From the National Magnetic Resonance Facility at
Madison, Department of Biochemistry, University of Wisconsin-Madison,
Madison, Wisconsin 53706-1544, the ¶ Department of Entomology,
University of Wisconsin-Madison, Madison, Wisconsin 53706, and the
Biochemical Laboratory, Institute of Low Temperature Science,
Hokkaido University, Sapporo 060-0819, Japan
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ABSTRACT |
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The structure of the recently identified
plasmatocyte spreading peptide from the moth Pseudoplusia
includens (PSP1) has been determined by NMR spectroscopy. This
novel insect cytokine consists of 23 amino acid residues and a single
disulfide bond. Torsion angle dynamics calculations utilizing a total
of 337 distance constraints yielded an ensemble of 30 structures with
an average backbone root mean square deviation for residues 7-22 of
0.18 Å from the mean structure. The structure consists of a disordered N-terminal region and a well defined core that is stabilized by numerous hydrophobic interactions and a short The insect immune response to parasites and pathogens involves the
action of different classes of blood cells (hemocytes), which adhere to
and spread across the surface of foreign targets. In phylogenetically
advanced insects like the Lepidoptera (moths and butterflies),
plasmatocytes and granular cells are the two most important classes of
hemocytes involved in cellular defense responses like encapsulation and
clotting (3). However, relatively little is known about the factors
mediating the movement and action of these immune cells (3). The
identification of a peptide that induces the adhesion and spreading of
plasmatocytes on foreign surfaces was recently reported (1). This
plasmatocyte-spreading peptide
(PSP1)1 of Pseudoplusia
includens is expressed as a preproprotein of 142 residues (2), but
the mature peptide consists of only the C-terminal 23 amino acids
containing one disulfide bond. Although the existence of cytokine-like
factors to regulate hemocyte activity has been proposed previously (3),
PSP1 is among the first soluble mediators of the insect cellular immune
response to be purified and functionally characterized.
The amino acid sequence of PSP1 shows no significant similarity to
vertebrate cytokines, but is related to two classes of insect peptides
previously identified (1). As shown in Fig. 1, the growth-blocking peptide (GBP) of
Pseudaletia separata and the paralytic peptides from
Manduca sexta, Heliothis virescens, and
Spodoptera exigua have high (>70%) sequence identity with PSP1, including the two cysteine residues and a highly conserved glycine. High sequence variabilty within this group is found only at
positions 4 and 8. Some similarity between the sequences of GBP and
human epidermal growth factor has been recently suggested (4), and this
would extend to PSP1 and the paralytic peptides.
-hairpin. Structural comparisons confirm that PSP1 adopts an epidermal growth factor (EGF)-like fold with close similarity to the C-terminal subdomain of
EGF-like module 5 of human thrombomodulin. The combination of the
three-dimensional structure of PSP1 and the extensive literature on
EGF-receptor interactions should accelerate the process of identifying
the specific residues responsible for receptor binding activity of this
family of immunoregulatory peptides.
INTRODUCTION
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Abstract
Introduction
References
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Fig. 1.
Comparison of the sequences of PSP1 from
P. includens, GBP from P. separata,
paralytic peptide from M. sexta
(PP1), and the C-terminal subdomains of human
epidermal growth factor (hEGF) and the hTM5.
Highlighted in boldface type are the cysteine,
glycine, and tyrosine residues conserved in the EGF-like domain family
and the corresponding residues of the insect peptides.
Chemically synthesized PSP1 was shown to induce an identical
plasmatocyte spreading and adhesion response to the peptide purified from the plasma of P. includens larvae and is therefore
suitable for detailed structure-function analysis (1). Here we report the three-dimensional structure of this novel insect cytokine determined by NMR spectroscopy and torsion angle dynamics calculations. The structure of PSP1 shows clear homology to the C-terminal subdomain of the EGF domain family. The backbone conformation of PSP1 is very
similar to that of the fifth EGF-like domain of the human anticoagulant
protein thrombomodulin (hTM5), despite sharing sequence identity at
only four positions, including the two cysteines of the conserved disulfide.
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EXPERIMENTAL PROCEDURES |
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Sample Preparation-- PSP1 was synthesized using tert-butyloxycarbonyl chemistry and purified by reversed-phase high performance liquid chromatography. Its identity was confirmed by matrix-assisted laser desorption/ionization time of flight mass spectrometry and NMR. The NMR sample consisted of 2 mg of pure lyophilized peptide, dissolved in 500 µl of a buffer containing 90% H2O/10% D2O, 20 mM sodium phosphate, pH 6.0.
NMR Spectroscopy--
All NMR spectra were recorded at 10 °C
on Bruker DMX750 and DMX500 spectrometers equipped with
triple-resonance (1H/13C/15N)
probes and with Z or three-axis pulsed field gradient capabilities. Quadrature detection in the indirectly detected dimensions was obtained
with the States-TPPI method (5). Water suppression was achieved using a
watergate sequence with a 3-9-19 selective inversion pulse (6). A
two-dimensional total correlation spectroscopy spectrum with an
isotropic mixing period of 67 ms was acquired at 499.84 MHz, with
spectral widths of 6009.6 Hz in both dimensions and time domain data
sizes of 2048 and 256 complex points in the direct and indirect
dimensions, respectively. A two-dimensional NOESY spectrum with a
300-ms mixing time was acquired at 750.13 MHz with spectral widths of
8892.8 Hz in both dimensions and time domain data sizes of 1024 and 256 complex points in the direct and indirect dimensions, respectively.
All Fourier transformations of NMR data were performed with Felix 95.0 (Molecular Simulations) or NMRPipe (7). Time domain data in both dimensions were apodized with a squared cosine bell prior to zero filling to the final matrix size, Fourier transformation, and phase correction. The initial value for the incremented delay was set in a manner that allowed predictable phasing in each dimension and minimized roll and offset of the base line (8). All 1H dimensions were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate.
Resonance Assignments and Structure Calculations-- Sequential resonance assignments for the backbone were obtained manually from analysis of the total correlation spectroscopy and NOESY spectra (9). Subsequent assignment of side chains and NOEs for structure calculations were obtained with the programs Garant (10) and XEASY (11).
Structures were calculated with the torsion angle dynamics approach of
the program DYANA 1.5 (12) using the anneal.dya macro with 4000 steps/structure in the early stages of refinement and 20,000 steps in
the final rounds, followed by 1000 steps of conjugate gradient
minimization. Distance constraint upper bounds were generated with the
CALIBA function of DYANA. For the initial round of structure calculations, sequential and intraresidue NOEs assigned by Garant, as
well as a small number of manually assigned additional medium and long
range NOEs were used as input restraints. Each round started with 60 randomized conformers, from which the 30 with the lowest target
function were used to analyze constraint violations and assign
additional NOE constraints (in Garant or XEASY) for the following round
of refinement. This process was repeated until all peaks in the
spectrum had been assigned and all consistent violations were
eliminated. In the later stages of refinement, the GLOMSA routine of
DYANA was applied to the set of restraints and ensemble of structures
to obtain a small number of stereospecific assignments based on
differences in NOE intensities for diastereotopic groups. From the
total of 60 structures calculated in the final round of refinement, the
30 conformers with the lowest target function were considered for
analysis. The mean structure calculated in MOLMOL (13) from this
ensemble of 30 structures was minimized in DYANA with 1000 steps of
conjugate gradient minimization and reported as the representative PSP1
structure. All analyses of root mean square deviation, secondary
structure, and hydrogen bonds were performed with MOLMOL.
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RESULTS AND DISCUSSION |
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Structure Determination--
Two-dimensional 1H NMR
spectroscopy was used to obtain complete resonance assignments for
chemically synthesized plasmatocyte-spreading peptide 1 (PSP1).2 As shown in Fig.
2, the chemical shift dispersion and
large number of NOEs observed indicate the presence of well defined
secondary and tertiary structure. A total of 699 cross-peaks were
assigned in the two-dimensional NOESY spectrum of PSP1, from which 333 nontrivial NOE distance constraints were produced after discarding duplicate and structurally redundant NOEs. Two additional pairs of
upper and lower bound distance constraints were included between the
S and C
atoms of Cys7 and
Cys19 to produce normal disulfide bond geometry (2.0 Å < dS
S < 2.1 Å, 3.72 Å < dC
C
< 3.99 Å). A summary of all 337 distance constraints by class is given in
Table I, and the distribution of
constraints by residue is shown in Fig.
3. Stereospecific assignments were
obtained from analysis of structures and NOE intensities for the
H
protons of Gly17, the H
protons of Tyr11 and Pro21, and the
H
and H
protons of Pro21.
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The ensemble of 30 PSP1 conformers, which resulted from the final stage of refinement, is shown in Fig. 4. The low average target function (0.12 ± 0.02 Å2) indicates good agreement between the ensemble of calculated conformers and the experimental constraints. Superposition of the well ordered residues (7-22) of this ensemble onto the corresponding residues of the mean structure produced an average RMSD to the mean of 0.18 Å for the backbone atoms and 0.84 Å for all nonhydrogen atoms. After minimization of the mean structure in DYANA to a final target function of 0.13, the average RMSD for residues 7-22 became 0.23 Å for the backbone and 0.98 Å for the heavy atoms. A summary of residual violations and RMSDs is given in Table I.
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The tertiary structure of PSP1 is stabilized by a combination of the
covalent disulfide linkage, hydrogen bonding within the -hairpin
structure, and hydrophobic side chain packing. All 30 torsion angle
dynamics conformers were consistent with a pair of hydrogen bonds
involving the backbone amide and carbonyl groups of Met12
and Lys20. Evidence for two additional hydrogen bonds
(Asp16 HN-Thr14 O
1,
Thr14 HN-Arg18 O) was observed in
a subset of the family of structures. Within the ordered portion of the
molecule, a subset of side chains forms a well defined hydrophobic core
centered around the side chain of Tyr11. This residue,
conserved throughout the family of EGF-like domains as a phenylalanine
or tyrosine (14), has NOE contacts to the side chains of
Cys7, Leu8, Ala9,
Cys19, Pro21, and Phe23. The
opposite side of the structure is largely composed of exposed and
disordered charged residues, including Arg13,
Asp16, Arg18, and Lys20.
As seen in Fig. 1, the sequences of PSP1, GBP from Pseudaletia separata, and a paralytic peptide from M. sexta differ in very few positions, and all may be expected to adopt the same three-dimensional structures. Although the structures of these related peptides have not been reported, preliminary analysis of the NOESY spectrum of GBP indicates that its structure will be very similar to that reported here for PSP1.3 Consistent with this result are functional assays using both PSP1 and GBP that show no significant differences in activity.4
Comparison with Epidermal Growth Factor Modules--
EGF-like
domains are peptides of approximately 40 residues in length, containing
six highly conserved cysteine residues. Three loops, designated A, B,
and C, are defined by the pattern of disulfide bonding. EGF modules are
alternatively described in terms of two subdomains, each containing a
-hairpin structure. The N-terminal EGF-like subdomain consists of
the first two disulfide-linked loops, and the smaller C-terminal
subdomain consists of the third disulfide and the C loop.
The structures of human epidermal growth factor (hEGF) (15) and the
hTM5 (16), which have been determined by NMR spectroscopy, share the
general structural features of the family of EGF-like domains. The
variability of the EGF family is illustrated by the differences between
these two structures, including alternative pairing for the first two
disulfides and a longer C loop in hTM5 than hEGF (16). Sequence
identity between PSP1 and these representatives of the family of
EGF-like domains is limited to the essential cysteine, glycine, and
tyrosine residues of the C-terminal EGF-like subdomain (Fig. 1), but
PSP1 has an extended C loop as found in hTM5. The backbone
conformations of these three peptides are compared in Fig.
5. Except for the displacement of the
-turn in hEGF, resulting from its shorter C loop, the structures of
all three molecules are quite similar. PSP1 and hTM5 align to a
remarkably high degree, with an average backbone atomic RMSD of ~1 Å for the 13 residues within the disulfide loop.
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Implications for Receptor Binding-- PSP1 was isolated as the peptide responsible for spreading of insect cell plasmatocytes on foreign surfaces, an important process in cellular defense. Hence, it will be of interest to determine the cellular receptor for this peptide. Given the striking similarity between the structure of PSP1 and the C-terminal subdomain of EGF-like modules, it is tempting to speculate that the ligand-receptor interactions of PSP1 may be analogous to those of the array of EGF domain-receptor complexes that have been investigated (17).
Fig. 6 shows the minimized mean structure of PSP1, including all side chains. The charged residues Arg13, Asp16, Arg18, and Lys20 are clearly clustered on one side of the molecule, and the patch of hydrophobic residues surrounding the Tyr11 side chain are located on the opposing side. No consensus receptor-binding site has been identified yet for EGF receptor ligands, despite intense efforts (17) stemming from the relevance of this system to the design of potential drugs for treating human cancers. However, the studies have determined that an arginine conserved in EGF receptor binding domains (Arg41 in hEGF) is essential for binding. Interestingly, Arg18 is the analogous residue in PSP1.
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Significant effort has been spent investigating the details of thrombin
recognition by thrombomodulin, including alanine-scanning mutagenesis,
NMR spectroscopy, and x-ray crystallography of the complexes between
small peptide fragments of hTM5 and thrombin (18-20). Although the
distinction between residues critical for proper folding and those
directly involved in binding may not be entirely clear from mutagenesis
alone, a structural interpretation clearly implicates the C loop of
hTM5, including the many acidic side chains on either side of
Cys421 (Fig. 1). None of these residues has a direct
correlation, but the cluster of charged side chains occurs in the same
vicinity of the PSP1 structure. In the absence of other information on the activity of PSP1, this concentration of acidic and basic residues is an obvious starting point for investigations of the receptor-binding determinants of this new class of insect cytokine peptides.
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ACKNOWLEDGEMENTS |
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We thank Dr. Milo Westler for assistance in acquiring and processing NMR data and Dr. Dorothee Kern for advice on using Garant and XEASY. We also thank Dr. Ed Mooberry and other members of the National Magnetic Resonance Facility at Madison staff for gracious help.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AI32917 and U. S. Department of Agriculture Grant 95-37302-1811. Equipment in the National Magnetic Resonance Facility at Madison was purchased with funds from the University of Wisconsin, National Science Foundation Biological Instrumentation Program Grant DMB-8415048, National Institutes of Health Biomedical Research Technology Program Grant RR02301, National Institutes of Health Shared Instrumentation Program Grant RR02781, and funds from the U. S. Department of Agriculture.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 (codes 1b1v and 1b5n) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
§ To whom correspondence should be addressed: National Magnetic Resonance Facility at Madison, Dept. of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706-1544. Tel.: 608-262-0459; Fax: 608-262-3759; E-mail: volkman{at}nmrfam.wisc.edu.
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ABBREVIATIONS |
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The abbreviations used are: PSP1, plasmatocyte-spreading peptide 1; GBP, growth blocking peptide; EGF, epidermal growth factor; hEGF, human EGF; hTM5, fifth EGF-like domain of human thrombomodulin; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; RMSD, root mean square deviation.
2 Chemical shift assignments of PSP1 have been deposited in the BioMagResBank Data Base (accession number 4292).
3 B. F. Volkman, unpublished results.
4 K. D. Clark, unpublished results.
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REFERENCES |
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