methyl protons were replaced by appropriate pseudo-atoms (17).

The phi  dihedral angle restraints were derived from the 3JHN-alpha coupling constants, estimated in the DQF-COSY spectra, from measurements of separations of extrema in dispersive and absorptive plots of rows through cross peaks (18). In these experiments, the digital resolution after zero-filling along omega 2 was 0.43 Hz/point; multiple solutions for each J value were selected.

Molecular Modeling

All calculations were carried out on a Silicon Graphics ONYX computer using the Consistent Valence Force Field (19, 20), implemented in the DISCOVER (BIOSYM Technologies Inc., San Diego, CA) software package, together with the INSIGHT II as graphic interface. The structures of fCT300-320 were computed using the simulated annealing methods (21) in the NMR refine module of the INSIGHT II package. The simulated annealing calculations included some distinct phases. Phase 1 involved randomization of all atomic coordinates followed by minimization of the starting structures using a quadratic potential and very low force constants for each term of the pseudo-energy function, including chiral and NOE constraints. Phase 2 involved simulated annealing with a progressive increment of the force constants up to their full values. Phase 3 involved cooling of the molecule from 1000 to 300 K over 10 ps. At the end of this protocol, the structures were energy minimized with full Consistent Valence Force Field (Morse and Lennard-Jones potentials, coulombic term) by steepest descents and conjugate gradients using several thousand iterations until the maximum derivative was less than 0.001 kcal/Å. Amide bonds were maintained as trans during the calculations. The quality of the final structures was analyzed on the basis of the number of NOE distance violations and of the root mean square deviation values of the backbone.


RESULTS

CD Spectroscopy

Fig. 1 shows the CD spectra for fCT300-320, as a function of concentration, in water, at pH 4.0 and 28 °C. At concentration higher than 0.8 mM, the occurrence of a transition from an extended conformation to a helix reach one is indicated by the shift of the minimum from 198 to 208 nm, together with the net increase of the negative band at 223-224 nm. On the basis of the EMBL structural prediction (22, 23)2 and according to the NMR data (see "NMR Spectroscopy"), the change of the CD spectra as a function of the peptide concentration can be indicative of the formation of helical aggregates. In fact, the slight red shift of the n-pi * transition would support this hypothesis. The possibility of formation of coiled coil structures is discarded as the negative band at 208 nm, due to the parallel-polarized pi -pi * transition, does not undergo any shift and the [theta ]220/[theta ]208 ratio is relatively constant for concentration above 0.8 mM (7, 24).


Fig. 1. CD spectra of fCT300-320 in water at 28 °C, pH 4.0, as a function of peptide concentration in the range 0.2-1.2 mM. 1, 0.2 mM; 2, 0.6 mM; 3, 0.8 mM; 4, 1.0 mM; and 5, 1.2 mM.
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Fig. 2, A and B, shows the CD spectra of 0.2 mM fCT300-320 at various H2O/TFE percentages (v/v), pH approx  4, at 28 and 5 °C, respectively. At both temperatures, upon addition of TFE as low as 10%, a significant change of the CD spectrum is observed. The shift of the negative band from 198 to 208 nm, together with the appearance of an additional negative band at 222 nm and of a positive band at 196 nm, suggest that the peptide is switching from an unordered state to a helix-rich one. The absence of any isodichroic point indicates the existence of an equilibrium between multiple conformations.


Fig. 2. CD spectra of 0.2 mM fCT300-320 in water at pH 4.0 upon addition of TFE in the concentration range 0-100% (v/v) at 28 °C (A) and 5 °C (B). 1, water; 2, 10% TFE; 3, 30% TFE; and 4, 100% TFE. Insets, alpha -helix content at (bullet ) 28 °C and (black-square) 5 °C as a function of TFE percentage, estimated as described under "Materials and Methods."
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As observed in the concentration dependence study, both at 28 and 5 °C, when increasing the TFE concentration, the behavior of the parallel-polarized pi -pi * transition at 208 nm does not show any red shift and any modification of the [theta ]220/[theta ]208 ratio is detected, thus confirming that the TFE-induced helical structures do not tend to aggregate in a coiled coil fashion.

The analysis of the spectra using the equation of Chen et al. (7) indicates that, at 28 °C, the alpha -helix content is approx  50% at 30% TFE, and here it stabilizes all the way through 100% TFE (Fig. 2A, inset). When the temperature is lowered at 5 °C, a plateau is reached at 40% TFE with a helix content of about 60% (Fig. 2B, inset).

Assignment of 1H-NMR Resonance Signals

Initially, one- and two-dimensional 1H-NMR experiments of fCT300-320 were recorded in water in the concentration range of 1.5-3.0 mM, varying the pH between 3.0 and 6.0 (data not shown). Under these conditions, the proton resonances line-width were larger than expected for a peptide of this size, in agreement with the presence of intermolecular aggregation as it was suggested by the CD data. Consequently, according to the CD results, the peptide was dissolved in 70% H2O, 30% TFE-d3 (v/v). In this condition, the NMR resonance lines presented the expected high resolution shape, allowing a proper analysis of the spectra. Following to the sequential assignment procedure (17), TOCSY spectra were used to identify the amino acids spin systems, and NOESY spectra were used both to obtain interresidue connectivities and to distinguish between equivalent spin systems. Assignments of the amino acids side-chain resonances were confirmed by DQF-COSY and double quantum-two-dimensional spectra; aromatic amino acids (Phe, Tyr) were identified using their beta -aromatic NOESY connectivities. The interpretation of the two-dimensional 1H-NMR spectra presenting a unique set of resonances suggests that the peptide is either in a monomeric state or in a symmetric dimeric one. All protons have been identified, and the chemical shifts are listed in Table I for 28 and 5 °C, together with the NH-alpha CH coupling constants (3JHN-alpha ).

Table I.

1H-NMR Chemical shiftsa (ppm) and 3JHN-alpha coupling constants (Hz) for fCT300-320, (1.6 mM) in 70% H2O, 30% TFE-d3, (v/v), pH approx  4, at 28 and 5 °C


28 °C
Residue 3JHN-alpha NH  alpha H  beta H  gamma H Others

Leu1 3.92 1.68, 1.42 1.54  delta H 0.75, 0.72 
Phe2 5.8 8.32 4.70 3.06, 3.01  delta H 7.15; varepsilon H 7.27; zeta H 7.23 
Try3 6.2 7.88 4.38 2.93, 2.77  delta H 7.02; varepsilon H 6.84 
Gly4 10.2 7.06 3.67
Phe5 5.6 7.66 4.53 3.13  delta H 7.21; varepsilon H 7.37; zeta H 7.30 
Leu6 6.7 7.69 4.18 1.68 1.52  delta H 0.88, 0.83 
Gly7 10.4 7.88 3.94
Lys8 7.0 8.05 4.14 1.95, 1.91 1.30  delta H 1.66, 1.61; varepsilon H 2.93 
Lys9 8.5 7.99 4.15 1.87, 1.75 1.40  delta H 1.65; varepsilon H 2.83 
Phe10 6.9 8.06 4.50 3.21, 3.13  delta H 7.19; varepsilon H 7.34; zeta H 7.25 
Lys11 5.7 7.94 4.03 1.93 1.47  delta H 1.74, 1.60; varepsilon H 3.01 
Lys12 5.6 7.92 4.02 1.86, 1.83 1.40, 1.31  delta H 1.66, 1.62; varepsilon H 2.95 
Tyr13 7.3 7.68 4.33 3.09, 3.02  delta H 7.05; varepsilon H 6.81 
Phe14 5.8 8.35 4.15 3.01, 2.97  delta H 7.17; varepsilon H 7.29; zeta H 7.20 
Leu15 5.7 8.17 3.96 1.87 1.53  delta H 0.93, 0.91 
Gln16 7.2 7.66 4.02 2.25, 2.16 2.51, 2.41 CONH2 7.12, 6.74 
Leu17 6.2 7.90 4.16 1.72 1.63  delta H 0.97 
Leu18 5.6 7.96 3.92 1.70 1.65  delta H 0.98, 0.84 
Lys19 5.8 7.66 3.97 1.80, 1.73 1.33, 1.26  delta H 1.61, 1.49; varepsilon H 2.94 
Tyr20 7.0 7.87 4.45 3.25, 3.07  delta H 7.20; varepsilon H 6.84 
Ile21 8.6 8.05 4.02 1.61, 1.50 1.30  gamma H3 0.99; delta H 0.85; CONH2 7.34, 6.82 
5 °C
Leu1 3.90 1.53 1.48  delta H 0.71 
Phe2 5.9 8.46 4.70 3.08, 3.00  delta H 7.13; varepsilon CH 7.25; zeta H 7.21 
Tyr3 4.7 8.03 4.35 2.94, 2.76  delta H 7.01; varepsilon H 6.79 
Gly4 7.3 7.05 3.65
Phe5 5.9 7.72 4.48 3.13  delta H 7.19; varepsilon H 7.33; zeta H 7.24 
Leu6 6.5 7.73 4.14 1.68 1.46  delta H 0.86, 0.79 
Gly7 8.2 7.89 3.92
Lys8 6.9 8.16 4.10 1.96, 1.92 1.32  delta H 1.62; varepsilon H 3.02; varepsilon NH 7.10 
Lys9 6.1 8.11 4.06 1.68 1.38  delta H 1.49; varepsilon H 2.90;
Phe10 5.9 8.17 4.41 3.21, 3.17  delta H 7.02; varepsilon H 7.17; zeta H 7.09 
Lys11 6.5 8.03 3.99 1.95 1.44  delta H 1.73, 1.58; varepsilon H 3.03 
Lys12 5.6 7.98 4.00 1.93, 1.85 1.38, 1.26  delta H 1.70, 1.66; varepsilon H 2.89 
Tyr13 5.8 7.75 4.30 3.13, 3.00  delta H 7.01; varepsilon H 6.76 
Phe14 5.8 8.57 4.12 3.05, 2.90  delta H 7.10; varepsilon H 7.22; zeta H 7.16 
Leu15 5.9 8.31 3.97 1.92 1.48  delta H 0.94 
Gln16 5.5 7.69 4.00 2.26, 2.16 2.48, 2.37 CONH2 7.18, 6.84 
Leu17 4.9 7.97 4.10 1.73 1.58  delta H 0.88 
Leu18 5.7 8.11 3.90 1.82 1.52  delta H 0.84, 0.70 
Lys19 5.5 7.75 3.95 1.82, 1.78 1.38, 1.26  delta H 1.68, 1.48; varepsilon H 2.92; varepsilon NH 7.04 
Tyr20 5.9 7.92 4.41 3.24, 3.10  delta H 7.14; varepsilon H 6.79 
Ile21 5.6 8.18 3.94 1.62, 1.48 1.29  gamma H3 0.96; delta H 0.83; CONH2 7.38, 6.91

a Chemical shifts are relative to the H2O resonance, located at 4.774 ppm at 28 °C and at 5.05 ppm at 5 °C downfield from 4,4-dimethyl-4-silapentane-1-sulfonate.

Evidence of Secondary Structure

Several NMR parameters provide information on the amount of secondary structure of peptides in solution. A fast and simple method, complementary to the analysis of the NOE connectivities, is based on the observed deviations of alpha CH proton chemical shifts with respect to the random coil values (25). The alpha -protons secondary shifts for fCT300-320 are reported in Fig. 3, and it is evident that, except for Phe2, Leu6, and Ile21 at 28 °C and only for Phe2 at 5 °C, all the alpha -protons resonances move upfield with a slightly higher deviation at 5 °C, thus suggesting the existence of a helical region extending between Lys8 and Tyr20 at both temperatures. The more pronounced deviation of the secondary alpha CH chemical shifts observed in the C-terminal part of the peptide indicates that the helical structure in that portion is more stable as compared with the N-terminal part.


Fig. 3. Plot of the alpha -protons secondary chemical shifts relative to the amino acid sequence at 28 (open bars) and 5 °C (solid bars). Delta delta alpha CH = observed delta alpha CH - random coil delta  value (25).
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The summary of the interresidue NOEs at 28 °C is reported in Fig. 4A, showing a good correlation with the alpha CH secondary shifts. In fact, medium and strong connectivities are found between the adjacent amide protons from Phe10 to Ile21 as can be seen also in the NH-NH region of the 150 ms NOESY spectrum displayed in Fig. 5A, indeed supporting the existence of a significant population of conformers containing a well defined alpha -helix in that part of the peptide.


Fig. 5. Contour plots of the NH-NH region of 600 MHz Watergate-NOESY spectra for fCT300-320 at 28 °C (A) and 5 °C (B). Data were collected in 70% H2O, 30% TFE-d3, pH approx  4, with 1.6 mM peptide concentration and using a mixing time of 150 ms. Some assignments are reported.
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On the other hand, the values of the 3JHN-alpha coupling constants, mostly around 6-7 Hz (Table I), together with the relatively weak intensities for the (i, i + 3) NOEs and the coexistence with the intense Calpha H-NH (i, i + 1) NOEs from the middle to the N terminus of the peptide suggest the presence of conformational fluctuations in that stretch.

The data collected at 5 °C show all the cross-peaks found at 28 °C, together with some new NOEs involving residues in the N-terminal region, Figs. 4 and 5B. The presence of extensive medium range Calpha H-NH (i, i + 3) and Calpha H-Cbeta H (i, i + 3) NOEs in the stretch between Phe5 to Tyr20 suggests that almost the entire peptide exists in an alpha -helical conformation. A large number of NH-NH (i, i + 1) NOEs from Tyr3 to Ile21, together with the majority of the 3JHN-alpha in the range of 4.7-6.0 Hz, strongly support this possibility.

Molecular Modeling

Qualitative analysis of NOE patterns only provides a crude description of the secondary structure of fCT300-320. The possible three-dimensional structures of the peptide were calculated following the molecular dynamics protocol described under "Materials and Methods," using a final set of 187 NOE-derived distance constraints at 28 °C and 220 distance constraints at 5 °C (Table II) together with 18 phi  dihedral angles restraints at both temperatures.

Table II.

Structural statistics of fCT300-320


28 °C 5 °C

Constraints
  Intraresidue 135 138
  Sequential  36  47
  Medium range  16  35
  Total 187 220
RMSDa
  Backbone 0.2-1.0 (res 12-19)d 0.2-0.9 (res 6-19)
  Heavy atoms 0.2-2.0 (res 12-19) 0.2-2.0 (res 6-19)
Energyb
  Leonnard-Jones-van der Waals  -48.6  -38.5
Hydrogen bondsc Donor NHa Acceptor COe Donor NH Acceptor CO

Leu17 Tyr13 Lys9 Phe5
Leu18 Phe14 Lys11 Gly7
Lys19 Leu15 Lys12 Lys8
Leu17 Tyr13
Leu18 Phe14
Lys19 Leu15
Tyr20 Gln16

a Root mean square deviation from pairwise comparison between all the structures (Å).
b In Kcal/mol.
c Hydrogen bonds were searched using the Measure Hbond facility of INSIGHT and were regarded as present if the following criteria were satisfied simultaneously: 1) the distance between the donor H and the acceptor O was less than 2.5 Å; 2) the angle between the heavy atom donor, the hydrogen, and the heavy atom acceptor (NH---O) was greater than 120 °; 3) the hydrogen bond occurred in at least 50% of the energy minimized structures.
d res represents residues.
e NH and CO represent backbone atoms.

A total of 100 structures were calculated for each temperature, of which 92 were energy minimized using the program DISCOVER; 8 structures were rejected because of the presence of D-amino acids, topological mirror images, and misfold. After minimization, 18 structures were selected, at both temperatures, based on the low residual distance violations (less than 0.4 Å) and low dihedral angles violations (less than 5°) and were used to represent the solution three-dimensional structure of fCT300-320. Table II shows the statistics for the selected structures as well as their energies.

Fig. 6, A and B, displays the 18 final Molecular Dynamics structures of fCT300-320, for both temperatures, superimposed for the minimum backbone deviation between residues Lys12-Lys19 at 28 °C and residues Leu6-Lys19 at 5 °C. As shown in Fig. 6A, at 28 °C, an alpha -helical folding is well defined in the C-terminal portion of the peptide. Indeed, at variance with the observed NOE pattern, Fig. 4A, on the basis of the analysis of the main chain hydrogen bonds and of the dihedral angles phi  and psi , carried out using the program DSSP (26), a stable alpha -helix results to extend only between residues Phe14 and Lys19. The wide conformational spread observed in the N-terminal portion of the peptide can be interpreted as indicative of the presence of a structurally flexible alpha -helix.


Fig. 6. Top, final 18 MD selected structures of fCT300-320 calculated from NMR-derived distance and phi  angle restraints at 28 °C (A) and 5 °C (B). The superimposition of the backbone atoms has been made from Lys12 to Lys19 at 28 °C and from Leu6 to Lys19 at 5 °C. The N terminus is shown at the top. Bottom, least-energy structure at both temperatures, showing the amphipathic character of the helix. Within positions 6-20, hydrophilic residues such as Lys8, Lys9, Lys11, Lys12, Lys19, and Gln16 are located on one side of the molecule, while hydrophobic residues such as Leu6, Phe10, Phe14, Leu17, and Leu18 are located on the other side of the molecule.
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Fig. 6B shows that, at 5 °C, a well defined alpha -helical conformation extends over the peptide stretch Leu6-Tyr20, confirming that the temperature exerts a stabilizing effect on the helical structure. The region containing residues 1-6 is still poorly defined due to the limited number of constraints available. In addition, it should be noted that, in this part of the peptide, there are two Gly residues known to promote an increase in phi  and psi  angular freedom and flexibility.

The hydrogen bonds observed for the converged structures of the peptide at both temperatures are listed in Table II and are in agreement with the other data.

The bottom of Fig. 6, A and B, presents a schematic representation of the least-energy structure of the peptide where the amphipathic nature of the alpha -helix at both temperatures is evident.


DISCUSSION

The results presented in this study allow delineating the preferential solution conformations of the synthetic linear peptide fCT300-320, corresponding to the fragment 300-320 of the CT cytoplasmic tail of the rat AT1A receptor and indicated to be essential for the interaction with the G-protein alpha -subunit. The obtained structures can be related to the active form in the AT1A receptor as it has been shown that the peptide is able to activate G-protein GTP binding (3).

fCT300-320 in 70% H2O, 30% TFE

d3 shows a rather stable helical conformation spanning from the central part of the peptide toward the C-terminal end as it is shown in Fig. 6. In acidic water, below 0.8 mM, the peptide is in a predominantly extended conformation, whereas at higher concentration, it tends to form helical aggregates (Fig. 1).

The molecular mechanism by which the AT1A receptor interacts with G-protein is yet to be clearly understood. However, all models proposed so far imply the need of a contact between some cytosolic fragments of the receptor and portions of the G-protein. To achieve this goal, the conformational properties of these fragments clearly play a crucial role and, in particular, the existence of a helix to coil transition seems to be the most probable molecular mechanism that may permit the association and subsequent dissociation between the AT1A receptor and the G-protein alpha -subunit.

Our results show that the conformation of fCT300-320 is sensitive both to the polarity of the medium and to the temperature in a way that the peptide can be forced to shift from an extended configuration to a helical one whose extension can be modulated by the strength of the external agents.

In the AT1A receptor, the 300-320 stretch is expected to be at the membrane interface, and the amphipathic property of the membrane could be an ideal factor to modulate its conformation. It has been suggested that AT1A receptor internalization might have conformational rather than functional requirements (27). In this respect, a variable interaction between the membrane and the AT1A CT tail could be favored by the conformational characteristics of that fragment.

Theoretical predictions of fCT300-320 structure, verified by our experimental results, have provided evidence for the amphipathic nature of the helix and concentration dependence studies have indicated the tendency to form helical aggregates. In addition, there is evidence, reported by Conchon et al. (27), indicating that the binding of the agonist to the receptor induces a lateral shift of the complex, and various authors are stressing the importance of the presence, in the receptor structure, of stretches that may utilize the charged surface of an amphipathic helix to achieve G-protein activation (28-30). All combined, it becomes quite tempting to speculate on a possible general molecular model for the many events triggered by ligand binding in the extracellular portion of the AT1A receptor.

The propagation of the agonist-induced conformational changes through the transmembrane segments results in a lateral shift of the receptor that might bring its cytoplasmic portions closer to the G-protein. This event would favor the interaction of the polar part of the receptor CT tail amphipathic helix with the G-protein alpha -subunit forming one of the anchoring points between the two proteins, the other being with the third intracellular loop (3). Concomitantly, affinity reasons would bring the hydrophobic part of the helix, still exposed to the medium, to interact with the nearby membrane surface, and this interaction might be responsible for the receptor internalization. Interestingly, one of the internalization motifs proposed for many G-protein coupled receptors is NPXXY (31) that, though in this case there are yet some doubts about its function, is present in the AT1A receptor, and it is located in position 298-302. The presence of Pro299 at the membrane interface is quite relevant since Pro residues are often observed at the N terminus of alpha -helices (32). In addition, similarly to what has been suggested for the internalization motif of an integral membrane protein of the trans-Golgi network (33), Pro299, being part of the internalization motif, could define the beginning of the helical stretch assuring, at the same time, a certain degree of flexibility that may be required for a better interaction with the membrane. Termination of the signal has been associated to a desensitization mechanism that can be induced by phosphorylation of the Ser and Thr residues of the receptor within the CT region (34). Indeed this event, changing the polarity of the surrounding, could induce unfolding of that fragment and decouple the receptor from the G-protein alpha -subunit.

In conclusion, there are no doubts that distinct opinions regarding the role of the AT1A CT tail are still present. In fact, while some authors are primarily stressing its role in the receptor internalization process induced by agonist binding (35, 36), others indicate a specific involvement in G-proteins interaction (1, 3, 4).

Based on our results, we believe that the receptor CT tail may be involved in all of these processes and that, due to the complexity of the events associated to the formation of the ternary complex, including the agonist, the receptor and the heterotrimeric G-protein, the experimental conditions used so far by the various researchers do not allow observing all its functions simultaneously, thus biasing the definition of a correct molecular model.

As suggested by Yeagle et al. (37) in relation to rhodopsin, we believe that, for the AT1A receptor as well, studying all the soluble peptides of the cytoplasmic portions of which the biological activity can be verified will allow delineating a profile of the receptor surface that is involved in the interaction with the G-protein alpha -subunit.

Studies are now underway to better define the flexibility of the CT region as well as to describe the structural properties of the other cytosolic portions of the AT1A receptor involved in G-protein coupling and signal transduction.


FOOTNOTES

*   This work was supported by MURST, CNR 95.02939.14/11519363 Italy and FAPESP, CNPq Brasil.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.
par    To whom correspondence should be addressed: Institute of Biological Chemistry, University of Parma, Via Gramsci, 14, 43100 Parma, Italy. Tel.: 39-521-290362; Fax: 39-521-988952; Email: aspin{at}ipruniv.cce.unipr.it.
1   The abbreviations used are: AT1A, type 1A angiotensin II receptor; CT, C-terminal portion of AT1A; fCT300-320, C-terminal fragment 300-320 of the rat AT1A receptor; DQF-COSY, double-quantum-filtered two-dimensional correlated spectroscopy; TOCSY, total correlated spectroscopy; NOE, nuclear Overhauser effect; NOESY, two-dimensional NOE spectroscopy; deg, degree; TFE, 2,2,2 trifluoroethanol.
2   E-mail: Predictprotein@EMBL-Heidelberg.DE.

ACKNOWLEDGEMENT

The Interfaculty Center for Measurements (CIM) is kindly acknowledged for the use of the NMR and computing facility.


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