From the Department of Applied Biological Chemistry,
Graduate School of Agricultural and Life Sciences, The University
of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan and
§ Biotechnology Research Center, The University of
Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
Received for publication, December 19, 2002, and in revised form, January 6, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molting in crustaceans is controlled by
molt-inhibiting hormone (MIH) and ecdysteroids. It is presumed that MIH
inhibits the synthesis and the secretion of ecdysteroids by the
Y-organ, resulting in molt suppression. The amino acid sequence of MIH
is similar to that of crustacean hyperglycemic hormone (CHH), and
therefore, they form a peptide family referred to as the CHH family.
Most of the CHH family peptides show no cross-activity, whereas a few peptides show multiple hormonal activities. To reveal the structural basis of this functional specificity, we determined the solution structure of MIH from the Kuruma prawn Marsupenaeus
japonicus and compared the solution structure of MIH with a
homology-modeled structure of M. japonicus CHH. The
solution structure of MIH consisted of five Molting is one of the most significant processes occurring during
the arthropod life cycle and is triggered by a molting hormone, ecdysteroids. In crustaceans, it is presumed that the synthesis and the
secretion of ecdysteroids by the Y-organ are suppressed by
molt-inhibiting hormone
(MIH)1 (1). MIH is produced
by the X-organ and released from the sinus gland located in the
eyestalk. Crustacean hyperglycemic hormone (CHH), gonad-inhibiting
hormone, and mandibular organ-inhibiting hormone are also
synthesized in and released from the X-organ/sinus gland complex. Most
of these peptides consist of 72-78 amino acid residues and exhibit
similar amino acid sequences. Therefore, these peptides form a peptide
family referred to as the CHH family (2). The CHH family peptides
commonly have six cysteine residues, which form three intramolecular
disulfide bonds. Circular dichroism spectral analyses of some CHH
family peptides demonstrated that they were rich in CHH family peptides show various biological activities: some peptides
that suppress molting, increase hemolymph glucose levels, and suppress
vitellogenesis in the ovary and others that suppress the synthesis of
methyl farnesoate in the mandibular organ (2). In general, each CHH
family peptide shows only one type of biological activity, although a
few peptides show multiple hormonal activities. For instance, CHH from
the American lobster Homarus americanus also exhibits
molt-inhibiting activity (6), and mandibular organ-inhibiting hormone
from the spider crab Libinia emarginata exhibits
hyperglycemic activity (7).
To reveal the structural basis of the functional specificity of the CHH
family peptides, we have elucidated the solution structure of MIH from
the Kuruma prawn Marsupenaeus japonicus by heteronuclear three-dimensional nuclear magnetic resonance. In this paper, we propose
the functionally important sites of the CHH family peptides presumed
from the comparison of the tertiary and surface structures between MIH
and CHH.
Sample Preparation--
A recombinant M. japonicus
MIH (r-MIH) was overproduced using a pET-28a(+) plasmid (Novagen) in
Escherichia coli BL21-CodonplusTM(DE3)-RIL (Stratagene) and
purified after the refolding reaction as described previously (5, 8).
Uniformly 15N-labeled and
13C/15N-doubly labeled r-MIHs were obtained by
growing the cells in M9 minimal medium.
Circular Dichroism Spectral Analysis--
The refolded r-MIH was
dissolved in 30% acetonitrile aqueous solution at the concentration of
100 µg/ml. The CD spectrum was recorded from 200 to 260 nm on a JASCO
J-720 spectropolarimeter at room temperature with a 1-mm path length cell.
NMR Experiments--
All NMR spectra were measured on a Varian
Unity INOVA500 spectrometer at 25 °C. Sample solution was prepared
at a concentration of 1-2 mM in 30%
C2H3CN, 70% 2H2O or
30% C2H3CN, 10% 2H2O,
60% 1H2O. Chemical shifts were referenced to
sodium 2,2-dimethyl-2-silapentanesulfonic acid. All of the non-labile
1H, 13C, and 15N atoms in r-MIH
were assigned by the standard triple resonance NMR experiments.
Data Processing and Structure Calculation--
All of the NMR
spectra were processed using the software NMRPipe (version 1.6) (9) on
an Octane work station (Silicon Graphics). Interproton distance
restraints were obtained from cross-peak intensities in
15N-edited and 13C-edited NOESY spectra (10,
11). Peak intensities on the13C-edited and the
15N-edited NOESY spectra were translated to interproton
distances based on the relation of NOE Structure Analysis--
Ramachandran Sample Preparation--
The r-MIH (8) was shown to be functionally
and structurally identical to native MIH by comparison of
molt-inhibiting activity, arrangement of the disulfide linkages, and
the CD spectra (5, 8).
It was not possible to dissolve r-MIH in water or phosphate-buffered
saline at high concentrations above 1 mM. However, r-MIH could be dissolved in 30% aqueous acetonitrile at concentrations over
2 mM. In addition, r-MIH in this solution showed
essentially the same CD spectrum as that in phosphate-buffered saline,
indicating that the conformation of r-MIH remained intact in this
solution (data not shown). Thus, we used 30% acetonitrile as the
solvent for the NMR experiments.
Quality of the Calculated Structure--
All of the non-labile
1H, 13C, and 15N atoms of r-MIH
were assigned based on the standard triple resonance NMR experiments
(Table I). A total of 2252 restraints including 2209 distance restraints and 43 torsion angle
restraints was obtained from the NMR data and used in the structural
calculations of r-MIH. A set of 10 structures was selected from 50 calculated structures based on the agreement with the experimental
data. In these selected structures, there were no violations greater
than 0.4 Å and 2° in interproton distances and torsion angles,
respectively. The solution structure of r-MIH was well defined with the
exception of the N- and C-terminal regions. The pairwise root mean
square deviations among the 10 structures were 0.51 ± 0.09 Å for
the backbone heavy atoms (N, C Tertiary Structure of r-MIH--
Fig.
1A shows the best-fit
superposition of the backbone atoms of r-MIH. The r-MIH molecule
consists of an N-terminal region, five
The conformation of r-MIH was stabilized by a number of hydrophobic
interactions in the cluster of nine hydrophobic residues (Leu16, Val20, Val23,
Phe46, Phe50, Leu54,
Phe66, Ile70, and Leu73) and the
three disulfide bonds (Fig. 1C). These hydrophobic and cysteine residues are conserved in most of the CHH family peptides as
shown in Fig. 2, suggesting that the CHH
family peptides harbor similar foldings.
We searched for proteins structurally similar to MIH using the DALI
server (17), and several were found to be similar to MIH including RNA
polymerase Structural Comparison of MIH with Other CHH Family
Peptides--
The tertiary structure of Pej-SGP-III, one of the
M. japonicus CHHs that has a 32% sequence identity
to M. japonicus MIH (19), was homology-modeled
using the SWISS model server (18). Fig. 3C shows the modeled structure
of CHH. This structure is very similar to the solution structure of
r-MIH with the exception that CHH lacks
Because the C-terminal sequence of CHH is shorter than that of MIH by a
few amino acids, the CHH lacked the C-terminal region present in
MIH. The C-terminal region of MIH was located close to Putative Functional Site of CHH Family Peptides--
Fig. 3,
E and F, shows the surface structures of MIH and
CHH. The residues conserved or homologous only within MIHs (positions 12, 13, 19, 20, 48, 56, 59, 61, 62, 69, 72, 73, and 76) are indicated in green, and the residues conserved or homologous only
within CHHs (positions 3, 4, 9, 11, 12, 19, 27, 42, 46, 54, 56, 57, 70, and 72) are indicated in blue. Thus, the green
residues are mainly located on the front in Fig. 3E,
and the blue residues are dispersed all over the molecular
surface. Therefore, the green-colored side in the MIH was
considered to be important for the molt-inhibiting activity. As this
side of the molecule includes
Our previous study demonstrated that the C-terminal amide moiety of CHH
is significant in conferring biological activity (21, 22). In addition,
the C-terminally truncated CHH from the South African spiny lobster
Jasus lalandii, which lacked six C-terminal amino acid
residues, showed no hyperglycemic activity (23). These results suggest
that the functional site of CHH may be located at the C-terminal
region. On the basis of our results from the present experiments, we
propose the hypothesis that the functionally important sites of CHH
family peptides may be located at the region containing
In conclusion, we have determined the solution structure of MIH and
have demonstrated the existence of a novel structural motif. This is
the first report on the tertiary structure analysis of a crustacean
neuropeptide. The elucidation of this solution structure is expected to
provide new insights, shedding light not only on structure-activity
relationships but also on the molecular evolution of the CHH family
peptides
-helices and no
-structures, constituting a novel structural motif. The
homology-modeled structure of M. japonicus CHH was very
similar to the solution structure of MIH with the exception of the
absence of the N-terminal
-helix and the C-terminal tail, which were
sterically close to each other. The surface properties of MIH around
this region were quite different from those of CHH. These results
strongly suggest that this region is a functionally important site for
conferring molt-inhibiting activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helices (3-5).
However, there has so far been no report on the determination of the
tertiary structure of any of the CHH family peptides.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(distance)
6.
The upper bound distance restraints were set to the calculated distances plus 1.0 Å considering internal mobility.
-Angle
restraints were derived from 3JHNH
coupling constants obtained from HNHA spectrum (12) and
secondary structure predicted from the chemical shifts of
HN, C
, and C' by the program CSI (version
1.0) (13).
-Angles were restrained to the range of
90 to
40°
for the residues that satisfied both of the following criteria. 1) the
coupling constant (3JHNH
) was <6 Hz.
2) The residue was predicted to be
-helical by CSI.
Three-dimensional structure was calculated with the disulfide bond
distance restraints and the NMR-derived restraints including
interproton distance restraints and torsion angle restraints. 50 random
conformations were annealed in 10,000 steps by torsion angle dynamics
with the program DYANA (version 1.4) (14). Ten conformers having the
lowest DYANA target functions were used to present the solution
conformation of r-MIH.
-
-plot analysis was
performed with PROCHECK NMR (version 3.4) (15). Secondary structure was
defined, and tertiary structure was visualized with MOLMOL (version
2.6) (16). The DALI server (17) was used in the search for structurally similar proteins to r-MIH. Homology modeling of CHH was performed using
the SWISS model server (18).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, and C') and 1.24 ± 0.10 Å for all non-hydrogen atoms in the well defined region
(Asn5-Ala75). The conformation of the
N-terminal (Ser1-Asp4) and C-terminal
(Gly76-Gln77) regions was much less defined,
because only a few restraints were obtained for these regions. In the
Ramachandran
-
-plot for the well defined region of the 10 selected structures, 99.8%
-
plot was located in the
favored and allowed regions (Table I).
Structural statistics for the 10 DYANA structures of r-MIH
-helices (
1,
Val10-Asn13;
2,
Ile16-Phe31;
3,
Pro34-Cys40;
4,
Glu49-Lys55; and
5,
Glu62-Ile72), four loops between the
-helices (L1-2, L2-3, L3-4, and L4-5), and a C-terminal tail
region (Fig. 1B). The disulfide bonds linked N-terminal
region and L3-4,
2 and
3, and
2 and
4. This
-helical structure is
consistent with the prediction of the secondary structure by CSI.
View larger version (25K):
[in a new window]
Fig. 1.
A, stereopairs of the best-fit
superposition of the 10 selected structures of r-MIH. -Helical
regions are indicated in colors. B, ribbon model
of the energy-minimized average structure of r-MIH. C,
stereopairs of the backbone and non-hydrogen side chains involved in
the cluster of hydrophobic residues (indicated in red) and
cysteine residues (indicated in light blue) of r-MIH. These
figures were prepared using MOLMOL (16).
View larger version (38K):
[in a new window]
Fig. 2.
Amino acid sequence alignment of
the CHH family peptides. Shaded and open
squares indicate the residues involved in the cluster of
hydrophobic residues and cysteine residues forming three disulfide
bonds indicated at the bottom, respectively. Pej,
M. japonicus; Cam, Carcinus
maenas; Cap, Cancer pagurus; Orl,
Orconectes limosus; Hoa, H. americanus; Prb, Procambarus clarkii;
Mee, Metapenaeus ensis.
-factor, DNA ligase fragment, and acyl-CoA oxidase.
However, the Z-scores of these proteins for MIH, which indicate the
extent of similarity in three-dimensional structure, were very low (3.4 at highest); thus, we concluded that these proteins were not
structurally similar to MIH. These results indicated that CHH family
peptides would form a novel class of folds.
1. In the CHH
family peptides, MIHs have a glycine residue inserted at position 12 (20). The solution structure of r-MIH shows that this glycine residue
is located at
1. The absence of
1 in CHH
may be because of the lack of the glycine residue.
View larger version (50K):
[in a new window]
Fig. 3.
A, ribbon model of the energy-minimized
average structure of r-MIH. B, surface structure of r-MIH.
The view angle of the left figure is the same as
that of panel A, and that of the right figure is
the reverse. The acidic (aspartic acid and glutamic acid), basic
(arginine, lysine, and histidine), and hydrophobic (leucine,
isoleucine, valine, tryptophan, phenylalanine, and tyrosine) residues
are colored red, blue, and green,
respectively. C, ribbon model of the homology-modeled
structure of Pej-SGP-III, one of M. japonicus CHHs.
D, surface structure of Pej-SGP-III. The view
angle of the left figure is the same as that of
panel C, and that of the right figure is the
reverse. E, surface structure of r-MIH. The view
angles are the same as those in panel B. The
characteristic residues for MIH are indicated in green.
F, surface structure of Pej-SGP-III. The view
angles are the same as those in panel D. The
characteristic residues for CHH are indicated in blue. These
figures were prepared using MOLMOL (16).
1
in the tertiary structure (Fig. 3A). The surface properties including electrostatic potential and hydrophobicity of these peptides
were different in this terminal region (Fig. 3, B and D). Therefore, it was presumed that the region containing
1 and the C terminus was important for molt-inhibiting activity.
1 and the C terminus, these results were consistent with the presumption described above.
1 and the C terminus.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. M. N. Wilder of the Japan International Research Center for Agricultural Sciences for critical reading of this paper.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grants-in-aid 11460054 and 1208200 for Scientific Research and the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Uehara Memorial Foundation.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 the structure factors (code 1J0T) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Chemical shifts for this peptide were deposited in the BioMagResBank under the entry number 5653.
¶ To whom correspondence should be addressed. Tel.: 81-3-5841-5132; Fax: 81-3-5841-8022; E-mail: anagahi@mail.ecc.u-tokyo.ac.jp.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212962200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MIH, molt-inhibiting hormone; r-MIH, recombinant M. japonicus MIH; CHH, crustacean hyperglycemic hormone; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; SGP, sinus gland peptide.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Soumoff, C., and O'Connor, J. D. (1982) Gen. Comp. Endocrinol. 48, 432-439[Medline] [Order article via Infotrieve] |
2. | Keller, R. (1992) Experientia 48, 439-448[Medline] [Order article via Infotrieve] |
3. | Huberman, A., Aguilar, M. B., Brew, K., Shabanowitz, J., and Hunt, D. F. (1993) Peptides 14, 7-16[CrossRef][Medline] [Order article via Infotrieve] |
4. | Chung, J. S., and Webster, S. G. (1996) Eur. J. Biochem. 240, 358-364[Abstract] |
5. | Katayama, H., Ohira, T., Nagata, K., and Nagasawa, H. (2001) Biosci. Biotechnol. Biochem. 65, 1832-1839[CrossRef][Medline] [Order article via Infotrieve] |
6. | Chang, E. S., Bruce, M. J., and Newcomb, R. W. (1987) Gen. Comp. Endocrinol. 65, 56-64[Medline] [Order article via Infotrieve] |
7. | Lei, L., and Laufer, H. (1996) Arch. Insect. Biochem. Physiol. 32, 375-385[CrossRef] |
8. | Ohira, T., Nishimura, T., Sonobe, H., Okuno, A., Watanabe, T., Nagasawa, H., Kawazoe, I., and Aida, K. (1999) Biosci. Biotechnol. Biochem. 63, 1576-1581[Medline] [Order article via Infotrieve] |
9. | Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, 277-293[Medline] [Order article via Infotrieve] |
10. | Muhandiram, D. R., Farrow, N. A., Xu, G.-Y., Smallcombe, S. H., and Kay, L. E. (1993) J. Magn. Reson. 102, 317-321[CrossRef] |
11. | Marion, D., Driscoll, P. C., Kay, L. E., Wingfield, P. T., Bax, A., Gronenborn, A. M., and Clore, G. M. (1989) Biochemistry 28, 6150-6156[Medline] [Order article via Infotrieve] |
12. | Vuister, G. W., and Bax, A. (1993) J. Am. Chem. Soc. 115, 7772-7777 |
13. | Wishart, D. S., and Sykes, B. D. (1994) J. Biomol. NMR 4, 171-180[Medline] [Order article via Infotrieve] |
14. | Güntert, P., Mumenthaler, C., and Wüthrich, K. (1997) J. Mol. Biol. 273, 283-298[CrossRef][Medline] [Order article via Infotrieve] |
15. | Laskowski, R. A., Rullmann, J. A., MacArthur, M. W., Kaptein, R., and Thornton, J. M. (1996) J. Biomol. NMR 8, 477-486[Medline] [Order article via Infotrieve] |
16. | Koradi, R., Billeter, M., and Wüthrich, K. (1996) J. Mol. Graph. 14, 51-55[CrossRef][Medline] [Order article via Infotrieve] |
17. | Holm, M., and Sander, C. (1993) J. Mol. Biol. 233, 123-138[CrossRef][Medline] [Order article via Infotrieve] |
18. | Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714-2723[Medline] [Order article via Infotrieve] |
19. | Yang, W.-J., Aida, K., and Nagasawa, H. (1995) Aquaculture 135, 205-212[CrossRef] |
20. | Lacombe, C., Grève, P., and Martin, G. (1999) Neuropeptides 33, 71-80[CrossRef][Medline] [Order article via Infotrieve] |
21. | Katayama, H., Ohira, T., Aida, K., and Nagasawa, H. (2002) Peptides 23, 1537-1546[CrossRef][Medline] [Order article via Infotrieve] |
22. | Ohira, T., Katayama, H., Aida, K., and Nagasawa, H. (2003) Fisheries Sci. 69, 95-100 |
23. | Marco, H. G., Brandt, W., Stoeva, S., Voelter, W., and Gäde, G. (2000) Peptides 21, 19-27[CrossRef][Medline] [Order article via Infotrieve] |