Download - Conformational properties of a cyclic peptide bradykinin B2 receptor antagonist using experimental and theoretical methods

K. KrauseL.F. PinedaR. PeteranderlS. Reissmann

Authors' af®liations:

K. Krause and L.F. Pineda, Institut fuÈ r Biochemie

und Biophysik, Friedrich-Schiller-UniversitaÈ t

Jena, Philosophenweg 12, D-07743 Jena,

Germany.

R. Peteranderl, Institut fuÈ r Organische Chemie

und Biochemie, Technische UniversitaÈt

MuÈ nchen, Lichtenbergstraûe 4, D-85747

Garching, Germany.

S. Reissmann, Institut fuÈ r Biochemie und

Biophysik, Friedrich-Schiller-UniversitaÈ t Jena,

Philosophenweg 12, D-07743 Jena, Germany.

Correspondence to:

Luis Felipe Pineda De Castro

Institut fuÈ r Molekulare Biotechnologie e.V.

Postfach 100813

D-07708 Jena

Germany

Tel.: 49-3641-656491

Fax: 49-3641-609818

E-mail: [email protected]

Dates:

Received 9 April 1999

Revised 25 May 1999

Accepted 24 July 1999

To cite this article:

Krause, K., Pineda L.F., Peteranderl, R. & Reissmann, S.

Conformational properties of a cyclic peptide bradykinin

B2 receptor agonist by experimental and theoretical

methods.

J. Peptide Res., 2000, 55, 63±71

Copyright Munksgaard International Publishers Ltd, 2000

ISSN 1397±002X

Conformational properties ofa cyclic peptide bradykinin B2

receptor antagonist usingexperimental and theoreticalmethods

Key words: Molecular dynamics simulations; NOE; peptide

hormones; simulated annealing

Abstract: The solution conformation of the cyclic peptide J324

(cyclo0,6-[Lys0,Glu6,D-Phe7]BK), an antagonist targeted at the

bradykinin (BK) B2 receptor, has been investigated using

experimental and theoretical methods. In order to gain insight

into the structural requirements essential for BK antagonism, we

carried out molecular dynamics (MD) simulations using simulated

annealing as the sampling protocol. Following a free MD

simulation we performed simulations using nuclear Overhauser

enhancement (NOE) distance constraints determined by NMR

experiments. The low-energy structures obtained were compared

with each other, grouped into families and analyzed with

respect to the presence of secondary structural elements in their

backbone. We also introduced new ways of plotting structural

data for a more comprehensive analysis of large conformational

sets. Finally, the relationship between characteristic backbone

conformations and the spatial arrangement of speci®c

pharmacophore centers was investigated.

Abbreviations: BK, bradykinin; DMSO, dimethylsulfoxide; MD,

molecular dynamics; NOE, nuclear Overhauser effect; RMS, root

mean square; RMSD, root mean square deviation; SA, simulated

annealing; SPL, SYBYL programming language; TPPI, time

proportional phase incrementation.

Bradykinin (BK) is a peptide hormone with the sequence

RPPGFSPFR, found in tissues and plasma. It is implicated in

numerous pathophysiological processes, for example per-

ipheral pain and in¯ammation. Owing to these observa-

tions, great efforts have been undertaken in order to develop

63

BK antagonists as potential therapeutic agents. In the past a

large number of peptide antagonists has been synthesized by

various groups (1), while peptide design has gone through

several iterations.

In the last three decades several groups have begun

attempts to describe the conformation of BK and some of its

analogs. After initial studies focused on the conformation of

agonistic BK analogs and partial sequences, the situation

changed with the discovery of the ®rst BK antagonist. The

goal of these investigations was to describe the bioactive

conformation(s) of BK antagonists and de®ne the differences

between the agonists and antagonists. Whereas, in the past,

conformational analysis was performed by means of con-

ventional chemical and physical methods, such as thin ®lm

dialysis, H-exchange, ORD-, CD-, Raman-, ESR- and

¯uorescence spectroscopy, more recently NMR spectro-

scopy combined with molecular modeling has been increas-

ingly applied (2).

The results of those numerous investigations were rather

insuf®cient for an accurate description of the bioactive

conformations. Thus, different conformational shapes have

been proposed for agonists and antagonists, e.g. random

conformations (3, 4), conformations stabilized by up to three

hydrogen bonds (5) or quasicyclic structures (6, 7) have been

proposed for agonists. As a result of studies on antagonists

containing different amino acid substitutions at position 7

and additional replacements at other sequence positions,

turn structures have been postulated, either in the N-

terminal sequence (8), the C-terminal sequence (9±12) or

both (13, 14).

The conformation of the linear nonapeptide changes

through interaction with its receptor, a large membrane-

bound protein. Two approaches to characterizing the

receptor-bound conformation have been used considering

this conformational change and the lack of an isolated and

crystallized hormone receptor complex, suitable for X-ray

analysis. Ottleben et al. (15) used a monoclonal antibody to

mimic the receptor-binding area. The conformation of

antibody-bound BK was studied after labeling with 13C

and 15N by NMR spectroscopy. Nevertheless, this model ®ts

the binding requirements of the receptor to only a limited

extent. Thus, the C-terminal Arg is not necessary for

binding to the antibody, but is essential for binding to the B2

receptor. Therefore, the conformation found seems to

correlate only partially with the receptor-bound conforma-

tion. A second approach was used by Kyle et al. (16) to

describe the receptor-bound conformation. Based on the

results of structural homology modeling and computer

simulation, the authors generated a model of the ligand

bound to the rat B2 receptor, which, even though it is highly

speculative, is supported by mutagenesis data. Therefore,

conformational analysis of the hormone±receptor complex

remains a great challenge for biochemistry and also a

prerequisite for the real rational design of potent agonists

and antagonists.

Most conformational studies were performed on linear

agonists and antagonists, i.e. on peptides with high

conformational ¯exibility. In some analogs this ¯exibility

was reduced through the introduction of constrained

nonproteinogenic amino acids (17, 18) or by cyclization

between the N- and C-termini (19, 20). We attempted to

reduce the conformational ¯exibility by intramolecular

cyclization of the side chains. Owing to conformational

constraints, the solution conformation of a biologically

active cyclic peptide is expected to be close to the bioactive

conformation. For this reason we synthesized a series of BK

antagonists with lactam bridges in either the N- or C-

terminal part of the molecule. These bridges were built

between the side chains of Lys and Glu residues added to or

inserted into the sequence (21). A second series contained

`backbone cyclisized' (22) analogs with lactam bridges built

between peptide bonds modi®ed with aminoalkyl and

carboxyalkyl residues (23), respectively.

In spite of the intramolecular cyclization, many of the

analogs are still too ¯exible for conformational analysis by

NMR spectroscopy and exist in solution as an equilibrium of

many conformers. Few peptides ful®lled both requirements:

biological potency and suf®ciently reduced ¯exibility. In

this paper we report the results of our ®rst conformational

studies on cyclic BK analogs. We started with the analog

cycloo,6-[Lys0,Glu6,d-Phe7]BK (J324), a ®rst-generation

cyclic antagonist with a lactam bridge between the side

chains of a Lys residue that had been introduced as an N-

terminal extension and Glu residue that replaced the Ser

residue at position 6 in the native sequence. This antagonist

shows only modest antagonistic activity in the isolated rat

uterus assay (pA2 = 5.78 u 0.02). Nevertheless, its activity

is higher than that of the corresponding linear peptide (rat

uterus: no activity; guinea-pig ileum: pA2 = 4.90 u 0.32;

21).

Preliminary studies by means of energy minimization

calculations and MD simulations of J324, as well as of a

second antagonist that contains a disul®de bridge in the N-

terminal region, showed that a b-turn in this segment of the

peptide appears to be a de®ning structural feature, although

it is not the sole determinant of its antagonistic activity (21,

24).

Krause et al . Conformational properties of bradykinin B2 receptor agonist

64 | J. Peptide Res. 55, 2000 / 63±71

An additional approach towards the elucidation of the

essential structural requirements for BK antagonism

focused, not on the backbone conformation, but rather on

the spatial arrangement of functional groups and their

speci®c properties (charge, hydrophobicity, aromaticity,

etc.). This led to the development of three-dimensional

pharmacophore models with the help of expert systems such

as Catalyst (25), and consensus MD simulations. Based on

these models, several proprietary three-dimensional data-

bases were screened for new potential BK antagonists (26).

As a direct result of these studies, a novel class of nonpeptide

lead structures for BK B2-receptor antagonists has already

been suggested (Pineda et al., unpublished results).

Continuing our previous studies on the conformational

properties of the cyclic BK B2-receptor antagonists synthe-

sized in one of our laboratories (SR), we report here the

results of the investigation into the structure of J324 in

dimethylsulfoxide (DMSO) solution using NMR spectro-

scopy and both restrained and free MD simulations, always

assuming that there is only a single preferred conformation.

The low-energy structures sampled were analyzed using

novel plots, and the relationship between the resulting

backbone conformations, which are characteristic for BK

antagonism, and the speci®c spatial arrangement of the

pharmacophore centers is addressed.

Experimental Procedures

Samples for NMR spectroscopy were prepared by dissolving

the HPLC-puri®ed peptide in perdeuterated DMSO without

any further adjustment of the pH. The one- and two-

dimensional spectra were recorded on a Brucker AMX500

instrument at 300K. One-dimensional spectra were recorded

with 32 000 data points. Data for the TOCSY (27) or NOESY

(28) experiments were recorded with 4096 points in the

direct and 1024 points in the indirect dimension with four

scans. Time-proportional phase incrementation (TPPI) (29)

was used in the direct dimensions in both cases. Coupling

constants were extracted from a PE-COSY experiment (30)

with 4096 3 1024 data points. In all two-dimensional

spectra the sweep width in both dimensions was

4545.45 Hz. The different data sets were processed using

UXNMR xwin-nmr 1.3 on a Silicon Graphics Indy/4600

workstation.

The mixing time in the NOESY experiment was 200 ms,

and the volumes of the NOE cross-peaks were translated

into distances using a geminal proton pair as an internal

standard. In the TOCSY experiment an MLEV sequence was

used to transfer coherence. The mixing time was 62 ms,

leading to TOCSY as well as ROESY (31) peaks in the

resulting two-dimensional spectra. This combination of

through-space and through-bond transfer simpli®ed the

spin-system assignment as well as the sequential assign-

ment of the spectra.

All force ®eld calculations were performed on Silicon

Graphics (Crimson/R4000) and Evans & Sutherland (ESV3/

32) graphics workstations operating under IRIX 5.3 and ES/

os 2.3, respectively. The molecular modeling software sybyl

V6.2±6.3 was used throughout the study (32). Calculations

were performed using the sybyl implementation of the

amber all-atom force ®eld (33, 34) with default parameters.

Electrostatic interactions were included in all calculations

assuming a distance-dependent dielectric constant (e = r)

and completely ionized groups. This rather simpli®ed

dielectric model seems to be appropriate for a ®rst approach

and although it could lead to an overestimation of those

interactions in the absence of explicit solvent molecules, the

inclusion of a relatively large number of experimental

constraints into the calculations is expected to compensate

for this effect. The starting conformation was built and

minimized by consecutive steepest descent, conjugate

gradient (Powell) and quasi-Newton (BFGS) energy mini-

mization steps until a ®nal root mean square (RMS) gradient

not larger than 0.05 kcal/mol.AÊ was reached. A simulated

annealing (SA) MD protocol was used to sample the

conformational space accessible to the selected molecule

(NTV ensemble). One hundred and twenty SA cycles

consisting of a 5 ps high temperature interval at 900 K

followed by a 5 ps annealing interval in which the

temperature was decreased exponentially from 900 to

300 K were carried out during each simulation. An integra-

tion time step of 1 fs was used. Conformers were sampled at

the end of each cycle and stored in a database for further

minimization. The resulting 120 low-temperature confor-

mers were minimized following the same scheme as used for

the starting structure.

These calculations were performed both as free simula-

tions without any constraints and as restrained simulations

with NOE distance constraints. For the latter simulations

158 distance constraints were used. These distances

correspond to the unequivocally assigned NOE cross-peaks

of the main conformation of the peptide. Values of 50 and

1 kcal/mol.AÊ 2 were used for the force constant in the

additional distance range constraint energy terms.

Low-energy structures, i.e. those within an energy range of

20 kcal/mol above the lowest energy conformation, were

compared with each other and clustered into families prior


J. Peptide Res. 55, 2000 / 63±71 | 65

to analysis. The classi®cation into structure clusters was

based on the calculation of the RMS deviation (RMSD)

values for the best superposition of equivalent atomic

centers in two conformers. Custom-written sybyl program-

ming language (SPL) scripts allowed a graphical representa-

tion of RMSD values calculated for the set of sampled

conformers (clustergraph). The value of the RMSD threshold

(resolution level) was typically varied from 2.0 to 0.5 AÊ until

we arrived at a manageable number of families (between 10

and 20).

Using this approach, the 120 conformers were grouped

into structural families according to the position of peptide

backbone atoms. These families were subsequently ana-

lyzed to clarify the presence of secondary structural

elements such as b- (35) and c-turns (36, 37). In addition to

the analysis of representative structures derived from the

clustering, we also used a novel approach of plotting the

structural data of all 120 conformers to arrive at a more

complete analysis of such large conformational sets.

Furthermore, we examined the general shape of the back-

bone and tried to identify characteristic hydrogen bond

patterns that stabilize the conformation of the molecule.

Shifting the focus of our investigation towards the role of

the side chains, we then used the clustergraph representa-

tions described above to compare the three-dimensional

arrangement of ®ve speci®c pharmacophore centers, two

positive ionizable (basic) groups, two hydrophobic groups

and a ring-aromatic group centered on the side chains of

residues Arg1 and Arg9, Pro3 and Phe5 and d-Phe7,

respectively, (26) in the generated sets of low-energy

conformers.

Finally, the relationship between the spatial arrangement

of those speci®c pharmacophore centers and the established

characteristic backbone conformations was explored on the

basis of RMSD values and b-turn distribution patterns.

Results and Discussion

The free MD simulation generated 45 low-energy structures

(20 kcal/mol range). It should be noted, however, that the

results concerning the structure of the C-terminal part of the

molecule have to be examined with caution, since this

region is expected to be highly ¯exible due to the lack of

conformational restriction (cyclization). This region was

therefore excluded in the classi®cation of the structures.

The clustering based on the positions of 19 atoms in the

cyclic part of the peptide backbone yielded 12 families of

similar conformations using an RMSD threshold of 1.3 AÊ .

Examination of the b-turn types for the members of any

family shows that a similar position for the backbone atoms

does not necessarily lead to the same b-turn type distribu-

tion pattern. This clearly demonstrates that the pattern in

the conformer with the lowest potential energy of each

family (root conformer) is not always representative of all its

members, as has been implicitly assumed in numerous

conformational studies.

b-turns form in different tetrapeptide segments of the 45

low-energy structures (Fig. 1). They are frequently found

between residues Arg1 and Gly4, Pro2 and Phe5, and Glu6 and

Arg9. The formation of speci®c b-turn types in certain

segments seems to be favored. Examples are type VI turns

involving residues Arg1-Gly4 (in 21 of the 45 low-energy

conformations) and type I turns in the segment Pro2-Phe5 (in

seven conformations).

In order to avoid a more or less arbitrary clustering of

conformations into structural families, we introduced novel

graphical representations of the data to be analyzed that

made use of the procedure of Chou & Fasman (35). Plotting

the backbone angles allows a more complete analysis of

secondary structural elements in large conformational sets.

Figure 2 shows all b-turn candidates, characterized by an

upper limit of 7 AÊ for the distance between the Ca-atoms of

the residues i and i + 3 of the various tetrapeptide segments.

Elucidation of a type III b-turn between residues Pro2 and

Phe5 of conformer 70 (identi®ed as a candidate in Fig. 2)

using two different graphical representations of relevant

torsion intervals is illustrated in Figs 3 and 4, respectively.

30

25

20

15

10

5

0

Num

ber

of β

-tur

ns

6–95–84–73–62–51–40–3

Tetrapeptide segment

VII VI V' V IV III ' III II ' II I ' I

Figure 1. Number and types of b-turns found in the corresponding

tetrapeptide segments of 45 low-energy conformers generated by the

free MD simulation.


66 | J. Peptide Res. 55, 2000 / 63±71

All 45 low-energy structures show a compact, loop-shaped

backbone conformation with both termini in close proxi-

mity to each other, stabilized by numerous hydrogen bonds.

In many cases both of the Arg residues are involved in these

bonds. Hydrogen bonds within tripeptide segments as well

as c-turns are typically found in the C-terminal part of the

molecule. b-turns comprising residues Arg1-Gly4 and Glu6-

Arg9 are often stabilized by hydrogen bonds. We also found a

large number of hydrogen bonds in the pentapeptide

segments Lys0-Gly4 and Arg1-Phe5.

We have not been able to ®nd any consistent three-

dimensional arrangement of the ®ve pharmacophore centers

listed above among the 45 low-energy structures generated

using the unrestrained simulation. In view of the similarity

shown by the backbone structures this can only be due to

the high degree of ¯exibility of the side chains where these

centers are located.

In the SA calculations with restraints the distances

derived from the NOEs of the dominant conformation in

the NMR spectra were used. An initial restrained SA

simulation was carried out using a value of 50 kcal/

mol.AÊ 2 for the force constant of the additional distance

constraints energy terms. This led to an extremely high

Tet

rape

ptid

e se

gmen

t

6–9

5–8

4–7

3–6

2–5

1–4

0–3

0 20 40 60 80 100 120

Conformer number

Figure 2. b-turn candidates [d(Cai±Ca

i+3) # 7AÊ ] in the corresponding


free MD simulation. Conformer 70 is indicated by a larger triangle.

180

0

_180

_180 0 180

w3+w4

D(°)

y3+

y4D(°

)

Figure 3. Backbone torsion angles of two central residues of the

tetrapeptide segment Pro2-Phe5 (w3,4, y3,4) used for type assignment in

19 low-energy conformers (free MD simulation) that contain a b-turn

candidate in this segment. The gray circle represents 508 tolerance

intervals for an ideal type III turn. Conformer 70 is indicated by larger

symbols.

180

0

_180

180

0

_180

0 20 40 60 80 100 120

w3+

y3D(°

)w

4+y

4D(°

)

Conformer number

Figure 4. Backbone torsion angles of two central residues of the

tetrapeptide segment Pro2-Phe5 used for type assignment in 19 low-

energy conformers (free MD simulation) that contain a b-turn

candidate in this segment. The gray zones represent 508 tolerance

intervals for an ideal type III turn. Conformer 70 is indicated by larger

symbols.


J. Peptide Res. 55, 2000 / 63±71 | 67

contribution of the angle-bending term to the total energy.

Consequently, additional calculations with different values

for this force constant were performed, after which a value of

1 kcal/mol.AÊ 2 was chosen. Sixty-two low-energy structures

were then sampled by the MD simulation applying NOE

distance constraints. The average violation of these con-

straints in the lowest energy structure was 0.8 AÊ .

Since the backbone conformations of the generated low-

energy structures were very similar to each other, clustering

based on the position of all 30 backbone atoms was carried

out with an RMSD threshold of only 0.5 AÊ . Nevertheless,

analysis of the b-turn types found in different members of a

structure family shows that, even at this high resolution,

similar positions of backbone atoms do not necessarily

correspond to the presence of the same b-turn type

distribution pattern due to the discrete character of type

assignment.

Including NOE distance constraints from the main

structure in the simulation leads to a strong restriction of

the conformational space, as demonstrated by the results of

b-turn identi®cation and type assignment (Fig. 5). In the

low-energy structure ensemble we found a type VI b-turn (a

cis Pro2±Pro3 bond) comprising residues Arg1-Gly4, a type I

b-turn between residues Pro2 and Phe5 and different types of

b-turn in the segment Glu6-Arg9 in all, 94% and 69% of the

structures, respectively. A higher structural diversity with

respect to the presence of b-turns and their type is observed

in the C-terminal part of the backbone compared with the

N-terminal part. This is partially due to the cyclization of

the N-terminal region of the peptide, but is also a

consequence of the relatively small number of NOE distance

constraints found for residues located in the C-terminal

region. The difference in structural determination between

these two regions of the peptide is illustrated in Fig. 6,

which displays the best superposition of the corresponding

backbone atoms of the conformers containing a b-turn

between residues Pro2-Phe5 and Glu6-Arg9, respectively.

This illustration also shows that the N-terminal turn, rather

than the C-terminal one, should be considered a de®ning

structural feature of J324.

Compared with investigations on the bioactive conforma-

tion of antagonists published in the last decade, the results

of the present study, as stated above, support the importance

of a b-turn in the N-terminal (2±5) segment as a key

structural feature for the BK antagonism. This is also a

con®rmation of the results of our earlier investigations.

However, these results agree only partially with those of

(A)

(B)

Figure 6. Best superposition of low-energy conformers (restrained MD

simulation) that contain a b-turn involving the residues (A) Pro2-Phe5

and (B) Glu6-Lys9, respectively, based on the corresponding backbone

atoms. For greater clarity only the peptide backbones are shown.

70

60

50

40

30

20

10

0

Num

ber

of β

-tur

ns

6–95–84–73–62–51–40–3

Tetrapeptide segment

VII VI V' V IV III ' III II ' II I ' I

Figure 5. Number and types of b-turns found in the corresponding


MD simulation including NOE distance constraints.


68 | J. Peptide Res. 55, 2000 / 63±71

other previous investigations. Although some of these

studies also found different types of b-turn comprising

residues Pro2-Phe5, most of them proposed a (frequently type

II') b-turn in segment Glu6-Arg9 to be the dominant

structural motif of peptide BK antagonists (7, 38). In contrast

to this conclusion Liu et al. (8) postulate a strained

conformation in the C-terminal part and a b-turn for the

segment 2±5 for an antagonist with cyclopentyl glycine at

position 7. Other authors (13, 14), however, assume turns in

both the C- and in the N-terminal part of the sequence.

Among our newly synthesized series of antagonists with

cyclization in the C-terminal part of the molecule only one

analog with a lactam bridge between the modi®ed peptide

bond Phe8-Arg9 and a Glu residue at position 6 is biologically

active and even more potent than the corresponding linear

analog (39). This ®nding agrees with the publications

mentioned above and indicates that both the N- and the

C-terminal parts of the sequence are of comparable

importance for the antagonistic activity.

The backbone of the 62 low-energy structures of J324

generated by restrained MD also shows a compact loop

conformation stabilized by a characteristic hydrogen bond

pattern (Fig. 7). Hydrogen bonds between Arg1 and Arg9

stabilize this loop conformation in every structure of the set.

Hydrogen bonds within tripeptide segments and c-turns are

found mainly in the C-terminal part of the molecule. More

than 90% of all b-turns involving residues Arg1-Gly4 and

more than 60% in the segment Glu6-Arg9 are stabilized by

hydrogen bonds. Most structures also show hydrogen bonds

within the pentapeptide segments Lys0-Gly4 and Arg1-Phe5.

Cluster analysis of the 62 structures based on the ®ve

pharmacophore centers listed above, using a RMSD thresh-

old of 1.5 AÊ , resulted in 31 conformational families with

similar spatial arrangements of these centers. We examined

the members of these families in order to identify

characteristic backbone conformations on the basis of

RMSD values and b-turn distribution patterns. No relation,

however, could be established between the spatial arrange-

ment of pharmacopore centers and the backbone conforma-

tion of structures within the same family. Frequently the

backbones of structures belonging to different families

showed a greater degree of similarity than those of members

belonging to the same family.

Finally, six representative consensus conformations of

J324 from a previous study (26) were compared with the low-

energy conformations obtained from free and restrained

simulations with respect to the position of the ®ve

pharmacophore centers. However, no similar three-dimen-

sional arrangements of these centers were apparent at an

RMSD up to 1.5 AÊ (Fig. 8).

Conclusions

The conformation of the peptide B2 receptor antagonist

cyclo0,6-[Lys0,Glu6,d-Phe7]BK was examined using a free

MD simulation, as well as a restrained MD simulation with

158 NOE distance constraints. The representative, low-

energy conformations generated by both simulations show

similarities concerning the frequency and distribution

pattern of secondary structure elements in the peptide

Con

form

er n

umbe

r

160

140

120

100

80

60

40

20

0

0 20 40 60 80 100 120 140 160

Conformer number

Figure 8. Comparison of low-energy conformers obtained by

consensus (structure nos 1±6), free (structure nos 7±51) and restrained

MD simulations using a distance range constraint force constant of

50 kcal/mol.AÊ 2 (structure nos 52±103) and 1 kcal/mol.AÊ 2 (structure

nos 104±165), respectively, based on the position of ®ve

pharmacophore centers. The dots in the clustergraph correspond to

pairs of conformers, whose best superposition yields RMSD values not

larger than 1.5 AÊ .

Figure 7. Overall backbone shape (shaded tube) and hydrogen bond

pattern (dashed lines) of the lowest energy conformation obtained

from the restrained MD simulation.


J. Peptide Res. 55, 2000 / 63±71 | 69

backbone, which are mostly b-turns of various types. The

topology of the backbone is dominated by a compact loop

conformation stabilized by a characteristic hydrogen bond

pattern.

Structures containing a type VI b-turn between residues

Arg1 and Gly4 (with a cis Pro2±Pro3 bond), a type I b-turn

between Pro2 and Phe5, and/or b-turns of different type in

the segment Glu6-Arg9 occur very frequently among the

low-energy conformational states of the studied cyclic BK

analog. The inclusion of distance constraints into the

simulations leads to a substantial restriction of the

conformational ¯exibility of the structure, especially in

the N-terminal region, as could be expected. Thus, the

present study supports the importance of a b-turn in the N-

terminal (2±5) segment as one of the key structural features

for the BK antagonism, as proposed by our earlier investiga-

tions.

In contrast, characteristic spatial arrangements of phar-

macophore centers, which one would expect to be of crucial

importance for receptor binding af®nity, do not seem to be

correlated to, or induced by, a speci®c backbone conforma-

tion. It is necessary to bear in mind, however, that while

more sophisticated sampling techniques, such as consensus

MD, might be able to throw light on these questions, the

relatively straightforward conformational search procedure

used in this investigation fails to cover the regions of the

conformational space related to side-chain degrees of free-

dom, even if experimental data are taken into account.

Acknowledgments: We wish to thank Prof. Horst Kessler,

Technische UniversitaÈ t MuÈ nchen, for critical reading of the

manuscript and for his valuable suggestions. We are indebted to

Dr JuÈ rgen SuÈ hnel who kindly allowed us to use the computa-

tional facilities at IMB Jena. This work was performed with the

®nancial support of the Deutsche Forschungsgemeinschaft (Re-

853/2-1).

References

1. Stewart, J.M. (1995) Bradykinin B2 receptor

antagonists: development and applications.

Can. J. Physiol. Pharmacol. 73, 787±790.

2. Kotovych, G., Cann, J.R., Stewart, J.M. &

Yamanaoto, H. (1998) NMR and CD

conformational studies of bradykinin and its

agonists and antagonists: application to

receptor binding. Biochem. Cell Biol. 76, 257±

266.

3. Bodanszky, A., Bodanszky, M., Jorpes, E.J.,

Mutt, V. & Ondetti, M.A. (1970) Molecular

architecture of peptide hormones. Optical

rotatory dispersion of cholecystokinin-

pancreozymin, bradykinin and 6-glycine

bradykinin. Experientia 26, 948±950.

4. Brady, A.H., Ryan, J.W. & Stewart, J.M. (1971)

Circular dichroism of bradykinin and related

peptides. Biochem. J. 121, 179±184.

5. Cann, J.R., Stewart, J.M. & Matsueda, G.R.

(1973) A circular dichroism study of the

secondary structure of bradykinin.

Biochemistry 12, 3780±3788.

6. Ivanov, V.T., Filatova, M.P., Reissmann, S.

et al. (1975) The solution conformation of

bradykinin. In Peptides: Chemistry,

Structure, and Biology (Walter, R. &

Meienhofer, J., eds). Ann Arbor Science

Publishers, Ann Arbor, MI, pp. 151±157.

7. Sejbal, J., Cann, J.R., Stewart, J.M., Gera, L. &

Kotovych, G. (1996) An NMR, CD, molecular

dynamics and ¯uorometric study of the

conformation of the bradykinin antagonist B-

9340 in water and in aqueous micellar

solutions. J. Med. Chem. 39, 1281±1292.

8. Liu, X., Stewart, J.M., Gera, L. & Kotovych, G.

(1993) Proton magnetic resonance studies of

bradykinin antagonists. Biopolymers 33,

1237±1244.

9. Kyle, D.J., Blake, P.R., Smithwick, D. et al.

(1993) NMR and computational evidence that

high-af®nity bradykinin receptor antagonists

adopt C-terminal b-turns. J. Med. Chem. 36,

1450±1460.

10. Young, J.K. & Hicks, R.P. (1994) NMR and

molecular modeling investigations of the

neuropeptide bradykinin in three different

solvent systems: DMSO, 9 : 1 dioxane/water,

and in the presence of 7.4 mm lyso

phosphatidylcholine micelles. Biopolymers

34, 611±623.

11. Lee, S.C., Russel, A.F. & Laidig, W.D. (1990)

Three-dimensional structure of bradykinin in

SDS micelles. Study using nuclear magnetic

resonance, distance geometry, and restrained

molecular mechanics and dynamics. Int. J.

Peptide Protein Res. 35, 367±377.

12. Sawutz, D.G., Salvino, J.M., Seoane, P.R.

et al. (1994) Synthesis, characterization, and

conformational analysis of the d/l-Tic7

stereoisomers of the bradykinin receptor

antagonist d-Arg0[Hyp3,Thi5,d-

Tic7,Oic8]bradykinin. Biochemistry 33,

2373±2379.

13. Guba, W., Haessner, R., Breipohl, G. et al.

(1994) Combined approach of NMR and

molecular dynamics within a biphasic

membrane mimetic: conformation and

orientation of the bradykinin antagonist Hoe

140. J. Am. Chem. Soc. 116, 7532±7540.

14. Chakravarty, S., Wilkins, D. & Kyle, D.J.

(1993) Design of potent, cyclic peptide

bradykinin receptor antagonists from

conformationally constrained linear peptides.

J. Med. Chem. 36, 2569±2571.

15. Ottleben, H., Haasemann, M.,

Ramachandran, R., MuÈ ller-Esterl, W. &

Brown, L.R. (1997) NMR investigations of

recombinant 15N/13C/2H-labeled bradykinin

bound to a Fab mimic of the B2 receptor.

Recept. Channels 5, 237±241.

16. Kyle, D.J., Chakravarty, S., Sinsko, J.A. &

Stormann, T.M. (1994) A proposed model of

bradykinin bound to the rat B2 receptor and

its utility for drug design. J. Med. Chem. 37,

1347±1354.

17. Kyle, D.J., Green, L.M., Blake, P.R.,

Smithwick, D. & Summers, M.F. (1992) A

novel b-turn mimic useful for mapping the

unknown topology of peptide receptors.

Peptide Res. 5, 206±209.

18. Welsh, J.H., Zerbe, O., Von Philipsborn, W. &

Robinson, J.A. (1992) b-turns induced in

bradykinin by (S)-a-methylproline. FEBS Lett.

297, 216±220.

19. Saulitis, Yu B., Liepinsh, E.E., Mutulis, F.K. &

Chipens, G.I. (1986) Interpretation of1H NMR spectra of the cyclic analog of

kallidin in solutions. Bioorg. Khim. 12, 1317±

1328.

20. Krstenansky, J.L., Ho, T., Tahilramani, R.

et al. (1994) Cyclic hexapeptide antagonists

of the bradykinin B2 receptor: receptor

binding and solution backbone conformation.

Lett. Peptide Sci. 1, 229±234.


70 | J. Peptide Res. 55, 2000 / 63±71

21. Seyfarth, L., Pineda, L.F., Liepina, I.,

Paegelow, I., Liebmann, C. & Reissmann, S.

(1995) New cyclic bradykinin antagonists

containing disul®de and lactam bridges at the

N-terminal sequence. Int. J. Peptide Protein

Res. 46, 155±165.

22. Gilon, C., Halle, D., Chorev, M., Selinger, Z.

& Byk, G. (1991) Backbone cyclization: a new

method for conferring conformational

constraint on peptides. Biopolymers 31, 745±

750.

23. Reissmann, S., Greiner, G., Jezek, J. et al.

(1994/95) Design, synthesis and

characterization of bradykinin antagonists

via cyclization of the modi®ed backbone.

Biomed. Peptides, Proteins and Nucleic

Acids 1, 51±56.

24. Abdelrahim, F., Pineda, L.F., Seyfarth, L.,

Reissmann, S. & Paegelow, I. (1995)

Conformational properties of new cyclic

bradykinin antagonists. In Peptides 1994.

Proceedings 23rd European Peptide

Symposium, Sept. 4±10, 1994, Braga,

Portugal (Maia, H.L.S., ed.). ESCOM, Leiden,

The Netherlands, pp. 527±528.

25. CATALYST. Molecular Simulations Inc., San

Diego, CA.

26. Pineda, L.F. (1999) Formulation of 3D-

pharmacophore models for bradykinin B2-

receptor antagonists. Z. Phys. Chem.- Int. J.

Res. Phys. Chem. Chem. Phys. 209, 111±131.

27. Braunschweiler, L. & Ernst, R.R. (1983)

Coherence transfer by isotropic mixing:

application to proton correlation

spectroscopy. J. Magn. Reson. 53, 521±528.

28. Jeener, J., Meier, B.H., Bachmann, P. & Ernst,

R.R. (1979) Investigation of exchange

processes by two-dimensional NMR

spectroscopy. J. Chem. Phys. 71, 4546±4553.

29. Marion, D. & WuÈ thrich, K. (1983)

Application of phase sensitive two-

dimensional correlated spectroscopy (COSY)

for measurements of 1H±1H spin±spin

coupling constants in proteins. Biochem.

Biophys. Res. Commun. 113, 967±974.

30. MuÈ ller, L. (1987) P.E.COSY, a simple

alternative to E.COSY. J. Magn. Reson. 72,

191±196.

31. Bothner-By, A.A., Stevens, R.L., Lee, J.,

Warren, C.D. & Jeanloz, R.W. (1984)

Structure determination of a tetrasaccharide:

transient nuclear Overhauser effects in the

rotating frame. J. Am. Chem. Soc. 106, 811±

813.

32. SYBYL & Molecular Modelling Software.

Tripos Ass. Inc., St. Louis, MO.

33. Weiner, S.J., Kollman, P.A., Case, D.A. et al.

(1984) A new force ®eld for molecular

mechanical simulation of nucleic acids and

proteins. J. Am. Chem. Soc. 106, 765±784.

34. Weiner, S.J., Kollman, P.A., Nguyen, D.T. &

Case, D.A. (1986) An all atom force ®eld for

simulations of proteins and nucleic acids. J.

Comp. Chem. 7, 230±252.

35. Chou, P.Y. & Fasman, G.D. (1977) Beta-turns

in proteins. J. Mol. Biol. 115, 135±175.

36. Milner-White, E.J., Ross, B.M., Ismail, R.,

Belhadj-Mostefa, K. & Poet, R. (1988) One

type of gamma-turn, rather than the other

gives rise to chain-reversal in proteins. J. Mol.

Biol. 204, 777±782.

37. Smith, J.A. & Pease, L.G. (1980) Reverse turns

in peptides and proteins. CRC Crit. Rev.

Biochem. 8, 315±399.

38. Filizola, M., Centeno, N.B., CartenõÂ-Farina,

M. & Perez, J.J. (1998) Conformational

analysis of the highly potent bradykinin

antagonist Hoe-140 by means of two different

computational methods. J. Biomol. Struct.

Dynam. 15, 639±652 [and references therein].

39. Schumann, Ch., Seyfarth, L., Greiner, G.,

Paegelow, I. & Reissmann, S. (1999) Synthesis

of new building units with arginine and their

incorporation into backbone cyclized

bradykinin analogues. In Peptides

Proceedings 25th European Peptide

Symposium, Aug. 30±Sept. 4, 1998, Budapest,

Hungary, in press.


J. Peptide Res. 55, 2000 / 63±71 | 71

Download - Conformational properties of a cyclic peptide bradykinin B2 receptor antagonist using experimental and theoretical methods

Top Related