Eur. J. Biochem. 270, 2163–2173 (2003) FEBS 2003
doi:10.1046/j.1432-1033.2003.03573.x
Comparison of the solution structures of angiotensin I & II Implication for structure-function relationship Georgios A. Spyroulias1, Panagiota Nikolakopoulou1, Andreas Tzakos3, Ioannis P. Gerothanassis3, Vassiliki Magafa1, Evy Manessi-Zoupa2 and Paul Cordopatis1 1
Departments of Pharmacy, and 2Chemistry, University of Patras, Greece; 3Department of Chemistry, University of Ioannina, Greece
Conformational analysis of angiotensin I (AI) and II (AII) peptides has been performed through 2D 1H-NMR spectroscopy in dimethylsulfoxide and 2,2,2-trifluoroethanol/H2O. The solution structural models of AI and AII have been determined in dimethylsulfoxide using NOE distance and 3 JHNHa coupling constants. Finally, the AI family of models resulting from restrained energy minimization (REM) refinement, exhibits pairwise rmsd values for the family ensemble 0.26 ± 0.13 A˚, 1.05 ± 0.23 A˚, for backbone and heavy atoms, respectively, and the distance penalty function is calculated at 0.075 ± 0.006 A˚2. Comparable results have been afforded for AII ensemble (rmsd values 0.30 ± 0.22 A˚, 1.38 ± 0.48 A˚ for backbone and heavy atoms, respectively; distance penalty function is 0.029 ± 0.003 A˚2). The two peptides demonstrate similar N-terminal and different C-terminal conformation as a consequence of
the presence/absence of the His9-Leu10 dipeptide, which plays an important role in the different biological function of the two peptides. Other conformational variations focused on the side-chain orientation of aromatic residues, which constitute a biologically relevant hydrophobic core and whose inter-residue contacts are strong in dimethylsulfoxide and are retained even in mixed organic-aqueous media. Detailed analysis of the peptide structural features attempts to elucidate the conformational role of the C-terminal dipeptide to the different binding affinity of AI and AII towards the AT1 receptor and sets the basis for understanding the factors that might govern free- or bounddepended AII structural differentiation.
The octapeptide angiotensin-II (H-Asp–Arg–Val–Tyr–Ile– His–Pro–Phe-OH, AII) is one of the oldest peptide hormones, known for its multiplicity of biological actions related to endocrine or connected to the central and peripheral nervous system. It is produced by the conversion of angiotensin-I (H-Asp–Arg–Val–Tyr–Ile–His–Pro–Phe– His–Leu-OH, AI) to AII by the action of the angiotensin-I converting enzyme (ACE) of the vascular endothelium. AI is generated in the circulation by the action of rennin from the kidneys on its substrate, called alpha2-globulin or angiotensinogen, produced in the liver [1]. AII is a potent pressor agent, which has a vital role in the regulation of blood pressure, in the conservation of total blood volume and salt homeostasis. Furthermore, it
is involved in the release of alcohol dehydrogenase (ADH), cell growth and the stimulation of the sympathetic system. Several antagonists of AII are efficient antipressor agents. Inadequate functioning of the renin– angiotensin system contributes substantially to the development of hypertension and cardiovascular and renal pathology (including left ventricular hypertrophy, structural alternations of the vasculature, neointima formation, nephrosclerosis, etc.) [2]. Structure–activity relationships studies from several laboratories have revealed the topological contribution of the individual amino acid residues of the active AII molecule [3–5]. These studies have included theoretical [6,7] physicochemical [8,9], and spectroscopic investigation [10–16] and have led to several models for the AII structure in solution. However, a great variety of structural features such as a helix [8,17,18,] b pleated sheet, b and c turn [15,19], charge relay [20,21] and other structures [6,22] have been proposed. The plethora of conformational features for the eightresidue peptide of AII is beyond any doubt disproportional to its length. Most importantly, the various AII structural characteristics reported are, in many cases, in contradiction. On the other hand, no experimental structural model for AI has been reported so far. Although AI does not exhibit the same biological or pharmacological interest as AII, it is the vassopressor AII’s precursor peptide and the substrate peptide to ACE catalytic pocket. Upon ACE hydrolysis, the inert AI is converted to the biologically active AII. The two peptides differ in the length and nature of their C-terminus and AII lacks the His-Leu terminal dipeptide. Thus, the
Correspondence to G. A. Spyroulias, Department of Pharmacy, University of Patras, GR-26504 Patras, Greece. Fax: + 30 610997714, Tel.: + 30 610997721, E-mail:
[email protected] Abbreviations: AI, angiotensin I; AII, angiotensin II; REM, restrained energy minimization. Note: AI and AII atomic coordinates of the 20-structure ensemble of conformers and the mean energy minimized structure have been deposited to the Protein Data Bank (accession codes 1n9u and 1n9v, respectively). Model 21 in each one of the above entries is the corresponding minimized average structure. (Received 31 October 2002, revised 4 March 2003, accepted 14 March 2003)
Keywords: angiotensin; NMR; renin-angiotensin system; solid phase peptide synthesis; solution structure.
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2164 G. A. Spyroulias et al. (Eur. J. Biochem. 270)
presence or absence of this terminal fragment should possess a key role in the structure-activity relationship in both peptides. Consequently, the aim of this research is focused not only in the solution structure determination of two biologically interesting peptides but also in the comparative conformational analysis of AI and AII, in their unbound forms. The NMR studies have been performed in dimethylsulfoxide and 2,2,2-trifluoroethanol/H2O mixture. Dimethylsulfoxide as 2,2,2-trifluoroethanol, EtOH, etc., have been reported to favor the fold of the angiotensineamide in a rather compact structure determined through circular dichroism measurements [15]. Additionally, dimethylsulfoxide has been widely used in the past for NMR measurements of various angiotensin analogues in an attempt at nonpolar receptor environment simulation [16,23,24] and elimination of possible multiple conformational isomers, which could exist in fast exchange [25]. 2,2,2-Trifluoroethanol/H2O was also used in order to investigate the conformation of the peptides in mixtures of organic solvents with water.
Materials and methods Sample preparation Synthesis was performed using solid phase chemistry on a 2-chlorotrityl chloride resin [26] via the Fmoc/tBu methodology [27]. The peptides were cleaved from the resin after treatment with trifluoroacetic acid/dichloromethane (8 : 2) and purified via gel chromatography and RP-HPLC (98% purity). Peptides were dissolved in dimethylsulfoxide-d6 and 2,2,2-trifluoroethanol-d2/H2O (2 : 1, v/v %) to a concentration of 2–2.5 mM in order to record 1D and 2D NMR spectra. NMR spectroscopy Data were acquired at 298K on a Bruker Avance 400-MHz spectrometer. One-dimensional NMR spectra were recorded using spectral width of 12–14 p.p.m. with or without presaturation of the H2O signal (Fig. 1A–D). 1H-1H
Fig. 1. 1D 1H 400-MHz NMR spectra of the AI and AII. Data recorded in dimethylsulfoxide-d6 (A and C, respectively) and 2,2,2trifluoroethanol/H2O (B and D, respectively) at 298K. DMSO, dimethylsulfoxide.
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Solution structure of angiotenins I & II in dimethylsulfoxide (Eur. J. Biochem. 270) 2165
Fig. 2. Analysis of NMR-derived data. Schematic representation of the sequential and medium-range NOE connectivities of AI and AII in dimethylsulfoxide-d6 (A and G, respectively) and in 2,2,2-trifluoroethanol/ H2O (D and J, respectively). Number of NOE constraints per residue for AI (B and E) and AII (H and K). White, gray, and dark gray vertical bars represent, respectively, intraresidue, sequential, and medium-range connectivities. Short and medium range (i, i + 2,3,4) side-chain NOE for AI (C and F) and AII (I and L) are indicated by arrows. All diagrams illustrate meaningful NOE constraints extracted from sm ¼ 400 ms NOESY. DMSO, dimethylsulfoxide.
DQF-COSY [28], TOCSY [29] (Fig. 2), using the MLEV-17 spin-lock sequence and sm ¼ 80–100 ms, and 1H-13C HSQC [30] (with 200 p.p.m. spectral width in F1) experiments were recorded. 1H-1H TPPI NOESY [31,32] spectra were acquired using sm 200–800 ms applying water suppression during the relaxation delay and mixing time [33]. All 2D spectra were acquired with 12 p.p.m. spectral width, consisting of 2K data points in the F2 dimension, 16–32 transients and 1024 complex increments in the F1 dimension. Raw data were multiplied in both dimensions by a pure cosine-squared bell window function and Fouriertransformed to obtain 2048 · 2048 real data points. A polynomial base-line correction was applied in both directions. For data processing and spectral analysis, the standard Bruker software and XEASY program (ETH, Zu¨rich) [34] were used. NOE constraints 552 and 433 NOESY (sm ¼ 400 ms) cross-peaks were assigned in both dimension for AI and AII, respectively, in dimethylsulfoxide. The number of unique NOESY crosspeaks were 299 and 244 (29.5 and 29.6 constraints per
residue for AI and AII, respectively) and their intensities were converted into upper limit distances through CALIBA [35]. The total number of assigned cross-peaks in 2,2,2trifluoroethanol/H2O NOESY spectra (sm ¼ 400 ms) were 233 and 206 for AI and AII, respectively, while the unique NOEs were 131 (AI) and 116 (AII). Sequential constraints, and number of NOEs are shown at Fig. 2. Structure calculations and refinement in dimethylsulfoxide Appropriate pseudoatom corrections were applied to methylene and methyl hydrogens that were not stereospecifically assigned [36,37]. 225 for AI and 180 for AII (22.1 and 21.6 per residues, respectively) NOE constraints were found meaningful and used in DYANA [38] calculations, together with seven 3JHNHa coupling constants for AI and six for AII. Upper (2.40 A˚) distance limits between HN and O atoms together with upper (3.30 A˚) and lower (2.60 A˚) limits between the corresponding N and O atoms, were set for two H-bonds which occurred in all the 20 calculated DYANA models and used as additional constraints. The u angles were restrained to )120 ± 50 for 3JHNHa ¼ 7.5
2166 G. A. Spyroulias et al. (Eur. J. Biochem. 270)
(Arg2 for AI and AII) or 9.0 Hz (Val3-Tyr4 for AI and AII, and His9 for AI) and )120 ± 30 for 3JHNHa > 9.0 Hz (Ile5-His6 for AI and AII, Leu10 for AI and Phe8 in AII). Also, 3JHNHa for Phe8 in AI has measured around 7.0 Hz and u has not been restrained [36]. The 20 best structures (out of 300 calculated) in terms of target function and NOE violations 1) NOEs have been observed, from which 18 are of the (i, i + 3,4) type. Among them more characteristic are those which are detected between the side-chains of: (a) Arg2 and Ile5 (b) Tyr4 aryl ring and His6 a and b protons, and (c) His6 imidazole ring (Hd2) and Phe8 and His9 backbone protons (Fig. 2C). The AI’s NOE number is dramatically reduced in 2,2,2-trifluoroethanol/H2O mixture and no Ha-HN (i, i + 2) type connectivities were observed (Fig. 2D). Meaningful medium range (i, i + n; n > 1) constraints in NOESY with sm ¼ 400 ms are only nine (Fig. 2E). However, the characteristic NOEs between His6 Hd2 and Phe8 HN, Ha, Hb are still present in 2,2,2-trifluoroethanol/ H2O, suggesting similar C-turn conformation in the two media (Fig. 2F). Angiotensin II. Analysis of AII NOESY maps exhibits similar to the AI NOE cross-peak pattern with 46 (i, i + n; n > 1) of which nine are of (i, i + 3) type, meaningful NOEs. Medium range NOE connectivities, not observed in the case of AI, of the Ha-HN(i, i + 3) type between Arg2 and Ile5 have been detected in the case of AII (Fig. 2G,I). The above NOE together with the HN-HN and Ha-HN (i, i + 2) type NOEs between Val3-Ile5 suggest a turnlike N-terminus structure of AII. Moreover, AII NOEs observed between Asp1/Arg2 and Tyr4/Ile5 side-chains further support this assumption. No NOEs were observed between the side-chains of Val3 and Ile5 while Val3
Fig. 3. Twenty models ensemble and mean structure of AI (A) and AII (B). The molecular surface electrostatic potentials are color coded: potentials less than )10 kT are illustrated in red, potentials larger than 10 kT in blue and neutral (0 kT) potentials are white (k is the Boltzmann constant, 1.380622 · 10)23 JÆK)1; T is the absolute temperature). Figure was generated with MOLMOL [41]. Rmsd values of the mean structure to the family ensemble are also illustrated.
8.581
7.813
8.023
7.883
8.328
Arg2
Val3
Tyr4
Ile5
His6
AII
8.107
Asp1
AI
8.020
7.874
8.303
Tyr4
Ile5
His6
Phe8
8.306
7.814
Val3
Pro7
8.576
Arg2
8.215
Leu10
8.109
8.278
His9
Asp1
8.128
Phe8
Pro7
HN
AI
4.110 49.789 4.356 53.307 4.173 58.137 4.496 54.760 4.137 57.542 4.753 50.908 4.369 60.178 4.420 54.268
4.118 49.646 4.355 53.312 4.178 57.980 4.494 54.766 4.121 57.415 4.769 51.064 4.308 60.606 4.453 54.878 4.643 52.228 4.221 51.247
Ha
Dimethylsulfoxide-d6
2.768, 2.643 36.481 1.623 29.772 1.928 31.684 2.795, 2.633 37.270 1.647 37.420 3.017, 2.920 28.127 2.016 29.937 3.016, 2.934 37.386
2.788, 2.649 36.403 1.620 29.812 1.926 31.787 2.787, 2.621 37.267 1.637 37.396 2.983, 2.931 28.182 1.978 29.921 2.973, 2.839 37.448 3.086, 2.971 27.927 1.531 40.616
Hb
Hc 1.479; Hd 3.080; He 7.649 29.770; 41.213 cCH3 0.747, 0.740 19.944, 15.963 Hd 7.017; He 6.610; OH 9.160 130.910; 115.621 Hc 1.349, 1.048; cCH3 0.781; dCH3 0.755 24.951; 11.657; 18.441 Hd2 7.317, He1 8.797 118.156; 130.910 Hc 1.795; Hd 3.628, 3.446 29.861; 47.614 Hd 7.231; He 7.248; Hf 7.286 127.210; 130.020; 128.907
Hc 1.479; Hd 3.077; He 7.674 29.817; 41.208 cCH3 0.759, 0.743 19.983, 15.894 Hd 7.015; He 6.608; OH 9.120 130.865; 115.642 Hc 1.341, 1.042; cCH3 0.782; dCH3 0.746 25.035; 11.583; 18.491 Hd1 7.212; Hd2 7.329; He1 8.839 117.907; 134.778 Hc 1.772; Hd 3.610, 3.459 29.946; 47.714 Hd 7.233; He 7.206; Hf 7.184 127.116; 129.819; 128.877 Hd2 7.323; He1 8.925 117.711; 134.661 Hc 1.622; dCH3 0.900, 0.841 25.035; 23.685, 22.023
Other
7.767
7.886
7.681
7.995
7.802
8.606
7.971
7.932
7.947
7.993
7.665
7.957
7.793
8.569
HN
4.376 50.829 4.438 54.207 4.177 58.766 4.716 54.742 4.177 59.912 4.995 50.577 4.495 61.004 4.702 55.191
4.401 50.426 4.442 54.115 4.146 59.867 4.713 55.078 4.155 58.968 5.003 50.587 4.466 61.058 4.643 55.650 4.692 52.672 4.427 51.938
Ha
3.016, 2.921 36.196 1.824 28.730 2.056 30.935 3.096, 2.962 36.717 1.825 36.868 3.261, 3.155 26.520 2.277, 2.025 29.449 3.288, 3.187 36.900
3.072, 2.988 35.506 1.795 28.748 2.034 30.913 3.067, 2.939 36.678 1.812 36.783 3.246, 3.135 27.308 2.280, 2.031 29.479 3.140, 3.130 37.363 3.300, 3.176 26.412 1.674 40.052
Hb
H2O/2,2,2-trifluoroethanol-d2, 34/66%
Table 1. 1H and 13C Chemical shifts (p.p.m) for AI and AII residues at 298K in 400-MHz AVANCE Bruker Spectrometer.
Hc 1.633; Hd 3.228; He 7.119 24.650; 40.984 cCH3 0.963, 0.904 17.647, 14.445 Hd 7.165; He 6.846 130.774; 115.822 Hc 1.480, 1.169; cCH3 0.901; dCH3 0.926 24.767; 9.732; 18.358 Hd2 7.252, He1 8.460 118.470; 133.712 Hc 1.964, Hd 3.769, 3.556 24.603; 48.093 Hd 7.307; He 7.325; Hf 7.364 127.272; 129.569; 128.760
Hc 1.622; Hd 3.218; He 7.117 24.579, 40.957 cCH3 0.957, 0.926 18.336, 14.472 Hd 7.149; He 6.833 130.746; 115.723 Hc 1.443, 1.156; cCH3 0.906; dCH3 0.888 24.731; 9.715; 17.682 Hd2 7.269; He1 8.513 118.378; 133.675 Hc 1.964; Hd 3.769, 3.556 29.470; 48.167 Hd 7.296; He 7.273; Hf 7.377 127.468; 129.355; 128.888 Hd2 7.254; He1 8.525 117.900; 133.595 Hc 1.703; dCH3 0.900, 0.841 24.787; 21.785, 20.477
Other
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2168 G. A. Spyroulias et al. (Eur. J. Biochem. 270)
side-chain is within NOE distance with the Tyr4 Ha, Hb (strong interaction) and aryl protons (weak interaction). Tyr4-His6 Ha-HN NOE observed in sm ¼ 400 ms but not in sm < 400 ms for AI and AII, is probably due to spin diffusion effects. A striking difference between AI and AII in terms of NOEs concerns the absence in AII NOESY, of the cross-peaks between His6 Hd2 and Phe8 HN and His6Phe8 HN-HN and Ha-HN. As in the case of AI, AII’s medium range NOE of (i, i + n; n > 1) type is limited in 2,2,2-trifluoroethanol/H2O (six in NOESY with sm ¼ 400 ms) and the only Ha-HN (i, i + 2) NOE was observed between Arg2 and Tyr4 (Fig. 2J,L). This turn-like Nterminal conformation is further supported by the Hc-CHc3 NOE between Arg2 and Ile5. NMR analysis and structure calculations No NOE signals were observed in both peptides in dimethylsulfoxide and 2,2,2-trifluoroethanol/H2O mixtures that involve main- or side-chain protons between C- and N-terminus protons excluding possible proximity of the two ends. However, analysis of conformational properties of the peptides in 2,2,2-trifluoroethanol/H2O suffers by the limited number of detectable NOEs, even in NOESY with sm ¼ 800 ms. Another intriguing point concerning the AI/ AII conformation, is the Tyr4 and His6 aromatic ring orientation [3–5,16,21]. These two residues are involved in a network of NOE interactions that accounts for 18 and 15 medium range dipolar proton–proton interactions for AI and AII, respectively. These NOEs reflect the similar sidechain orientation for these residues and the close proximity of the Tyr4 aromatic ring to the backbone, to the Cb atom and to the imidazole of His6. The average number of NOEs per residue for the tripeptide fragment Tyr4-His6 is remarkably large (Fig. 2B,H) for both peptides indicating a compact clustering of side-chains. The clustering of these residues in AI/AII is further supported by the data acquired in 2,2,2-trifluoroethanol/H2O where the tripeptide Tyr4His6 possesses the larger number of NOE constraints (Fig. 2E,K), as well. These data indicate also that the hydrophobic core is still formed in mixtures of aqueous and nonaqueous media. Regardless of the limited NOEs observed in 2,2,2-trifluoroethanol/H2O, four constraints for AI and AII between Tyr4 aryl protons and His6 HN, Hb and Hd2 indicate that the Tyr4 aryl ring is within NOE distance with His6 backbone, Cb atom and imidazole ring. The interest in the AI His6-Leu10 and AII His6-Phe8 C-terminal fragment is mainly focused on the backbone turn structure and the conformational characteristics of Phe8. However, phenyl ring o-, m- and p-protons possess three, nearly degenerated resonances (in both solvents used; Table 1) indicating that the phenyl ring rotation occurs in the fast motion limit. The fast rotation of the aromatic nucleus suggests absence of any steric interaction with vicinal amino acids. This aspect is also supported by the complete absence of any intra or inter-residue NOESY peaks with the protons of any adjacent residue. Motional and conformational restriction of Phe8 side-chain would be anticipated only if the bulky phenyl ring oriented towards the hydrophobic core of the peptide, close to His6 and Tyr4. In this case, the phenyl ring rotation
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should occur in the slow motion limit and should yield detectable intra- and inter-residue NOEs. Nevertheless, the only observed Phe8 inter-residue NOEs emerged through the interaction of HN, Ha, Hb with His6 Hd2 and He1 protons. A total number of 20 NOEs between AI His6 imidazole and Phe8-Leu10 protons in dimethylsulfoxide suggest that His6 points towards the C-terminus. On the other hand, no NOEs were observed between the His9 imidazole protons and the Tyr4 aromatic protons, but it should be considered that no intra or inter-residue NOEs involving the protons of the His9 ring, other than the ill-defined Hd2-He1 cross-peak, are observed in NOESY spectra. Additionally, His9 and Leu10 yield a relatively large number of NOE distances (His9/Leu10 constraints: 27/25 from which 21/11 are interand 6/14 intraresidue, respectively) assisting the calculation of their conformation. Thirty-nine NOEs (34 inter- and five intra residue constraints), involving Phe8 backbone and Hb protons, have been detected in AI in contrast to only 12 NOEs in AII, suggesting that the longer C-terminus diminishes its orientational freedom. Additionally, the close proximity of His6 side-chain and Phe8 backbone has been also verified in 2,2,2-trifluoroethanol/H2O due to three NOEs between the His6 Hd2 and Phe8 backbone and b-protons. Despite the fact that the numerous NOE constraints in dimethylsulfoxide have not been afforded in 2,2,2-trifluoroethanol/H2O, these data strongly suggest that the conformation of Tyr4, His6 and Phe8 is probably retained upon differentiation of the hydrophobic/hydrophilic character of the media. AII possess Phe8 as terminal residue, which is expected to exhibit enhanced conformational freedom. As a result, there is a loss of the NOE cross-peak between the Hd2 His6 and HN Phe8 protons which suggests that Phe8 moves far from His6, at a distance larger than in AI. Additionally, no NOE was observed between His6 and Phe8 in 2,2,2-trifluoroethanol/H2O. NMR models in dimethylsulfoxide. The average target function for the DYANA family of 20 calculated models (Fig. 3A) was found 0.048 A˚2 for AI and 0.0192 A˚2 for AII models (Fig. 4B). The final AI REM models exhibit pairwise rmsd values 0.26 ± 0.13 A˚ (BB), 1.05 ± 0.23 A˚ (HA) for the 20 structures and 0.19 ± 0.06 A˚ (BB), 0.73 ± 0.13 A˚ (HA) to the mean structure (Fig. 3A), respectively. The rmsd values for AII REM ensemble are 0.30 ± 0.22 A˚ (BB), 1.38 ± 0.48 A˚ (HA) for the 20 models and 0.25 ± 0.06 A˚ (BB), 1.00 ± 0.13 A˚ (HA) to the mean structure (Fig. 3B). NMR models in 2,2,2-trifluoroethanol/H2O. Angiotensin models calculated using NMR-derived data acquired in 2,2,2-trifluoroethanol/H2O suffer from low resolution due to the limited number of NOE constraints. The resulting DYANA family of 20 models for AI and AII has rmsd values of 2.36 ± 0.85 (BB), 3.82 ± 0.93 (HA) (average target function 0.037 A˚2) and 1.05 ± 0.46 (BB), 2.29 ± 0.72 (HA) (average target function 0.023 A˚2), respectively, indicating low resolution especially in calculation of sidechains. Therefore, detailed conformational analysis presented below is based on data acquired and structures calculated in dimethylsulfoxide.
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Solution structure of angiotenins I & II in dimethylsulfoxide (Eur. J. Biochem. 270) 2169
Fig. 4. Chemical shift variation and structural differences in terms of rmsd, between AI and AII. (A) Best fit of mean, energy minimized, structures of AI (blue) and AII (grey). (B) Chemical shift variation (in p.p.m) plots between the Ha and HN resonances of AI and AII, and (C) rmsd values between AI and AII mean, energy minimized, structures, extracted for the common residues Asp1-Phe8.
Structure validation. Ramachandran plot analysis of the AI and AII ensemble of 20 conformers, showed that 58.3% of the nonglycyl residues fall into the most favored regions of torsion angle space, and 41.7% of the residues are in other allowed regions. No residue falls in the generously allowed region or in disallowed region. The families of conformers have no distance constraint violations greater than 0.10 A˚. Evaluating the impact of various classes of NOEs (i.e. intraresidue, sequential and medium range) of the calculated structure in such short peptides, we performed various calculations with reduced NOE data set. Original AI NOE data set (225 meaningful), was used to generate NOE data set excluding the following groups of NOEs: (a) 24 constraints involving backbone (BB) intraand inter-residue (i, i + 1) NOE (namely group 1) (b) additionally to group 1, 11 BB inter-residue (i, i + n; n > 1) NOE (group 2) (c) additionally to group 2, 24 BB-SC (sidechain) intraresidue NOE (group 3), and (d) additionally to group 3, 58 BB-SC sequential NOEs. Each one of the above NOE data set was used as input constraint file in DYANA calculations. In any of the above cases, 20 conformers, having the lowest overall energy were selected for further refinement through REM. The pairwise rmsd of the 20structure ensemble of conformers calculated with the first three NOE data set described above, range among 0.53 ± 0.30 A˚ and 0.62 ± 0.31 A˚ measured for all residues. Also, the pairwise rmsd for the conformers calculated with the fourth NOE data set was found 0.59 ± 0.19 A˚ for Arg2-His9 fragment. The above data, together with the analysis of mean energy minimized models, indicate that the backbone conformation and side-chain of residues Arg2His9 exhibit slightly higher rmsd while the NOE constraints are limited. Nevertheless, these models practically exhibit the same conformational features with those in models calculated with the entire number of NOEs. For AII
conformers are calculated with NOE data sets generated as described above in (a) (c) and (d) and same data has been afforded. Finally, the structure calculations, which were performed without inclusion of u angle constraints, have only resulted to higher conformational flexibility especially for the two terminal dipeptide fragments.
Discussion Previous structural studies focused on AII by J.M. Matsoukas et al. [23] reported that the aromatic residues, Tyr4, His6 and Phe8, common in both molecules, comprise a hydrophobic core at the middle of the molecule (for recent structure-activity relationships reports of AII analogues see [23–25,42–53]). It is also suggested that the existence of a p-stacking structure [16,20,23], might allow a charge transfer process between the two peptide termini having a great impact on its biological activity. Additionally, absence or protection of the Tyr4 hydroxyl group diminishes, to a great extent, the pressor activity of AII [25,54], while activity is gradually diminished in parallel to the progressive destruction of His6 imidazole when it is irradiated by UV light [55]. According to the existing AII models, the Tyr4/His6 interaction varied considerably [16,25,45–50,56] and their rings were found parallel in favorable p-stacking condition [51] or far apart at a distance of 7.5 up to 10–12 A˚ [25,52,53,56]. However, among these AII models, there should be mentioned: (a) the X-ray AII structure when the hormone is complexed to a high-affinity monoclonal antibody (Mab) [57], and (b) the various models of AII bound to the AT1 receptor models [58–62], which provide valuable information about the peptide conformation in its bound form. The former study revealed that the bound AII
2170 G. A. Spyroulias et al. (Eur. J. Biochem. 270)
exists in a rather compact U-shape conformation possessing two turns, one at each terminus. Additionally, the termini are at a close distance and salt-bridged, while the side-chain of Asp1 and the backbone carbonyl of His6 are H-bonded [57]. Among the latter reports, one describes the AII bound to AT1, where the peptide retains a b strand extended conformation with the two termini distant [58]. The best fit of the backbone atoms for AI and AII mean energy minimized structures is illustrated in Fig. 4A. Both structures exhibit a similar S-shape backbone conformation with the N-end amide nitrogens and C-end carboxyl oxygens at distances measured up to 21.8 A˚ for AI and 17.8 A˚ for AII, respectively. Chemical shift differences and rmsd values for the Asp1-Phe8 common residues (Fig. 4B,C) reveal great similarities to the backbone conformation of the Arg2-His6/Pro7 penta- or hexapeptide fragment and to the side-chain orientation of the Val3-His6 tetrapeptide. Structural differences implied by rmsd values and chemical shift differences for the dipeptide fragment Pro7 and Phe8. Variation in conformational features of this C-terminus region is related to the different length of the two peptides. On the other hand, comparison in great detail between the AI and AII N-termini is avoided. Asp1 related NOESY cross-peaks are limited for both peptides, and this is the major drawback in ambiguous conformational investigation of the N-terminus. Possibly, dimethylsulfoxide accounts for the loss of backbone amide proton of Asp1 [63] resulting in considerable loss of intraresidue scalar or inter-residue NOE contacts. As far as the AI hydrophobic core is concerned, Tyr4 and His6 rings form an angle of 35, while the distance between their centers is 6.1 A˚. The distance between the phenolic oxygen and the b- and c-carbons of the imidazole nucleus of His6 was calculated as being equal to 4.0 and 4.7 A˚. No hydrogen bonding occurred during the course of DYANA calculation between Tyr4 OH and the His6 imidazole ring. Tyr4, His6 and His9 side-chains are far apart from Phe8 phenyl ring while the latter is oriented towards the hydrophobic core formed by Tyr4 and His6. The His9 imidazole arrangement close to the aromatic rings of His6 and Tyr4 (interring distances 5.2 A˚ and 5.8 A˚, respectively) is certainly assisted by the existence of b-turn-like structure of the C-terminal tetrapeptide. Analysis of the AII to AI superposed structure (Fig. 4), indicates that, apart from overall resemblance, the Tyr4 aryl ring in AI structures is slightly rotated, moving farther from the His6 imidazole ring. Indeed, in the presence of the AI His9-Leu10 dipeptide, the interring distance between Tyr4 and His6 residues becomes 1 A˚ larger, while the distance between their Cb atoms remains constant ( 7.1 A˚). Practically intact remain also two out of the three backbone torsion angle (wAI fi AII 123.1 fi 86.1, xAI fi AII )177.9 fi )178.8, uAI fi AII )146.6 fi )148.9). The p–p* interaction between Tyr4 and His6 rings is perturbed and the hydrophobicity of the core is diminished while the exposure of the tyrosyl OH to the solvent is increased. On the other hand, the two termini possess a hydrophilic character due to negative charge density at the C-termini and positive charge density at the N-termini (Fig. 4). This property imposes on AI and AII a dipole-like character, which plays a fundamental role in enzyme/receptor-peptide molecular recognition and complex formation processes.
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Overall, the S-shape skeleton of AII observed in our NMR models is different from the U-shape AII model in solid state presented by Amzel et al. [57] in a Fab-AII complex, Carpenter et al. [56] in phospholipid environment and computer modeled receptor-bound conformation by Joseph et al. [25]. On the other hand, the skeleton of the present AII model is closer to twisted-extended conformation proposed by Nikiforovich et al. [52,53,64], Matsoukas et al. [23] and the model of AII bound to AT1 receptor by Leduc et al. [58]. Regarding the spatial arrangement of Tyr4, His6 and Phe8, the present AII model differs remarkably from AII bound to Fab where Tyr4 and His6 rings are far apart. Our AII model also excludes the possibility of three aromatic residues clustering suggested by Matsoukas et al. [23], which is also overruled by Nikiforovich et al. [52,53]. However, our model resembles the clustering of Tyr4 and His6 in Matsoukas et al. [23] AII NMR models in dimethylsulfoxide. Finally, it should be noted that the structural feature of this NMR AII model exhibits great similarity with the model proposed by Fermandjian et al. [15] which exhibits one b-turn at each terminus while Tyr4 and His6 rings are parallel and Phe8 ring is far apart [15]. Concluding remarks – biological implications The active conformation of AII has been long debated among the U-shape and the twisted-extended [57,64,65]. According to the structure analysis the existence and orientation of AI His9 and Leu10 side-chains affect the conformation of Tyr4 ring and differentiate the magnitude of His9-Tyr4/His6 contact. Furthermore, His9-Tyr4 interaction provides a measure for His6-Tyr4 conformation/ interaction and for Tyr4 tendency to move out of the hydrophobic pocket. Phe8 in AII could play a similar role to that of His9 in AI. This residue is the last in the sequence and it possesses remarkable orientational freedom. Phe8, in the AII model presented here, orients far from the hydrophobic core but could reorient its aromatic ring towards His6/Tyr4 through intermolecular interactions, which are usually evolved in the spatially restricted interior of a receptor’s pocket [49,50]. Then Tyr4 or His6 would be reinforced to minimize steric interactions by modifying not only their side-chain but also their main-chain conformation through rotation of u and/ or x torsion angles in the middle of the AII sequence. Such a rotation in our AII model results in an S to U conformational transition with the two termini oriented towards the same direction approaching close spatial proximity (data not shown). Although it is difficult to elucidate the various steps of such a transition, the above data could provide a reasonable explanation for the question why AII in its Fab-complexed structure [57] exhibits a U-shaped structure with Tyr4 and His6 placed far apart from each other. Considering the various AII models reported hitherto, extended conformation is suggested in organic media while U-shape conformation resulted from studies in which intermolecular interactions between AII and the environment (water/lipid interface [56], or receptor’s cavities [57]) are raised. Transformation of the S-shape, of the free AII, to a functionally cooperative U-shape conformation in bound state would result in: (a) eliminating the possibility
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Solution structure of angiotenins I & II in dimethylsulfoxide (Eur. J. Biochem. 270) 2171
of p)p* stacking geometry between Tyr4 and His6, and (b) allowing the so far protected His6, in the hydrophobic core, and the pharmacophore Tyr4 [23,25,47–50] to emerge and stimulate receptor response demonstrating their biological activity. On the other hand, AI and AII NMR models exhibit conformational features similar to the AII twisted-extended bioactive conformation suggested by Marshall and Nikiforovich [64]. In this conformation a functionally important structural difference between the two peptides is the length and the conformation of the C-terminus, whose AI His9Leu10 dipeptide shifts the COO– by two residues while masks the important free AII Phe8-COO– group. It is well established that AII binds tightly to the receptor’s cavity while AI more likely does not. AII possesses the unprotected Phe8 free carboxyl C-terminus that in AT1 environment interacts with Lys199 [59–62,66]. According to studies on the interaction of AII with AT1 receptor, a four–point interaction has been postulated [59,60,62,66] between: (a) the C-terminal carboxylate anion and Lys199 (b) the His6 and Asp263 (c) Tyr4 and Arg167, and (d) Arg2 and Asp281. The model proposed by Inagami et al. [59] suggests that the distance between the AII COO– terminus interaction site of AT1 and the Asp263/Thr260 site, which interacts with His6 imidazole, is 16 A˚ and the deducing distance between AII COO– and His6 imidazole is 7.0–7.5 A˚. In our AII S-shaped model this distance is found to be 7.1 A˚ (6.7 A˚ in AI) and indicates favorable arrangement for the peptide binding important residues. On the other hand, in AI, the AII’s inactive precursor, the turn-like C-terminal could also hinder a favorable peptide C-terminus-receptor pocket fit due to steric interactions and shield with their side-chains (His9) the His6 imidazole from interaction with the Asp263/ Thr260 AT1 binding site.
Acknowledgements General Secretariat of Research and Technology of Greece (PC) and University of Patras for a K. Karatheodoris Research Grant (PC & GAS), are acknowledged for financial support. We also thank Instrumental Analysis Center (University of Patras) and EC’s Access to Research Infrastructures Action of the Improving Human Potential Program (PARABIO, Contract No. HPRI-CT-1999-00009) for further support.
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Supplementary material The following material is available from http://www. blackwellpublishing.com/products/journals/suppmat/EJB/ EJB3573/EJB3573sm.htm Table S1. Experimental NOESY cross-peaks intensities (upper distance constraints) for AI in dimethylsulfoxide. Table S2. Experimental NOESY cross-peaks intensities (upper distance constraints) for AII in dimethylsulfoxide. Table S3. Experimental NOESY cross-peaks intensities (upper distance constraints) for AI in 2,2,2-trifluoroethanol/H2O. Table S4. Experimental NOESY cross-peaks intensities (upper distance constraints) for AII in 2,2,2-trifluoroethanol/H2O. Table S5. Summary of statistical data for REM models and mean structure. Fig. S1. Cluster of aromatic residues in AI and AII and interring distances.
