Conformational Analysis of Bradykinin by Annealed

Molecular Dynamics and Comparison to NMR-

Derived Conformations

Joseph M. Salvino,* Peter R. Seoane, and Roland E. Dolle

Medicinal Chemistry Department, Pharmaceutical Research Division, Sterling Winthrop, Inc., Malvern,
Pennsylvania 19355

Received 5 March 1992; accepted 10 September 1992

Conformational analysis of bradykinin (BK), a nonapeptide of the sequence RPPGFSPFR, was accomplished
using annealed molecular dynamics (AMD) at 1000 K in BIOGRAF 2.2. One hundred anneal cycles produced
100 conformations over approximately 2000 ps. These conformations were compared to structures derived
by nuclear magnetic resonance (NMR) methods for similar shape and energy. Energy minimization of relevant
conformations using both BIOGRAF 2.2 and AMBER 3.0a revealed that the AMD-determined conformations
are in the same energy range as the NMR-determined structures. Also, the shape of the relevant conformations
appeared similar, suggesting that AMD is a good tool for the conformational analysis of small peptide ligands.
0 1993 by John Wiley 8. Sons, Inc.

INTRODUCTION the nondegenerative purification of membrane re-

The nonapeptide bradykinin (BK), Arg-Pro-Pro-Gly-
Phe-Ser-Pro-Phe-Arg, is a potent pain-producing
agent and has been the subject of intensive biological
investigation for over 30 years.'-7 BK is released
upon tissue iqjury or trauma via the rapid enzymatic
cleavage of kininogens by the proteolytic processing
of the high-molecular-weight kallikreins?JOThe ac-
tion of BK proceeds through G-protein-mediated
mechanisms that activate phospholipase C and leads
to increased synthesis of inositol triphosphate
(IP3).11-17
To gain insight into a possible bioactive confor-
mation, the solution conformations of BK, BK frag-
ments, and several related peptide analogs have been
studied extensively by several spectroscopic tech-
niques. These techniques include circular dichroism
(CD),'s-20 13C and 'H nuclear magnetic resonance
(NMR),19-26and laser Raman spectros~opy.'~ The
general conclusion from these studies is that BK
possesses a large degree of conformational flexibil-
ity and does not adopt a single preferred stable con-
formation in solutio11?~2~
To better correlate the active conformations of
polypeptide hormones with their biological func-
tions, it is essential to characterize the structures of
these hormones in their respective hormone-recep-
tor complexes. BK binds to a G-protein-coupledre-
ceptor of the family of receptors containing seven
transmembrane-spanning a-helices. Unfortunately,
'Author to whom all correspondence should be addressed.
ceptors and their crystallization still remain a prob-
lem. In the absence of suitable receptors, lipid sys-
tems have been used to mimic the amphiphilic
membrane-embedded receptor environment. In
many cases, physical studies of the conformations
of peptides bound to vesicles and micelles have pro-
vided valuable insight into the molecular basis of
peptide activities. These studies include enkephalin
in sodium dodecyl sulfate (SDS) and lysophospha-
tidylcholine (WC) m i c e l l e ~ and
~ ~ -gramicidin
~~ A in
SDS micelles.3° BK has been examined by NMR in
SDS micelles using 2-D experiments at 500 MHzF6
More recently, BK and a related BK receptor antag-
onist were examined by NMR at 500 MHz in 9: 1
dioxane-d6/H20solvent mixture as a mimic of the
amphiphilic membrane-embedded BK receptor en-
vironment.3l
In the present study, a conformational analysis of
BK was undertaken by using annealed molecular dy-
namics (AMD) in the absence of solvent. The re-
sulting conformations were then compared to struc-
tures generated using distance geometry methods
from NMR data of BK in SDS micelles.* The con-
formations generated from both AMD methods and
NMR data were energy minimized in AMBER 3.0a
and BIOGRAF 2.2 to evaluate the energies of these
structures and then superimposed and analyzed for
rms fit.
'These coordinates were kindly supplied by S.C. Lee of the
Proctor and Gamble Co., Miami Valley Laboratories (Cincinatti,
OH) (see ref. 26).

Journal of Computational Chemistry, Vol. 14, No. 4, 438-444 (1993)

Q 1993 by John Wiley & Sons, Inc. CCC 0192-8651/93/040438-07
ANALYSIS OF BRADYKININ 439

AMD as a conformational analysis tool is becom- Table I. MIN1BK.macro.

ing more available and widely used?’>%The advan- beginmacro
tages of using this type of computational tool are In-Out
rapid analysis time, leading to faster design of new In-Out/read
ligands, and a rank order of the conformations gen- BioDesign
“minbk.bgf’
erated by energy. In contrast to distance geometry % minbk.bgf
methods, which force a molecule to fit NMR con- In-Out/ return
straints,32 this method allows the molecule under Energy
study to freely sample conformational space. Cali- Energy/setup ee
bration of the AMD against the NMR structures is bk
then used to extract relevant conformations. bk

bk
METHODS
Energy/constrain
AMD was used as a tool for conformational analysis Cnstrain/prot cnst
Prot cst/all 1
of BK, a nonapeptide of the sequence RPPGFSPFR. Prot cst/return
The BK structure was built in BIOGRAF 2.2 from a Cnstrain/return
library of standard amino acid residues. This linear Energy/dynamics
starting conformation was energy minimized to an Dynamics/annealed
rms gradient less than 0.1, constraining the C a asym- Annealed/temp var
Temp var/init tem
metrical centers to remain in the L configuration. “1000”
This resulted in a linear starting conformation with Temp var/final tern
good bond length and bond angle geometries. The “0”
exact protocol of the dynamics run is contained in Temp var/return
the macrofile MIN1BK.macro listed in Table I. Annealed/anneal cy
“100”
The dynamics were run in the absence of water Annealedlwrite trj
solvent and a dielectric constant of 1,4, and 25 was Annealed/execute
used. A dielectric constant of 4 is in general regarded “MIN1BK.trj”
as the bulk dielect.ric of a membrane; variation up “minbk.bgf starting structure”
to E = 25 may reflect an effective dielectric around “minimized with ALL L constraint”
“ 1,

the charges as polar groups attempt to solvate them. Annealedheturn

An anneal temperature range of 1000-0 K was
needed to adequately sample conformational space.
This was determined by experimenting with various
annealing temperatures and comparing the resulting
structures with the starting linear conformation. For
example, 600 K did not move the structure away from
the linear conformation adequately and 1500 K put
too much energy into the molecule, resulting in
an extremely distorted structure. The asymmetric
centers ( C a ) must be constrained to avoid racemi-
zation to D-amino acids while annealing the mole-
cule. The starting conformation is in effect heated
to 100 K over about 5 ps and then cooled to 0 K over
approximately 5 ps, resulting in the first conforma-
tion. This structure is then used as the starting point
for another annealing cycle. This process is repeated
altogether 100 times. Each time, the structure at the
end of the annealing cycle is saved, resulting in a
total of 100 conformations. The one-hundredth con-
formation is the result of about 2000 ps of dynamics.
We and others33 observed that within about 1000-
2000 ps of dynamics small peptide systems sample
a diverse range of conformational space.
In this analysis, the use of an all-atom or united-
atom model made little difference qualitatively.How-
ever, the united-atom model has the advantage of
shorter computational time, and because the results
Dynamics/return
Energylreturn
Exit
Yes
%
endmacro
of the AMD study wdl be used as a qualitative tool
this method was preferred. Atomic partial charges
were generated in BIOGRAF 2.2 using the charge
equilibration procedure ( q e q ~ i l ) ?The
~ overall
+
charge on the starting BK structure was 2. For the
initial study, a dielectric constant of 1 was used to
keep the simulation protocol simple. For complete-
ness, AMD studies were also carried out using a
dielectric constant of 4 and 25.* The initial study was
undertaken using a dielectric constant of 1 to keep
the simulation protocol simple. The dielectric con-
stant of 4 is in general accepted as the bulk dielectric
of a membrane. The dielectric constant of 25 was
thought to simulate an effective dielectric around
the charges as polar groups solvate them. A complete
analysis was performed on the trajectory file gen-
erated where E = 1. The two additional trajectory
*The AMD studies at dielectric constants of 4 and 25 were
conducted at the request of the reviewers.
440
b t.
CONF 21
BKa and CONF 21
Ca backbone Rh4S = 2.703 A
Q Q
BKa
Figure 1. Lowest-energy conformation from NMR data,
BKa, compared to the lowest-energystructure from AMD
method, CONF 21.
files ( E = 4 and 25) were then analyzed by using the
low-energy backbone conformations from both AMD
and NMR structures, CONF 21, BKa, and BKb.
Analysis of the trajectory file where E = 1 gave a
list of the conformations within 5 kcal of the lowest-
found energy gave 10 conformers. Each of these 10
conformers were energy minimized to a gradient
rms < 0.1 without constraints in BIOGRAF 2.2.%
This procedure was applied to choose the lowest-
energy structure from the set of similar low-energy
conformations. The conformer CONF 21 (shown in
Figures 1 and 2) was found to be the lowest-energy
structure from this dynamics run.
RESULTS
Recently published NMR structures, generated by
using a set of interproton distances from the quan-
titative analysis of nuclear Overhauser enhance-
ments, which were then used in distance geometry
and restrained molecular dynamics calculations to
yield a set of structures that were refined using re-
SALVINO, SEOANE, AND D O L E
& b
C O W 21
BKbandCONF21
C a backbone RMS = 2.177 A
d d
BW
Figure 2. Second-lowest-energy conformation from
NMR data, BKb, compared to the lowest-energy structure
from AMD method, CONF 21.
strained energy minimization (see the footnote on p.
439), were used to calibrate the AMD procedure. To
use these NMR structures in a meaningful way, rel-
ative energy values were needed to determine which
structures were the lowest-energy conformers. It is
reasonable to assume that the BK receptor will rec-
ognize a low-energy conformation as a preferred
binding conformation. Therefore, the NMR struc-
tures, referred to as BKa-BKg, were energy mini-
mized using BIOGRAF 2.2 and AMBER 3.0a?6 The
energy values are listed in Table 11. The relative order
of the energy of the conformations is more important
than the absolute value given for each conformer
because the absolute value will change from pro-
gram to program.
Although the rank order of the conformations was
not exactly the same in BIOGRAF 2.2 and AMBER,
they did find the identical lowest- and highest-energy
conformations. The differences in the energy from
the AMBER minimizations were striking. It singled
out two low-energy conformations BKa and BKb,
which look significantly different from the rest of
the structures (see Figs. 1 and 2). These structures,
ANALYSIS OF BRADYKININ 441

Table 11. Total energies of NMR-derived BK structures calculated in AMBER 3.0a and BIOGRAF 2.2.
BKa BKb BKc BKd BKe BKf BKg
AMBER - 145.6 - 138.2 - 88.0 - 63.9 - 62.4 - 48.0 - 25.1
Biograf 117.4 138.7 147.6 161.3 148.3 143.0 168.2
The total energies are calculated at E = 1.

as do the other higher-energy NMR-derived struc-
tures (BKc-BKg), adopt a p-turn-like structure in the
carboxy terminal region, S6P7F8R9.This turn has
been a feature common to conformational features
proposed for BK in other studies and in some cases
is suggested as the recognition element toward its
re~eptor.~~~”~~”~
The minimized NMR structures, BKa-BKg, were
used individually as amide backbone templates and
matched against the 100 structures in the AMD tra-
jectory file. Only the C a atoms were used to obtain
the rms values. The overlays of the NMR BK struc-
tures with the best matches from the AMD trajectory
file ( E = 1) are shown in Figures 3 and 4.Figure 5
shows the results of matching the amide backbones
of the lowest-energy NMR BK structures, BKa and
BKb, with their best match from the AMD trajectory
files where the dielectric constant was varied at
E = 4 and 25. Figure 6 shows the overlay of the
amide backbones of CONF 21, the lowest-energy
AMD structure from the trajectory file where E = 1,
with its best match from the trajectory files run at
E = 4 and 25.
By examination of the overlays in Figures 1-6, it
is evident that in this instance the AMD procedure
produced conformations that bear a striking resem-
blance to the structures known from the experi-
mental data. Variation of the dielectric constant us-

%% BKd and CONF 40

Ca backbone RMS = 2.049A

BKa and CONF 41

Ca backbone RMS = 2.216A

BKe and CONF 33

Ca backbone RMS = 3.001 A

BKf and CONF 1

Ca backbone RMS = 3.075A
HKb and CONF 36
Ca backbone RMS = 1.140A

E3Kc and CONF 40 BKg and CONF 1

Ca backbone RMS = 2.049A Ca backbone RMS = 3.463A
Figure 3. Superimpositions of the amide backbones of Figure 4. Superimpositions of the amide backbones of
BKa-BKc (NMR derived) and CONF structures (AMD de- BKd-BKg (NMR derived) and CONF structures (AMD de-
rived) after energy minimization. rived) after energy minimization.
442 SALVINO, SEOANE, AND DOLLE

BKa with C O W 15 from AMD trajectory file run at &=4

Ca backbone RMS = 2.17A
CONF 2 1 with CONF 2 from AMD trajectory file run at e=4
Ca backbone RMS = 1.35 A

BKa with CONF 15 from AMD trajectory ffie run at &=25

Ca backbone RMS = 2.16A

CONF 2 1 with CONF 39 kom AMD trajectory file r u n a t &=25

BKb with CONF 28 from- trajectory 81e run at &=4
Ca backbone RMS = 2.28 A
Ca backbone RMS = 0.998 A
Figure 6. Stereoviews of amide backbone overlays of
AMD structure CONF 21 from AMD trajectory file run at
E = 1 with its best match from AMD trajectory files run
at E = 4 and 25.

can reveal gross structural similarities or differ-

BW with CONF 82 from AMD trajectory file run at &=25
Ca backbone RMS = 2.24 A
Figure 5. Stereoviews of amide backbone overlays of
NMR-derived BKa and BKb with their best match from
AMD trajectory files run at E = 4 and 25.
ing the values E = 1, 4, and 25 made a small
difference in the conformations,mainly in the direc-
tion in which the C-terminal carboxylate group
pointed. The overall shapes of the conformations
remained qualitatively similar. For example, a large
percentage of the conformations in the trajectory
fiies had a @turn-like feature near the C-terminal
end of the molecule. This method is useful for ligand
design on flexible short linear peptides because it
ences.
The AMD conformers were energy minimized in
AMBER and BIOGRAF 2.2 to compare these con-
formers energetically with the BK NMR structures.
The total energies are given in Table 111. In addition,
CONF 21, the lowest-energy conformer from the
AMD trajectory files,was minimized in BIOGRAF 2.2
at various dielectric constants, E = 1,4, and 25, and
the total energy as well as a partition of the total
energy into its components is given in Table IV.It is
interesting to point out that of the various internal
and nonbonded energy components the electrostatic
term was the only one to vary significantly.However,
the van der Waals and H-bonded components of the
total energy have the greatest absolute value.
The lowest-energy conformation from the AMD
procedure, CONF 21, looked remarkably like the two

Table 111. Total energies of representative conformations from AMD trajectory files run at dielectric constants of E =
1, 4,and 25 calculated in AMBER 3.0a and BIOGRAF 2.2.
CONF ( E = 1) 21 40 36 41 33 1
AMBER - 158.0 - 129.9 - 126.0 - 125.4 - 122.7 - 99.6
Biograf 62.5 80.6 79.3 94.2 91.82 114.7
CONF ( E = 4) 15 28 2
Biograf 89.8 107.74 90.62
CONF ( E = 25) 15 82 39
Bioaaf' 94.92 77.91 97.24
The total energies are all calculated at E = 1.
ANALYSIS OF BRADYKININ 443

Table IV. CONF 21 minimized at E = 1,4, and 25. experiments suggest that the protocol in Table I is
Enerm decomDosition in general applicable to other small peptide systems.
Internal Nonbonded
= 1 (total energy = 62.59)
The authors thank Drs. Adi Treasurywala, John Wen-
Bonds 19.6071 van der Waals 85.0687 doloski, and Edward Jaeger for stimulating discussions
Angles 31.4631 Electrostatic - 35.5282 and technical assistance. The coordinates of all the AMD
Torsions 33.9587 H bond - 74.6547 structures are available in PDB format from the authors.
Inversions 2.6804
= 4 (total energy = 88.1642)
Bonds 19.4421 van der Waals 82.6585 References
Angles 31.0179 Electrostatic - 8.2556
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