COMMUNICATION
Solution Structure of an Insect Growth Factor, Growth-blocking
Peptide*
Tomoyasu
Aizawa
,
Naoki
Fujitani
,
Yoichi
Hayakawa§,
Atsushi
Ohnishi§,
Tadayasu
Ohkubo¶,
Yasuhiro
Kumaki
,
Keiichi
Kawano
**,
Kunio
Hikichi
, and
Katsutoshi
Nitta
From the
Division of Biological Sciences, Graduate
School of Science, Hokkaido University, Sapporo 060-0810, Japan, the
§ Institute of Low Temperature Science, Hokkaido University,
Sapporo 060-0819, Japan, the ¶ Japan Advanced Institute of Science
and Technology, Ishikawa 923-1292, Japan, and the
Faculty of
Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University,
Toyama 930-0194, Japan
 |
ABSTRACT |
Growth-blocking peptide (GBP) is an insect growth
factor consisting of 25 amino acid residues that retards the
development of lepidopteran larvae at high concentration while it
stimulates larval growth at low concentration. In this study, we
determined the solution structure of GBP by two-dimensional
1H NMR spectroscopy. The structure contains a short
segment of double-stranded
-sheet involving residues 11-13 and
19-21 and a type-II
-turn in the loop region (residues 8-11),
whereas the N and C termini are disordered. This is the first report of
the three-dimensional structure of the peptiderigic insect growth factor, and the structure of the well defined region of GBP was found
to share similarity with that of the C-terminal domain of the epidermal
growth factor (EGF). Because GBP has been reported to stimulate DNA
synthesis of not only insect cells but also human keratinocyte cells at
the same level with EGF, the structural similarity between GBP and EGF
may lead to the interaction of GBP to EGF receptor.
 |
INTRODUCTION |
Endoparasitic wasps can alter the development of their hosts from
larvae to pupae. This developmental disturbance allows the wasps to
complete their larval growth and to emerge while the host is still in
the larval stage, otherwise the wasp larvae would be trapped in the
sclerotized pupal cuticles. Growth-blocking peptide (GBP)1
was originally isolated from the larval
hemolymph of the host armyworm
Pseudaletia separata whose development is halted in the last
larval instar stage from parasitization by the parasitoid wasp
Cotesia kariyai (1-3). The fact that injection of 20 pmol of GBP into early last instar larvae of the armyworm retards larval growth and causes more than a few days delay in pupation clearly shows
its growth blocking activity (2, 3). We have since determined that GBP
is a host gene product and that the change of titers of this peptide
correlate well with the growth rate of insect larvae (4, 5). Recently,
injection studies of GBP indicated that low concentrations (lower than
1 pmol/larva) stimulated larval growth, whereas high concentrations
(higher than 10 pmol/larva) retarded growth (6). Furthermore, bioassay data revealed that several pmol/ml of GBP stimulated DNA synthesis of
SF-9 insect cells and human keratinocyte cells, although several nmol/ml of GBP did not stimulate cell proliferation at all. Therefore, it is thought that GBP can act as a growth factor controlling the
proliferation of a broad range of cells and the growth rate of whole larvae.
To examine structural features of GBP, NMR was used in this study. The
solution structure that we report here was obtained by two-dimensional
1H NMR spectroscopy with the aid of distance geometry and
simulated annealing. The three-dimensional structure of GBP contains a
short double-stranded
-sheet and a
-turn in the C-terminal
region, while six residues in the N terminus and three residues in the C terminus are highly disordered.
 |
EXPERIMENTAL PROCEDURES |
1H NMR Spectroscopy--
The 25-residue peptide GBP
was prepared by the Peptide Institute (Osaka, Japan) using an Applied
Biosystem Model 430A peptide synthesizer with
tert-butyloxycarbonyl chemistry and was further purified by
high performance liquid column chromatography as described previously
(7). The two cysteines were oxidized to form an intramolecular
disulfide bond.
The GBP sample was dissolved at a final concentration of 1.5-3.0
mM in 350 µl of either 99% D2O or 90%
H2O/10% D2O at pH 4.4. The pH was adjusted to
4.4 by adding microliter increments of HCl and NaOH. The NMR
experiments were performed on either a JEOL JNM-Alpha 600 or a Varian
Unity-plus 750 spectrometer. The majority of NMR spectra were recorded
at a temperature of 30 °C, and some experiments were also recorded
at 10 °C to resolve ambiguities. DQF-COSY spectra (8) were collected
at 30 and 10 °C. TOCSY spectra (9) were obtained at 30 and 10 °C
with a mixing time ranging from 50 to 100 ms. NOESY spectra (10) were
collected at 30 °C with a mixing time of 100-500 ms. The water
signal was suppressed by the DANTE pulse (11). Each spectrum size was
1024-4096 complex points in the t2 dimension and 200-512 complex
points in t1. The NMR data were zero filled in both dimensions, and a shifted sine-bell was applied as a window function for resolution enhancement. Amide protons protected from solvent exchange were identified on a sample that had been lyophilized and resuspended in
D2O.
Structure Calculations--
A total of 117 distance restraints
for the structure calculations were obtained from the NOESY spectrum
acquired with a mixing time of 300 ms at 30 °C. The distance
restraints can be subdivided into 37 intraresidue, 43 sequential, 20 medium range, and 17 long range. The cross-peak intensities were
classified into three classes, strong (1.6-2.7 Å), medium (1.6-3.5
Å), and weak (1.6-5.0 Å). Seven dihedral angle restraints were
derived from the 3JHN-H
coupling
constants measured from the DQF-COSY spectrum. These coupling constants
greater than 8 Hz were used to estimate torsion angles
to be
constrained in the range from
160 ° to 80 °. The amide
resonances that were still visible after more than 30 min in
D2O were identified as hydrogen bond donors. Hydrogen bond
acceptors could be unambiguously determined on the basis of the
secondary structure and preliminary structure calculations. Three
hydrogen bond restraints identified from a series of one-dimensional NMR spectra of hydrogen-deuterium exchange experiments were used as
input for the calculations (1.7-2.7 Å (H-O) and 2.5-3.5 Å (N-O)). Three-dimensional structure calculations were carried out with the
computer program X-PLOR 3.1 (12) using the simulated annealing protocol. Initial structures were generated with fully extended conformation. The constraints from backbone NOEs were used as input for
the first distance geometry. Each structure was subjected to one round
of simulated annealing with all distance and angle constraints. The
simulated annealing consisted of 6000 cooling steps from 2000 K to a
final temperature of 100 K. A total of 100 structures were calculated.
All 100 structures were passed through the refinement, which consisted
of 15000 cooling steps from 2000 to 100 K. The 20 structures with the
lowest energy that contained no NOE restraint violations greater than
0.1 Å were selected. The RMSD values were calculated by using the
programs X-PLOR and MOLMOL (13).
 |
RESULTS AND DISCUSSION |
The GBP molecule was soluble in water and monomeric as determined
by NMR spectroscopy. At various peptide concentrations (3, 0.3, and
0.06 mM), there were no concentration-dependent
changes that indicate dimerization or aggregation in the chemical
shifts or peak line widths of one-dimensional 1H NMR
spectra of GBP. In addition, no NOE cross-peaks indicative of dimeric
or multimeric structure were detected.
The proton NMR signals were identified according to the standard
procedure of sequential assignment (14). The sequential assignment of
the residues was achieved by first identifying spin systems by a
combination of DQF-COSY spectra and TOCSY spectra acquired with
different mixing times. This procedure was followed by assigning
identified spin systems to particular residues in the peptide by the
observation of sequential NH-NH (dNN), C
H-NH (d
N), and C
H-NH (d
N) NOEs.
A nearly complete assignment of the proton NMR signals of GBP was
obtained. The NMR signals of the backbone protons for Glu1
and Asn2 were either not observed or not resolved. The
fingerprint region of the NOESY is shown in Fig.
1.

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Fig. 1.
Fingerprint region from a NOESY spectrum of
GBP recorded at pH 4.4 in H2O at 30 °C with a 300 ms
mixing time. The sequential d N connectivities are
indicated. Residue 15 and 21 are prolines. The alpha proton of
Met12 is not assigned. The cross-peak for Thr14
that is not visible in this spectrum is labeled because it was observed
and confirmed in other spectra.
|
|
Fig. 2 shows the pattern of sequential
and short range NOE cross-peaks, the
3JHN-H
coupling constants, and
the slowly exchanging amide protons for GBP. Elements of the secondary
structure were deduced from the pattern of sequential and medium
range NOE connectivities. Additional information was obtained from the
H-D exchange study and the values of
3JHN-H
coupling constants.
Slowly exchanging amide protons due to the formation of hydrogen bonds
were identified from a series of one-dimensional NMR spectra. In these
spectra, three backbone amide proton signals remained intensely after
more than 30 min and were classified as slowly exchanging amide
protons. From the DQF-COSY spectrum, nine residues were determined to
have 3JHN-H
greater than 8.0 Hz.
The secondary structures that were identified from these data are
summarized in Fig. 3. The secondary
structure elements of GBP consist of a double-stranded antiparallel
-sheet that involves Tyr11-Arg13 and
Cys19-Pro21 and a
-turn that involves
Val8-Tyr11. The regions of the
-sheet were
further confirmed by the interstrand d
NOEs between
Tyr11 and Pro21 and between Arg13
and Cys19 (Fig. 4). The
type-II
-turn (Val8-Tyr11) is identified by
patterns of NOE, a strong d
N NOE between Ala9 and Gly10, a strong dNN NOE
between Gly10 and Tyr11, and a weak
d
N NOE between Ala9 and Tyr11,
which is held together by a hydrogen bond between Val8 and
Tyr11. Position 3 of this
-turn is Gly10,
the preferred residue here in a type-II
-turn.

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Fig. 2.
Diagram of NOE connectivities between protons
of neighboring (d N, d N, and
dNN), J-coupling constants
(3JHN-H ), and slowly exchanging
amide protons in D2O. The strength of the observed NOE
is represented by the thickness of the bars. Residues with
3JHN-H of >8 Hz are indicated by
arrows. The backbone amide protons, which exchange slowly
with D2O, are indicated by filled circles. The
d N of Met12 is not identified because the
alpha proton of Met12 is not assigned (*). Glu1
and Asn2 are not assigned (#).
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Fig. 3.
Identification of the antiparallel -sheet
and the type II -turn. The NOE connectivities are indicated by
arrows. The broken line indicates hydrogen bonds
for which slowed amide-proton exchange was observed.
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Fig. 4.
H- H region from a NOESY spectrum of GBP
recorded at pH 4.4 in H2O at 30 °C with a 300 ms mixing
time. Cross-peaks between alpha protons are annotated.
|
|
A total of 130 NMR-derived distance and angle constraints were used as
input for distance geometry and simulated annealing calculations using
the program X-PLOR. A family of 20 accepted three-dimensional
structures were used to represent the solution structure of GBP (Fig.
5 and Table
I). The N-terminal and C-terminal residues, Glu1-Gly6 and
Phe23-Gln25, are not well defined in the NMR
structure because the NOE data are insufficient to ascertain a single
conformation. The mean pairwise RMSD of the well defined region
(Cys7-Thr22) is 0.89 Å for the backbone atoms
and 1.72 Å for all heavy atoms. The core structure of GBP, the small
antiparallel
-sheet and the
-turn were further confirmed by the
conformation. The
-sheet is twisted in a right-handed manner. The
loop region, which is composed of five residues
(Thr14-Arg18) with no secondary structure, is
less converged than the
-turn (Val8-Tyr11).
The mean pairwise RMSDs of the loop region are 0.88 and 1.80 Å for the
backbone and all heavy atoms, respectively. On the other hand, those
values of the
-turn are lower, 0.71 Å for the backbone and 1.01 Å for all heavy atoms. However, the disulfide bond between Cys7 and Cys19 appears to contribute
significantly to the stability of the loop region, as well as to
establish important conformational constraints for defining a single
conformation. Side chains in the
-turn were well defined, having
RMSD values lower than 1.50 Å. The side chains of Cys7 and
Cys19, and Pro21 also had low RMSD values. The
other side chains were not well resolved. In particular, the charged
side chains of Arg13, Arg18, Lys20,
and Asp16 had high RMSD values. However, it is uncertain
whether this result is due to conformational flexibility or a lack of
distance restraints of these residues.

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Fig. 5.
Three-dimensional structure of GBP.
A, stereo view of the ensemble of 20 NMR structures of GBP
superimposed for best fit over the backbone atoms of residues 7-22 of
the mean coordinate structure. B, ribbon drawing of the
minimized average structure of GBP. These diagrams were generated using
the program MOLMOL (13).
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Table I
Structural statistics of 20 structures of GBP
Represents the structural statistics for the 20 energy minimized
structures obtained through the refinement protocol in X-PLOR.
Eimpr, EVDW, and
ENOE are improper energy of torsion angles, van der
Waals' repulsion energy, and the square well NOE potential energy,
respectively. All energies and RMSD values were calculated using the
programs X-PLOR and MOLMOL.
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|
We recently reported that GBP and human EGF (epidermal growth factor)
induce an almost equivalent increase in DNA synthesis of a human
keratinocyte cell line (6). On the other hand, GBP induces a higher and
more persistent increase in SF-9 insect cell proliferation than does
EGF. These results suggest that GBP has an ability to stimulate cell
proliferation more broadly than does EGF. Fig.
6 compares the overall structure of GBP
with that of mouse EGF (15). The general folding of GBP, with the
exception of the N-terminal disordered region, clearly conforms to the
C-terminal domain of EGF with weak primary structural similarity (Fig.
7). The backbone RMSD between the average
GBP structure and the average mouse EGF structure over the region of
the
-sheet and the
-turn is 1.98 Å. In particular, the type II
-turn exists in GBP and EGF, is composed of
Val8-Tyr11 in GBP, and corresponds to
Val34-Tyr37 in EGF with high amino acid
sequence conservation. The three-dimensional structural similarity of
backbone between GBP and EGF raises the possibility that these two
growth factors may stimulate the same receptor. Moreover, some highly
conserved amino acid residues in EGF family are conserved in GBP. It is
known that these residues are very important in EGF receptor-ligand
association. For example, substitution of the highly conserved
Arg41 and Leu47 of EGF resulted in a marked
reduction in the affinity of the EGF receptor, which suggests that
these residues are essential for receptor affinity (16, 17). It is
likely that Arg18 of GBP corresponds to Arg41
of EGF in light of their alignments and three-dimensional structures. On the other hand, GBP does not have a Leu residue that corresponds to
Leu47 of EGF, although it is thought that the
hydrophobicity of Leu47 is important for interaction with
the EGF receptor. Thus, it is possible that Phe23 and/or
Tyr24 of GBP may act instead of Leu47 of EGF.
In fact, elimination of Phe23 leads to a reduction in the
growth blocking activity of GBP (18), suggesting that Phe23
of GBP may play an important role just as Leu47 of EGF
does. To our knowledge, such a small peptide as stimulates EGF receptor
at high level has not been found. If further studies elucidate the
interaction of GBP with EGF receptor clearly, the structure of GBP may
offer clues to the interaction between EGF and EGF receptor, which is
still not very clear.

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Fig. 6.
Comparison of the three-dimensional
structures of mouse EGF (A) and GBP (B).
The disulfide bonds, Cys33-Cys42 in EGF and
Cys7-Cys19 in GBP, are shown with
balls and sticks. The drawing of mouse EGF was
made using atomic coordinates obtained from the Brookhaven Protein Data
Bank (entry set 1EPG). This diagram was generated using the
WebLab Viewer computer program (Molecular Simulations Inc.).
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Fig. 7.
Sequence alignment of human EGF, mouse EGF,
and GBP. The amino acid sequence of GBP was aligned to the
C-terminal region of EGF. The partial C-terminal amino acid sequence of
human and mouse EGF shows 24-28% homology with the GBP. The disulfide
connectivity is shown by a solid line.
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|
GBP was initially identified as a hemolymphal peptide that delays the
normal development of the host insect larvae parasitized by parasitic
wasps (1-3). GBP cDNAs have been cloned from three lepidopteran
species, P. separata, Mamestra brassicae, and
Spodoptera litura, and the amino acid sequences of GBP in
these three species are highly homologous (6, 19). In addition, the
amino acid sequences of paralytic peptides and plasmatocyte-spreading
peptide isolated from insect hemolymph have been shown to be highly
homologous to those of GBP. Each of seven paralytic peptides found in
hemolymph of Manduca sexta, Spodoptera exigua,
and Heliothis virescens causes a temporary paralysis when
injected into larvae (20). On the other hand, plasmatocyte-spreading
peptide from the larvae of Pseudoplusia includens induces
the adhesion and spreading of plasmatocytes to foreign surfaces (21).
Although it has been reported that all these GBP and GBP-like peptides
vary in function, they share at least 14 completely conserved amino
acids. This is the first report on the three-dimensional structure of
these insect peptides. In addition, there has been no report on the
three-dimensional structure of a peptiderigic growth factor in insects.
Knowledge of the structure of GBP is important not only for elucidating the mechanism of its activity but also for clarifying the physiological roles of these homologous peptides and growth factors in insects.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1BQF) have
been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
**
To whom correspondence should be addressed: Faculty of
Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. Tel./Fax: 81-764-34-5061; E-mail: kawano{at}ms.toyama-mpu.ac.jp.
The abbreviations used are:
GBP, growth-blocking
peptide; EGF, epidermal growth factor; DQF-COSY, double quantum
filtered correlation spectroscopy; NOESY, nuclear Overhauser effect
spectroscopy; RMSD, root mean square difference; NOE, nuclear
Overhauser effect; TOCSY, total correlation spectroscopy; DANTE, delays
alternating with nutation for tailored excitation.
 |
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.