The Solution Structure of Molt-inhibiting Hormone from the Kuruma Prawn Marsupenaeus japonicus*

Hidekazu KatayamaDagger , Koji NagataDagger §, Tsuyoshi OhiraDagger , Fumiaki YumotoDagger , Masaru TanokuraDagger , and Hiromichi NagasawaDagger

From the Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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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 alpha -helices and no beta -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 alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha -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.

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 proportional to  (distance)-6. The upper bound distance restraints were set to the calculated distances plus 1.0 Å considering internal mobility. phi -Angle restraints were derived from 3JHNHalpha coupling constants obtained from HNHA spectrum (12) and secondary structure predicted from the chemical shifts of HN, Calpha , and C' by the program CSI (version 1.0) (13). phi -Angles were restrained to the range of -90 to -40° for the residues that satisfied both of the following criteria. 1) the coupling constant (3JHNHalpha ) was <6 Hz. 2) The residue was predicted to be alpha -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.

Structure Analysis-- Ramachandran phi -psi -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
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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, Calpha , 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 phi -psi -plot for the well defined region of the 10 selected structures, 99.8% phi -psi plot was located in the favored and allowed regions (Table I).

                              
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Table I
Structural statistics for the 10 DYANA structures of r-MIH

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 alpha -helices (alpha 1, Val10-Asn13; alpha 2, Ile16-Phe31; alpha 3, Pro34-Cys40; alpha 4, Glu49-Lys55; and alpha 5, Glu62-Ile72), four loops between the alpha -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, alpha 2 and alpha 3, and alpha 2 and alpha 4. This alpha -helical structure is consistent with the prediction of the secondary structure by CSI.


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Fig. 1.   A, stereopairs of the best-fit superposition of the 10 selected structures of r-MIH. alpha -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).

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.


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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.

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 sigma -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.

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 alpha 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 alpha 1. The absence of alpha 1 in CHH may be because of the lack of the glycine residue.


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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).

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 alpha 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 alpha 1 and the C terminus was important for molt-inhibiting activity.

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 alpha 1 and the C terminus, these results were consistent with the presumption described above.

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 alpha 1 and the C terminus.

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

    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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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