Comparison of the solution structures of angiotensin I & II
Implication for structure-function relationship
Georgios A. Spyroulias
1
, Panagiota Nikolakopoulou
1
, Andreas Tzakos
3
, Ioannis P. Gerothanassis
3
,
Vassiliki Magafa
1
, Evy Manessi-Zoupa
2
and Paul Cordopatis
1
1
Departments of Pharmacy, and
2
Chemistry, University of Patras, Greece;
3
Department of Chemistry,
University of Ioannina, Greece
Conformational analysis of angiotensin I (AI) and II (AII)
peptides has been performed through 2D
1
H-NMR spectro-
scopy in dimethylsulfoxide and 2,2,2-trifluoroethanol/H
2
O.
The solution structural models of AI and AII have been
determined in dimethylsulfoxide using NOE distance and
3
J
HNHa
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±0.48A
˚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 AT
1
receptor and sets the basis for under-
standing the factors that might govern free- or bound-
depended AII structural differentiation.
Keywords: angiotensin; NMR; renin-angiotensin system;
solid phase peptide synthesis; solution structure.
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 alpha
2
-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 sympa-
thetic system. Several antagonists of AII are efficient
antipressor agents. Inadequate functioning of the renin–
angiotensin system contributes substantially to the develo-
pment of hypertension and cardiovascular and renal
pathology (including left ventricular hypertrophy, struc-
tural 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] physico-
chemical [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
ahelix [8,17,18,] bpleated sheet, band cturn [15,19], charge
relay [20,21] and other structures [6,22] have been proposed.
The plethora of conformational features for the eight-
residue 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: G.A.Spyroulias@upatras.gr
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)
Eur. J. Biochem. 270, 2163–2173 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03573.x
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 conform-
ational analysis of AI and AII, in their unbound forms. The
NMR studies have been performed in dimethylsulfoxide
and 2,2,2-trifluoroethanol/H
2
O 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 measure-
ments [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 pos-
sible multiple conformational isomers, which could exist in
fast exchange [25]. 2,2,2-Trifluoroethanol/H
2
Owasalso
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 metho-
dology [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-d
6
and
2,2,2-trifluoroethanol-d
2
/H
2
O (2 : 1, v/v %) to a concen-
tration of 2–2.5 m
M
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 recor-
ded using spectral width of 12–14 p.p.m. with or with-
out presaturation of the H
2
O signal (Fig. 1A–D).
1
H-
1
H
Fig. 1. 1D
1
H 400-MHz NMR spectra of the
AI and AII. Data recorded in dimethyl-
sulfoxide-d
6
(A and C, respectively) and 2,2,2-
trifluoroethanol/H
2
O (B and D, respectively)
at 298K. DMSO, dimethylsulfoxide.
2164 G. A. Spyroulias et al.(Eur. J. Biochem. 270)FEBS 2003
DQF-COSY [28], TOCSY [29] (Fig. 2), using the MLEV-17
spin-lock sequence and s
m
¼80–100 ms, and
1
H-
13
C
HSQC [30] (with 200 p.p.m. spectral width in F1) experi-
ments were recorded.
1
H-
1
H TPPI NOESY [31,32] spectra
were acquired using s
m
200–800 ms applying water sup-
pression 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 dimen-
sion. Raw data were multiplied in both dimensions by a
pure cosine-squared bell window function and Fourier-
transformed to obtain 2048 ·2048 real data points. A
polynomial base-line correction was applied in both direc-
tions. 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 (s
m
¼400 ms) cross-peaks were
assigned in both dimension for AI and AII, respectively, in
dimethylsulfoxide. The number of unique NOESY cross-
peaks 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 2,2,2-
trifluoroethanol/H
2
ONOESYspectra(s
m
¼400 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 stereospe-
cifically 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
3
J
HNHa
coupling constants for AI and
six for AII. Upper (2.40 A
˚) distance limits between H
N
and
O atoms together with upper (3.30 A
˚)andlower(2.60A
˚)
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
3
J
HNHa
¼7.5
Fig. 2. Analysis of NMR-derived data.
Schematic representation of the sequential
and medium-range NOE connectivities of AI
and AII in dimethylsulfoxide-d
6
(A and G,
respectively) and in 2,2,2-trifluoroethanol/
H
2
O (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 s
m
¼400 ms
NOESY. DMSO, dimethylsulfoxide.
FEBS 2003 Solution structure of angiotenins I & II in dimethylsulfoxide (Eur. J. Biochem. 270) 2165
(Arg2forAIandAII)or9.0 Hz(Val3-Tyr4forAIandAII,
and His9 for AI) and )120 ± 30 for
3
J
HNHa
>9.0Hz
(Ile5-His6 for AI and AII, Leu10 for AI and Phe8 in AII).
Also,
3
J
HNHa
for Phe8 in AI has measured around 7.0 Hz
and uhas not been restrained [36]. The 20 best structures
(out of 300 calculated) in terms of target function and NOE
violations <0.1 A
˚comprise the family ensemble and are
refined through REM (
AMBER
5.0 [39];
SANDER
[40]
program). A force constant of 133.76 kJÆmol
)1
A
˚
2
is applied
for the distance constraints. AI and AII structures are
illustrated in Fig. 3 (figures are generated with
MOLMOL
[41]). Structural calculations have been performed on IBM
RISC6000 or on PentiumIII PC-Linux computers.
Results
Proton assignment
The downfield broad signal (9.20 p.p.m) observed in
dimethylsulfoxide spectra of both peptides, is assigned to
the Tyr4 OH group (instead of the previously reported
assignment to Arg2 H
e
proton [23]). OH disappears when
drops of H
2
O and/or D
2
O are added or when the water
signal is irradiated. Arg2 H
e
proton has been unambigu-
ously assigned, in both solvents, through its TOCSY
connectivities with Arg2 H
a
,H
d
and side-chain protons.
Chemical shifts for both peptides are reported in Table 1.
Angiotensin I. H
N
-H
N
,H
a
-H
N
and H
b
-H
N
connectivities
are detected in the region Arg2-His6, as well as, between
Phe8 and His9/Leu10 (Fig. 2A). Short range H
N
-H
N
and
H
a
-H
N
(i, i + 2) type connectivities are also identified
between residues Val3-Ile5 and His6-Phe8 indicating the
existence of two turns at each peptide terminus. In sum, 62
meaningful medium range (i, i +n;n>1)NOEshave
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 aand bprotons, and (c) His6
imidazole ring (H
d2
) and Phe8 and His9 backbone protons
(Fig. 2C). The AI’s NOE number is dramatically reduced
in 2,2,2-trifluoroethanol/H
2
OmixtureandnoH
a
-H
N
(i, i
+ 2) type connectivities were observed (Fig. 2D). Mean-
ingful medium range (i, i + n; n > 1) constraints in
NOESY with s
m
¼400 ms are only nine (Fig. 2E).
However, the characteristic NOEs between His6 H
d2
and
Phe8 H
N
,H
a
,H
b
are still present in 2,2,2-trifluoroethanol/
H
2
O, 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
thecaseofAI,oftheH
a
-H
N
(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 H
N
-H
N
and H
a
-H
N
(i, i + 2) type NOEs between Val3-Ile5 suggest a turn-
like 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)potentialsarewhite(kis the Boltzmann constant,
1.380622 ·10
)23
JÆK
)1
;Tis the absolute temperature). Figure was generated with
MOLMOL
[41]. Rmsd values of the mean structure to the family
ensemble are also illustrated.
2166 G. A. Spyroulias et al.(Eur. J. Biochem. 270)FEBS 2003
Table 1.
1
Hand
13
C Chemical shifts (p.p.m) for AI and AII residues at 298K in 400-MHz AVANCE Bruker Spectrometer.
AI
Dimethylsulfoxide-d
6
H
2
O/2,2,2-trifluoroethanol-d
2
, 34/66%
HN HaHbOther HN HaHbOther
AI Asp1 8.107 4.118
49.646
2.788, 2.649
36.403
4.401
50.426
3.072, 2.988
35.506
Arg2 8.581 4.355
53.312
1.620
29.812
Hc1.479; Hd3.077; He7.674
29.817; 41.208
8.569 4.442
54.115
1.795
28.748
Hc1.622; Hd3.218; He7.117
24.579, 40.957
Val3 7.813 4.178
57.980
1.926
31.787
cCH
3
0.759, 0.743
19.983, 15.894
7.793 4.146
59.867
2.034
30.913
cCH
3
0.957, 0.926
18.336, 14.472
Tyr4 8.023 4.494
54.766
2.787, 2.621
37.267
Hd7.015; He6.608; OH 9.120
130.865; 115.642
7.957 4.713
55.078
3.067, 2.939
36.678
Hd7.149; He6.833
130.746; 115.723
Ile5 7.883 4.121
57.415
1.637
37.396
Hc1.341, 1.042; cCH
3
0.782; dCH
3
0.746
25.035; 11.583; 18.491
7.665 4.155
58.968
1.812
36.783
Hc1.443, 1.156; cCH
3
0.906; dCH
3
0.888
24.731; 9.715; 17.682
His6 8.328 4.769
51.064
2.983, 2.931
28.182
Hd1 7.212; Hd2 7.329; He1 8.839
117.907; 134.778
7.993 5.003
50.587
3.246, 3.135
27.308
Hd2 7.269; He1 8.513
118.378; 133.675
Pro7 4.308
60.606
1.978
29.921
Hc1.772; Hd3.610, 3.459
29.946; 47.714
4.466
61.058
2.280, 2.031
29.479
Hc1.964; Hd3.769, 3.556
29.470; 48.167
Phe8 8.128 4.453
54.878
2.973, 2.839
37.448
Hd7.233; He7.206; Hf7.184
127.116; 129.819; 128.877
7.947 4.643
55.650
3.140, 3.130
37.363
Hd7.296; He7.273; Hf7.377
127.468; 129.355; 128.888
His9 8.278 4.643
52.228
3.086, 2.971
27.927
Hd2 7.323; He1 8.925
117.711; 134.661
7.932 4.692
52.672
3.300, 3.176
26.412
Hd2 7.254; He1 8.525
117.900; 133.595
Leu10 8.215 4.221
51.247
1.531
40.616
Hc1.622; dCH
3
0.900, 0.841
25.035; 23.685, 22.023
7.971 4.427
51.938
1.674
40.052
Hc1.703; dCH
3
0.900, 0.841
24.787; 21.785, 20.477
AII Asp1 8.109 4.110
49.789
2.768, 2.643
36.481
4.376
50.829
3.016, 2.921
36.196
Arg2 8.576 4.356
53.307
1.623
29.772
Hc1.479; Hd3.080; He7.649
29.770; 41.213
8.606 4.438
54.207
1.824
28.730
Hc1.633; Hd3.228; He7.119
24.650; 40.984
Val3 7.814 4.173
58.137
1.928
31.684
cCH
3
0.747, 0.740
19.944, 15.963
7.802 4.177
58.766
2.056
30.935
cCH
3
0.963, 0.904
17.647, 14.445
Tyr4 8.020 4.496
54.760
2.795, 2.633
37.270
Hd7.017; He6.610; OH 9.160
130.910; 115.621
7.995 4.716
54.742
3.096, 2.962
36.717
Hd7.165; He6.846
130.774; 115.822
Ile5 7.874 4.137
57.542
1.647
37.420
Hc1.349, 1.048; cCH
3
0.781; dCH
3
0.755
24.951; 11.657; 18.441
7.681 4.177
59.912
1.825
36.868
Hc1.480, 1.169; cCH
3
0.901; dCH
3
0.926
24.767; 9.732; 18.358
His6 8.303 4.753
50.908
3.017, 2.920
28.127
Hd2 7.317, He1 8.797
118.156; 130.910
7.886 4.995
50.577
3.261, 3.155
26.520
Hd2 7.252, He1 8.460
118.470; 133.712
Pro7 4.369
60.178
2.016
29.937
Hc1.795; Hd3.628, 3.446
29.861; 47.614
4.495
61.004
2.277, 2.025
29.449
Hc1.964, Hd3.769, 3.556
24.603; 48.093
Phe8 8.306 4.420
54.268
3.016, 2.934
37.386
Hd7.231; He7.248; Hf7.286
127.210; 130.020; 128.907
7.767 4.702
55.191
3.288, 3.187
36.900
Hd7.307; He7.325; Hf7.364
127.272; 129.569; 128.760
FEBS 2003 Solution structure of angiotenins I & II in dimethylsulfoxide (Eur. J. Biochem. 270) 2167