From the Department of Biochemistry and Molecular
Biophysics, Washington University School of Medicine, St. Louis,
Missouri 63110, the § Department of Cellular Biology and
Anatomy, Medical College of Georgia, Augusta, Georgia 30912, and the
¶ Department of Genetics, Washington University School of
Medicine, St. Louis, Missouri 63110
Received for publication, November 20, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Three-dimensional models of the catalytic
domains of Nudel (Ndl), Gastrulation Defective (Gd), Snake (Snk), and
Easter (Ea), and their complexes with substrate suggest a possible
organization of the enzyme cascade controlling the dorsoventral fate of
the fruit fly embryo. The models predict that Gd activates Snk, which in turn activates Ea. Gd can be activated either autoproteolytically or
by Ndl. The three-dimensional models of each enzyme-substrate complex
in the cascade rationalize existing mutagenesis data and the associated
phenotypes. The models also predict unanticipated features like a
Ca2+ binding site in Ea and a Na+ binding
site in Ndl and Gd. These binding sites are likely to play a crucial
role in vivo as suggested by mutant enzymes introduced into
embryos as mRNAs. The mutations in Gd that eliminate
Na+ binding cause an apparent increase in activity, whereas
mutations in Ea that abrogate Ca2+ binding result in
complete loss of activity. A mutation in Ea predicted to introduce
Na+ binding results in apparently increased activity with
ventralization of the embryo, an effect not observed with wild-type Ea mRNA.
Several genes in the dorsal group (1) are involved in
extracellular events that lead to dorsoventral polarization of the Drosophila melanogaster embryo. nudel,
pipe, and windbeutel are expressed by somatic
follicle cells during mid-oogenesis, whereas easter,
gastrulation defective, snake, and
spätzle are expressed by the nurse cells and oocyte.
These genes were identified in several large scale genetic screens for
maternal effect mutations that cause homozygous mutant females to
produce embryos with abnormal cell fates (2). Among the dorsal group
genes, nudel, gastrulation defective,
snake, and easter encode proteins containing
serine protease domains of the trypsin family. Easter
(Ea),1 Snk, Gd, and Ndl are
expressed and secreted during oogenesis as inactive zymogens into a
thin, fluid-filled perivitelline space that lies between the eggshell
and the oocyte. Genetic and molecular studies suggest that these
proteins act in a proteolytic cascade many hours later in the early
embryo (1, 3-5). The cascade resembles in its general organization
those controlling the innate immune response and blood coagulation (6).
Ovulation of the egg in some way triggers the self-activation of Ndl
into Ndl*. Gd can be activated either by Ndl* or by self-activation in
the presence of Snk. Subsequently, Gd* activates diffusible Snk and Snk* activates diffusible Ea. The result of this cascade is cleavage by
Ea* of the diffusible dimeric nerve growth factor-like Spz (7). The
processed Spz appears to function as a dimer to activate the
transmembrane receptor Toll only on the embryo surface that will become
ventralized through the Toll signaling pathway.
In contrast to the significant knowledge garnered from previous
in vivo studies, quantitative information on activity and specificity of various members of the cascade has so far eluded characterization involving purified proteins. Several questions remain
regarding the activation of Ndl and Gd (1, 3-5) and the specificity of
Gd* and Snk*. Elucidation of these timely and important questions would
benefit from the knowledge of the structural organization of the
enzymes involved in the cascade. However, none of the members of the
cascade has been crystallized so far or even expressed successfully for
detailed in vitro characterization. Hence, we felt that the
construction of three-dimensional models of Ndl*, Gd*, Snk*, and Ea* in
complex with their targets could fill a critical structure-function gap
in the field as recently shown for thrombin interactions with the
platelet receptors (8) and fibrinogen (9). The value of these models
stems from their timeliness and the new insight offered for future
mutagenesis studies, as illustrated in the present work by the effect
on embryo polarization when putative cation binding sites of examined
proteases were mutated.
Sequence Alignment and Comparative Modeling--
The fly
sequences came from the strain Berkeley in the Flybase (FB) and
Swiss Protein (SP) databases: Ea (FBgn0000533, SP-P13582), Gd
(FBgn0000808, SP-O62589), Ndl (FBgn0002926, SP-P98159), and Snk
(FBgn0003450, SP-P05049). These sequences were aligned with 1800 serine
proteases from the trypsin family pulled from the non-redundant data
base at the National Center for Biotechnology Information (NCBI)
(National Library of Medicine, National Institutes of Health, Bethesda,
MD) and the Flybase image at the NCBI using trypsin homologues as seeds
with the BLAST program and aligned together with ClustalX as described
recently (10). Sequences were clustered into 100 groups from a neighbor
junction tree accounting 500 bootstraps with ClustalX. One hundred
sequences were selected, one per cluster. Three-dimensional models of
70 structures were built by comparative modeling based on 12 of 20 crystal structures of serine proteases used in the sequence core. These
models were used to refine the alignment of the 100-sequence core (10). The theoretical three-dimensional models of activated protease domains
Ndl* (central or Ndl1*-(1146-1385) and C-terminal or
Ndl2*-(2017-2616)); Gd*-(256-528); Snk*-(191-430); and
Ea*-(127-392) were constructed by comparative modeling using the
program Modeller 4 (11). The following crystal structures of serine
proteases downloaded from the Protein Data Bank (PDB) (12) were
used as templates: trypsin (PDB code 1tld, 1.50 Å-resolution);
chymotrypsin (PDB code 4cha, 1.68 Å); tPA (PDB code 1rtf, 2.30 Å);
plasmin (PDB code 1bui, 2.65 Å); plasma kallikrein (PDB code 2pka,
2.05 Å); thrombin (PDB code 1ppb, 1.92 Å); factor Xa (PDB code 1hcg, 2.20 Å); factor IXa (PDB code 1rfn, 2.80 Å); factor VIIa (PDB code
1dan, 2.00 Å); and activated protein C (PDB code 1aut, 2.80 Å). These
proteases were chosen because they span the breadth of diversity of
trypsin-related domains and regulatory cation binding sites. Alignments
extracted from the 100-sequence core were optimized manually during
preliminary comparative modeling processes according to the distance
violation from templates provided by the Modeller program output files.
Two hundred models were built for each protease with different
frameshifts of alignment in the poorly conserved loops and different
seeds for the number generator. Models were then checked and ranked for
stereochemistry, structural topology features, and amino acid spatial
distribution with Procheck (13), WhatCheck (14), and Verify3D (15).
Conformers in the same clusters were pooled when the root mean square
deviations of their backbone was <2.5 Å. The best conformer was kept
for each cluster and optimized by molecular dynamics (fast annealing
50-600 K in 3 ps, slow cooling 600-50 K from 5 to 15 ps) and then
minimized (200 steepest descents and then 500 conjugate gradient
cycles) using the program Discover (Accelrys, San Diego, CA). The
following parameters were used in all of the procedures: force
field CFF91, dielectric constant set at 2, and cut off to threshold
non-covalent bonds was set at 14 Å during dynamics and set to
We screened the three-dimensional models of catalytic domains for
putative sites of cleavage by proteases. Only Arg, Lys, Ile, Leu, Phe,
and Val residues were selected if not followed by Pro provided that
their side chain was at least 50% exposed compared with same residue
in the tripeptide GXG. Every selected position i
in the model was scored for the solvent accessibility of neighbor
residues from i
Ca2+ and Na+ binding sites were identified with
the program VALE (16) using a grid of 0.1 Å, water molecule radius of
1.4 Å, and a minimum threshold for the sum of oxygen-cation
bond-strength contributions of 0.8.
Modeling of Enzyme-Substrate Complex--
Three-dimensional
models of protease-substrate complexes were built by comparative
modeling in a thorough or quick mode. In the thorough mode, the
protease-fragment complexes were threaded over thrombin-peptide crystal
structures (8, 9). We used the following templates:
peptide-Ac-DFLAEGGGVR from PDB (1bbr and 1ucy); PPACK from PDB (1ppb);
hirugen peptide-NGDFEEIPEEYL from PDB (1hah); and peptide-LDPR from PDB
(1nrs). 50 three-dimensional models were built and ranked in
terms of stereochemistry quality and lowest potential binding energies.
The accepted computer-generated models of protease-peptide substrate
complexes had root mean square deviations of <1.5 Å for the protease
backbone and peptide residues <10 Å from protease residues. Models
containing a ligand with root mean square deviations of <2 Å from a
higher ranked model were discarded. We selected the best ten models,
extracted the ligand, docked it on the best free protease
three-dimensional model as a starting point for a new modeling process,
and optimized the best complex as described above for the free
proteases. The thorough mode screened 1 of 50 three-dimensional models
of enzyme-target peptide complexes and was used to screen every
putative activation cleavage site of zymogens with every selected
protease to assess activator-activated pairs.
The quick mode was used to screen possible cleavage sites all along
sequence targets in and out of the catalytic domain. We used only one
of seven peptide three-dimensional models to template the position
P1-P11, chosen according to the length of the loop between P1 and the
closest hydrophobic side chain from P4 to P10 (seven possibilities).
Five three-dimensional models of the complex were provided by Modeler
runs, and then the best one was minimized as in the thorough mode.
Binding Free Energy Calculations--
We examined the relative
binding free energies of substrates on proteases by applying an
empirical method on bound and free components. We used the potential
energy of the system as an enthalpy term (force field CFF91), a
conformational entropy term based on solvent-accessible surface area
(SASA) of residues and a hydration free energy term based on finite
difference approximation of the Poisson-Boltzmann equation.
The predicted free energy of association between receptor (R) and
peptide (P),
The value of the conformational entropy
The size of the different ligands is very similar, and the resulting
loss of rotational and translational entropy upon binding
Based on the above definitions, the free energy for the
receptor-peptide complex becomes
Some of the terms cancel if we compare the association of same
length peptides bound to the same protease. The approximation of the
relative binding free energy is given by
This approach does not allow comparison of the binding of a
peptide to two different proteases unless the vibrational entropy variation upon binding is comparable.
The mRNA Preparation of Mutated gd and ea--
The plasmid
pNB-GD2 containing a full-length gd cDNA was obtained
from J. L. Marsh (University of California, Irvine, CA) (21). The
plasmid pGEM7Zf(+) containing a full-length ea cDNA was
obtained from K. V. Anderson (Sloan-Kettering Institute) (22). Mutations were introduced using the QuikChange Exchange kit
(Stratagene). We mutated Phe-225 to Ala and Pro in the putative
Na+ binding site of Gd. We also mutated separately Phe-225
to Ile, Ser, and Tyr to create the putative Na+ binding
site of Ea, and we mutated Glu-70 to Ala and Lys in the putative
Ca2+ binding site of Ea. mRNAs encoding wild-type and
mutant Gd and Ea were transcribed from plasmids by using the SP6
mMessage mMachine kit (Ambion, Austin, TX) and were dissolved in water
in a range of concentration from 0.06 to 1 mg/ml as estimated by UV
absorbance (4).
Fly Stocks and Embryo Injection--
The mutations and
allelic combinations used here were described previously:
gd7/gd7 (23) and
ea4/ea5022rx1 (24). Embryos
(0.5-1.5 h post-fertilization) were injected centrally at 40-60%
egg-length after the removal of the outer eggshell layer according to a
standard procedure (2). Injected embryos were visually examined during
gastrulation, and their cuticles were prepared for examination as
described previously (25, 26). The injection of mRNAs encoding
wild-type Gd or Ea was used as positive controls (4).
Identification of Cleavage Sites by Alignment of Primary
Sequences--
Zymogens Gd (amino acid 528), Snk (amino acid 430), and
Ea (amino acid 392) are organized in three domains: an N-terminal signal that is cleaved during protein secretion and a zymogen that
gives rise to A (N-terminal) and catalytic B (C-terminal) chains (Fig.
1). The A and B chains remain covalently
linked through disulfide bridges after proteolytic activation. The
topology of Ndl is more complex and unusual because it carries two S1a
protease domains. The first catalytic domain (Ndl1*-(1145-1385)) is
central, and the second (Ndl2*-(2017-2616)) is C-terminal. Eleven low
density lipoprotein (LDL) receptor-binding repeats intercalate the two protease domains (27). Four LDL receptor repeats are inserted in the
second protease catalytic domain.
To locate cleavage positions (
Ea and Snk show 25% identity overall, 33% within the B chain, and
feature the same potential disulfide bridges (Fig. 1). Alignment with
other proteases suggests that only one disulfide bridge, 1-122 in the chymotrypsin
numbering,2 links the A and B
chains. The disulfide bonds 42-58, 168-182, and 191-220 are
highly conserved in the catalytic B chain of serine proteases. Gd is
proposed to retain the disulfide bonds 42-58 and
168-182 as well as 1-122
linking the A and B chains (Fig. 1). Ndl could have five disulfide
bonds in Ndl1* (1-122 between the A and B chains
and 42-58, 136-201,
168-182, and 191-220
within the B chain). Ndl2* features only the
42-58 disulfide bond within the B chain and
1-122 between the A and B chains (Fig. 1).
Comparative Modeling of the Catalytic B
Chains--
Ndl1*-(1145-1385), Ndl2*-(2195-2616), Gd*-(212-528),
Snk*-(184-430), and Ea*-(128-392) were folded using the trypsin
scaffold (CATH 2.40.10.20; SCOP B.47.1.1) with two orthogonal
six-stranded
Fig. 2 displays the water-accessible surfaces of Ndl1*, Gd*, Snk*, and
Ea*. The overall architecture of the active site is similar in all
models, but their surfaces show notable differences in amino acid
composition. The four proteases feature the catalytic triad
His-57, Asp-102, Ser-195, and the
important ancillary residues Cys-42 and Cys-58
(SS-linked), Gly-193, Gly-196,
Gly-211, and Ser-214. The
Cys-168/Cys-182 disulfide bond stabilizes the
intervening loop that forms part of the binding site and is conserved
in all four proteases. The Cys-191/Cys-220
disulfide bond is present in Ndl1*, Snk*, and Ea* but not in Gd*. This
bond bridges the 186-loop and 220-loop that
shape the bottom of the primary specificity pocket. Binding site
pockets around residue 189 and the hydrophobic core around
residues 99, 174, and 215
differ among the four proteases.
The presence of Asp-189 in the S1 (31) pocket shows that
specificity is unambiguously trypsin-like for Ndl1* and Ea*, whereas Ser-189 suggests a chymotrypsin-like specificity for Gd*.
The presence of Gly-189 in Snk* makes the prediction of
specificity ambiguous. The shape and volume of the S1 pocket in Snk*
could accommodate a variety of side chains. Leukocyte elastase, which carries Gly189 and cleaves after Val in P1, shows a 23%
identity with Snk* in the catalytic B chain. The structure of elastase (PDB code 1ppg) complexed with the tetrapeptide AAPV (32) shows that
Val-190 defines the S1 specificity toward hydrophobic P1
residues. In the model of Snk*, the unusual His-190
(His-371) points out of the S1 pocket and interacts with
Asp-194 (Asp-375), thereby leaving the S1 pocket free to
interact with a variety of side chains besides hydrophobic residues.
Preferred Cleavage Sites from Enzyme-Substrate Three-dimensional
Models--
We predicted the position of potential protease targets in
every protease sequence based on 25-residue peptide binding energy to
each protease active site (Table I) or
the accessibility of the peptide within the catalytic domain (Fig. 1).
We computed the theoretical binding energy of all of the fragments
25-residue long with Arg or Lys at position 11 derived from the
sequence of Ndl1 (37 fragments), Ndl2 (78 fragments), Gd (60 fragments), Snk (42 fragments), Ea (38 fragments), and Spz (36 fragments) after docking them using the quick mode on Ndl1*, Snk*, and
Ea*. The same procedure was used for fragments containing Leu, Ile, Val, or Phe at position 11 derived from the sequence of Ndl1 (88 fragments), Gd (142 fragments), Snk (105 fragments), Ea (92 fragments), and Spz (74 fragments) and docked on Gd* and Snk*. All of the fragments
containing Pro at position 12 (~3% of total) and cleavage sites
inaccessible in protein three-dimensional models (score <0.5) were
discarded. We used an empirical relative binding free energy
The four proteases in complex with their primary targets were examined
further using the thorough mode. Relevant contacts of the best targets
with each enzyme are shown in Fig. 2, and the structure of the peptide and the
epitope of recognition are displayed in Fig. 3. The Ndl
fragment
1134SDSKEIVGDGR
The Gd* active site is characterized by very hydrophobic
properties of both primed and unprimed subsites (Fig. 2B).
Residue Ile-468 (Ile-194) replaces the canonical Asp in the
S1 pocket and contributes to the enhanced hydrophobicity of this site
together with Ile-463 (Ile-190) and the unusual Ile-511
(Ile-226) that replaces a highly conserved Gly. This
largely hydrophobic architecture of the S1 pocket is unusual in serine
proteases and probably compensates for the unusual Ala-488
(Ala-215) that replaces the highly conserved Trp at this
position. Residue Val-220 of Gd is potentially a good cleavage site for
Gd*. To test the possibility of Gd activation by Gd*, the Gd fragment
210PKSSDGITSPV
The Snk fragment
173SGKQCVPSVPL
The Ea fragment
116LPGQCGNILSNR
The Spz fragment
210NDLQPTDVSSR Putative Cation Binding Sites and Their Alteration in Vivo--
Many vertebrate serine proteases contain functional cation binding
sites that allosterically regulate activity and stability of the
enzymes (39, 40), but such sites have not previously been described in
invertebrate serine proteases. The inspection of the primary sequence
and screening of the dorsoventral protease three-dimensional models
with the program VALE (16) identified binding sites for Na+
in Ndl1* and Gd* and for Ca2+ in Ea*, each corresponding to
the positions of similar sites in the vertebrate proteases. The
Na+ binding sites of Ndl1* (Fig. 2A) and Gd*
(Fig. 2B) have an architecture similar to that described for
thrombin (36, 38). Two carbonyl O atoms from residues 221
and 224 contribute together with four buried water
molecules to the octahedral coordination of the cation: Arg-1361
(Arg-221) and Glu-1364 (Glu-224) for Ndl1*;
Cys-506 (Cys-221); and Gln-509 (Gln-224) for
Gd*. The Ca2+ binding site of Ea* (Fig. 2D) is
similar to that of trypsin (40) with two carboxylic side chains from
Glu-193 (Glu-70) and Glu-203 (Glu-80),
contributing to the octahedral coordination. In Ea*, three additional
carbonyl oxygens from the backbones of Thr-196 (Thr-73) and
Thr-198 (Thr-75) and the side chain of Asn-199
(Asn-76) contribute to Ca2+ binding. A water
molecule could provide the sixth oxygen in the coordination shell.
To determine whether these putative cation binding sites influence
protease function in vivo, we mutagenized key residues in Gd
and in Ea and then compared the ability of wild-type and mutant
proteases to rescue embryos lacking
maternal function for the respective proteins (Table II) (Figs.
4 and 5). In previous studies using the
same wild-type mRNAs and recipient embryos, Gd has been shown to
act in a dose-dependent
manner to cause an abnormal expansion of ventral pattern elements
("ventralization"), whereas Ea rescues to wild type but cannot
ventralize the embryo (4, 24). Similar studies could not be undertaken
for the putative Na+ binding site in Ndl, as wild-type Ndl
is unable to rescue in embryo RNA microinjection assays, presumably
because of the complex activation mechanism and early action of
this protease.3
For Gd, we injected synthetic mRNAs encoding wild-type Gd
protein or mutations Y510A (Y225A) and Y510P
(Y225P), expecting to disrupt Na+ binding (38,
39). At high doses (0.6 mg/ml), all three RNAs gave a strong
ventralization phenotype in which excess Gd activity causes too much
signaling through the Toll pathway (4), indicating that the mutant
proteins are active. At a 10-fold lower dose (0.06 mg/ml), the
wild-type RNA provides a broad range of phenotypes from strong
ventralization to moderate dorsalization (partial rescue in Table II)
while the mutant RNAs still show predominantly strongly and moderately
ventralized embryos. This finding suggests that mutants predicted to
lack Na+ binding have increased catalytic activity compared
with wild type, implying that Na+ binding to Gd* may
actually result in decreased catalytic activity.
For Ea, we compared the activity of wild type and two mutants of
Glu-193 (Glu-70): E193A (E70A) predicted to
abrogate Ca2+ binding, and E193K (E70K)
predicted to eliminate Ca2+ binding but with the charged
Lys partially substituting for Ca2+ (41, 42). These mutants
resulted in a complete loss of Ea activity equivalent to the injection
of the S337A (S195A) mutant lacking the catalytic serine,
indicating that the Glu-193 (Glu-70) residue and possibly
Ca2+ binding are critical for Ea function.
Engineering a Na+ Binding Site in
Ea--
Most invertebrate proteases contain Pro-225, which
is incompatible with Na+ binding rather than
Tyr-225 or Phe-225, which are compatible with
such binding (39). This usage dichotomy at residue 225 has
profound structural (38, 39) and evolutionary (6) implications. One
exciting possibility raised by the role of residue 225 in serine proteases (39) is the rational engineering of Na+
binding with the P225Y substitution. We surmised that Ea
would be an excellent candidate to engineer a Na+ site,
because it had already been shown that the P373S (P225S) substitution by an EMS mutation resulted in increased activity and
ventralized phenotype (24). Ser is an intermediate in the genetic
code between Pro and Tyr, and a saturation mutagenesis study has shown
that Ser is also intermediate in catalytic activity between Pro and Tyr
at position 225 in thrombin (38). We compared the
activities of Ea proteins containing the mutations P373Y
(P225Y) and P373S (P225S) with those of
wild-type Ea. We found that P373Y (P225Y), similar to P373S
(P225S), was capable of ventralizing easter-mutant embryos, something that the wild-type Ea
protein is not able to do even when injected at high levels (Table II) (24). The P373Y (P225Y) mutant had significantly stronger
ventralizing capacity than did P373S (P225S) and only
rarely resulted in wild-type gastrulation or embryo hatching, even when
titrated to a level (0.2 mg/ml) in which incomplete rescue was commonly
seen together with weak ventralization. This behavior differs from that
of previously described ventralizing easter alleles (24),
which can be titrated to give a significant level of wild-type rescue,
and suggests that this mutant enzyme may be less influenced by normal
regulatory controls (43). A P373I (P225I) substitution
resulted in a significant loss of activity with only weak partial
rescue seen. The corresponding mutation in thrombin drastically reduced
catalytic activity and did not provide Na+ binding from
the mutated thrombin crystal structure (38).
The primary cleavage sites of Ndl, Snk, Ea and Spz have been
proposed previously (3, 5, 7, 30) and are confirmed in the present
study. Ndl is secreted in the perivitelline space and is required for
the ventralization process upstream of Snk activation (30). The
activation of Gd remains controversial, but our models propose that
Ndl1* has trypsin-like activity and may bind Gd at its activation site
Lys-211 better than Snk, Ea, or Spz. We believe that the second
protease domain of Ndl*, Ndl2*, is inactive and plays no role in the
cascade. Therefore, although Gd can be activated at Val-220 by Gd*,
Ndl1* should be retained as a better potential actor in Gd
activation from predicted binding energies (3). Gd autoactivation might
be detected when Gd is overexpressed either in embryos (4) or in cell
culture (3, 5) as suggested by the predicted low affinity of Gd for its own activation site, but this leaky autoactivation might not be as
effective at physiologic expression levels of Gd. Hence, the proposed
three-dimensional models of Ndl1*, Gd*, Snk*, and Ea* are consistent
with the overall organization of the enzymatic cascade defining
dorsoventral polarity in the fruit fly as recently described from cell
culture and embryo studies (3, 5). The cascade is initiated by Gd
activation, more probably by Ndl1* as suggested in vivo (4),
and alternatively more weakly by Gd or Gd* as also proposed in
vivo previously (5). Gd* then activates Snk and Snk* activates Ea.
Ea* then processes Spz for signaling via the receptor Toll. The
three-dimensional models are also consistent with previous mutagenesis
studies of Ndl1* (33) and Ea* (24) and offer a structural explanation
of the observed mutant phenotypes.
Notably, the three-dimensional models reveal new structural features
that can be exploited in future in vivo studies. Of
particular importance is the unanticipated identification of a
Ca2+ binding site in Ea* and a Na+ binding site
in Gd* and Ndl1*. The binding of Ca2+ in trypsin stabilizes
the fold of the protease domain (40), and Na+ binding to
thrombin and many other serine proteases increases the catalytic
activity toward synthetic and natural substrates (36, 38, 39). Based on
the results presented here, it is highly likely that Ca2+
binding to Ea* plays a key role in the function of this enzyme in
vivo. Likewise, Na+ binding to Gd* and possibly Ndl1*
has functional significance. Interestingly, Na+ binding
to Gd* may actually result in the inhibition of the catalytic activity
of the enzyme in contrast to the effect observed in all other
Na+-dependent allosteric serine proteases
studied to date (39). The current knowledge on the role of residue
225 in serine proteases (39) predicts that Na+
binding can be introduced in proteases carrying Pro-225
using the Pro
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
during minimizations. The highest ranked models were used
for analysis. The in-and-out side chain distributions and the
sequence-structure compatibility analyzed with Verify3D gave the
following current score/expected score/threshold for the final
three-dimensional models: Ndl1* (118/116/52), Gd* (130/130/58), Snk*
(112/112/50), and Ea* (117/120/54). These values are comparable with
those obtained with the crystal structures used as templates. The
Ramachandran plot put
-
-dihedral angle pairs per residue mostly
in the favored and allowed regions as per the program Procheck: Ndl1*
(92.4%), Gd* (86.0%), Snk* (94.2%), and Ea* (91.4%). The
stereochemistry of the three-dimensional models satisfied Procheck and
WhatCheck requirements found for crystals of proteases solved at a
resolution lower than 2.5 Å. Solvent-accessible surfaces were
displayed with Insight II (Accelrys) using the Connolly's algorithm
with a 1.4-Å probe radius.
4 to i + 2. We defined a
protease cleavage site when [(rsc,i
4 + rsc,i
3 + rsc,i
2)/3 + ri + (rsc,i+1 + rsc,i+2)/2]/3 > 0.50, where
ri is the percentage of overall solvent
accessibility and rsc,i is the percentage of side
chain solvent accessibility.
G, was calculated considering that free R and P have the same conformation as in the complex RP from
G =
GRP
GR
GP with
Gx =
Gx,gas(
=1)
Gx,hyd(
=80) and x =RP, R, or P. The
value of
Gx,gas was calculated from its enthalpic
and entropic contributions expressed as
Gx,gas =
Hx,gas
T
Sx,gas with
Hx,gas = Ex,vdw + Ex,coul and
Sx,gas =
Sx,conf,gas +
Sx,rt,gas +
Sx,vib,gas. The enthalpy
Hx,gas is a function of the van der Waals
(Evdw) and coulombic (Ecoul)
components, whereas
Sx,gas is defined in terms of
the rotational, configurational, and vibrational components. Evdw and Ecoul were computed from
the CFF91 force-field without cut-off with
= 2.
Sx,conf,gas was computed from the loss of side
and main chain rotation freedom using the definition as shown in
Equation 1,
where
f1(rsc,i) = rsc,i8/(rsc,i8+0.5)
and rsc,i are the relative accessibility of the
ith residue side chain, rsc,i = SASAsc,i/SASAsc,i,GXG. SASAsc,i,GXG refers to
the side chain solvent-accessible surface area of amino acid
X in the tripeptide Gly-X-Gly. The empirical
scales of side chain rotation freedom,
(Eq. 1)
si were
taken from Pickett and Sternberg (17). The function
f1 decreases the entropy values when the
accessibility of the side chain is <50%. The loss of freedom of
residue i main chain dihedral angles
and
was roughly
considered as a function of the steric hindrance around residue
i
1 to i + 1, affecting the access of
allowed and core region in the Ramachandran graph.
ri is the smallest value of
SASAmc,i/SASAmc,i,GXG (accessibility of the main chains
only) or SASAi/SASAi,GXG (overall accessibility)
weighted by the attenuation function
f2(x) = x7/(x7 + 0.5). The
accessible area fraction,
i was fixed for each residue
dihedral pair
and
of X from the tripeptide
Ala-X-Ala in the Ramachandran graph: 0.28 for
X = Pro; 0.56 for X = Gly; and 0.40 for
all other amino acids according to the allowed and core region in
Procheck graphs (13).
Srt,gas between different ligands is
negligible. For 25-residue peptides associated to proteases modeled by
quick and thorough mode T
Srt,gas was
~18-20 kcal/mol at 298 K (18).
Svib,gas was not computed in the
absence of experimental data on the examined structures or their normal
mode vibrations. The main modes of vibrations are weakly affected for peptides of similar length, targeting the same site of a protease in a
slightly different conformation. The values of
Gx,hyd were calculated from their electrostatic
energy Ge and non-polar energy of hydration
Gn as
Ghyd =
Ge +
Gn. The electrostatic
energies
Ge were computed using the finite
difference Poisson-Boltzmann method implemented in the program DelPhi
(19) averaged from eight 1-Å resolution grids decayed by 0.5 Å in
one, two, or three of the x, y, and z directions. The choice of the
grid position and resolution affects final values (the mean ± S.D. is 0.8-1.8 kcal/mol). Ge values were computed
for the transfer of the solute in water from
= 2.0 to 80.0, Ge(80.0,2.0), and then in gas from
= 2.0 to
1.0, Ge(1.0,2.0), as
Ge = Ge(80.0,2.0)
Ge(1.0,2.0).
The radius was fixed to 1.4 Å for solvent molecules and 2 Å for ions.
Ionic strength was set at 145 mM, and the protonation state
and partial charge distribution were assigned by the program
Biopolymer according to the pH fixed at 7.0. The non-polar contribution
Gn was considered as linearly dependent on the
molecule solvent-accessible surface area using a surface tension
coefficient of 25 cal/mol/Å2 (20), i.e.
Gn = 25
SASA.
G =
Hgas
T
Srt,gas
T
Svib,gas
T
Sconf,gas +
Ghyd.
G ~
Hgas
T
Sconf,gas +
Ghyd.
G values refer to selected conformations and are
affected by the choice of the "best model" according to global
potential energy of the system and the goodness of its stereochemistry. The mean ± S.D. is ~2.4 kcal/mol between the
G
of the 10 best models of Snk* when it is bound to the activation site
of Ea. Lower deviations were estimated as 1.2 kcal/mol for Ea* with Spz peptide, 1.7 kcal/mol for Ndl1* with Gd peptide, and 1.4 kcal/mol for
Gd* with Snk peptide.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (47K):
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Fig. 1.
Sequence topology of Ndl, Gd, Snk, Ea, and
Spz. The sites of cleavage of signal peptide (S)
and predicted activation (scissors) are indicated.
Positions of putative secondary target sites are indicated in
pink (chymotrypsin-like target) or blue
(trypsin-like target) triangles. The secondary target sites
were predicted from high binding free energy scores and are >50%
accessible based on the three-dimensional models. Disulfide bonds
predicted by sequence alignment and from three-dimensional models are
shown in black. Also shown in the S1a domain in
orange are residues of the catalytic triad (red
circles), residues at the bottom of the S1 pocket (green
circles), and residues involved in Ca2+ or
Na+ binding (black circle). The Ndl1 and Ndl2
protease domains of Ndl are shown separately. The topologies of bovine
-trypsin (Try) and bovine
-chymotrypsin
(Chy) are reported for comparison. Low density lipoprotein
(LDL) marks the position of low density lipoprotein
receptor-like domain repeats (40 residues, Cys6-7) with
the number of repeats shown in parenthesis. Ea and Snk share a
disulfide-knot or "Clip" motif (1).
) of signal peptides, we used the
program SignalP (28). This yielded the following sites of cleavage: Ndl
1-47
48-2616 (VYH
GL, score 0.54, threshold 0.48); Gd
1-19
20-528 (TKA
VA, score 0.85, threshold 0.48); Snk
1-27
28-430 (LEA
LD, score 0.75, threshold 0.48); Ea
1-21
22-392 (SAG
QF, score 0.82, threshold 0.48), and Spz
1-25
26-326 (YEA
KE, score 0.93, threshold 0.48).
Alignment of Ea, Snk, Gd, and Ndl with other serine
proteases suggests the following cleavage sites for zymogen activation:
Ndl1* 48-1144
1145-2616 (GDGR
IVGG; trypsin-like cleavage); Snk*
28-183
184-430 (SVPL
IVGG; chymotrypsin-like cleavage); and
Ea* 22-127
128-392 (LSNR
IYGG; trypsin-like cleavage) (Fig. 1). The predicted underivatized Ea* A chain (106 amino acids, theoretical mass 12,086 Da), Ea* B chain (265 amino acids, theoretical mass 28,951 Da), Snk* A chain (156 amino acids, theoretical mass 17,372 Da), and Snk* B chain (247 amino acids, theoretical mass 27,319 Da)
agree with Western blots described by Dissing et al. (5). In
the case of Gd, no basic or hydrophobic residue occupies the canonical
position, and the closest putative cleavage site is either 30 or 22 residues upstream, specifically Gd* 20-211
212-528 (GEPK
SSDG;
trypsin-like cleavage) or Gd* 20-220
221-528 (TSPV
FVDD; chymotrypsin-like cleavage). The fragment 212-528 expressed in S2
insect cells is an active protease (3). DeLotto (29) proposed the
cleavage site Gd* 20-136
137-528 (EHIR
KLSF; trypsin-like cleavage) located 83 residues upstream of the canonical activation site. The proposed cleavage site is 12 residues upstream of a type A
von Willebrand repeat motif
(LLLDXXEXXVRXXD) as described for
complement factor B and C2. With such a cleavage, the predicted underivatized Gd* A chain (116 amino acids, theoretical mass 13,396 Da)
and Gd* B chain (390 amino acids, theoretical mass 43579 Da) agree with
the Western blots described by Dissing et al. (5). The
alignment of Spz with the protease activation sites proposes 26-220
221-326 (VSSR
VGGS; trypsin-like cleavage) as the best site of cleavage to produce the fragments documented by
SDS-polyacrylamide gel electrophoresis (5). Ndl* could also be
processed to release only the central catalytic domain Ndl1*
(241 amino acids, theoretical mass 26,862 Da) by a second cleavage
1145-1385
1386-2616 (TTPR
LLPK; trypsin-like cleavage) as shown
by LeMosy et al. (30). The cleavage site
1386-2016
2017-2616 (NLMR
LLNV; trypsin-like cleavage) is also
detectable in the C-terminal domain Ndl2 (600 amino acids).
-barrels flanking the active site groove hosting the
catalytic triad (Fig. 2). Insertions or
deletions relative to chymotrypsin occur in loops at the protein
surface and outside the active site. Although the identity between
Ndl2* and other trypsin-like proteases is low, it spreads uniformly
among all domains and especially at the level of the two
-barrels.
Ndl2* features an unusual catalytic triad, where the nucleophile
Ser-195 is coupled to Glu-102 and Ser-57 that replace the canonical Asp-102 and
His-57. There is no other example of a Ser-Glu-Ser
catalytic triad among 1800 other serine proteases in the NCBI
(www.ncbi.nlm.nih.gov) and MEROPS (www.merops.co.uk) databases,
which suggests that Ndl2* may not participate in the cascade as an
active protease. The LDL domain is inserted away from the potential
active site in the 186-loop, where insertions of various
length also exists in thrombin and tissue- plasminogen activator.
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Fig. 2.
Three-dimensional homology models of Ndl1*
(A), Gd* (B), Snk*
(C), and Ea* (D). Enzyme
residues are numbered according to chymotrypsin for ease of
comparison. The models are shown as ribbons on the
left side or as solvent-accessible surface areas on the
right side, color-coded according to amino acid
properties (Asp and Glu in red; Lys and Arg in
blue; His in purple; Ala, Ile, Leu, Met, and Val
in yellow; Phe, Trp, and Tyr in green; Asn, Cys,
Gln, Gly, Pro, Ser, and Thr in white). The Na+
(orange ball) and Ca2+ (purple ball)
binding sites are detailed in the insets. Also shown are
main residue contacts between primary targets (listed
vertically in black as 25-residue peptides) and enzymes
(with individual residues color-coded). Cleavage sites are
indicated by scissors.
G as a criterion to select optimal cleavage sites.
Values were computed for all possible cross-activation to test
protease-target specificity (Table I). The best cleavage sites for
Ndl1* are Arg-1144 in Ndl and Lys-211 in Gd. The best cleavage site for Gd* is Leu-183 in Snk. The best cleavage site for Snk* is Arg-127 in
Ea. The best cleavage site for Ea* is Arg-220 in Spz (Fig. 1, Table I).
Interestingly, the other fragments are predicted to bind with high
scores and can be regarded as secondary target sites. Fig. 1 reports
potential cleavage sites within catalytic domains sorted by
trypsin-like cleavage sites (blue, Arg or Lys in P1) or
chymotrypsin-like cleavage sites (pink, Leu, Val, Ile, and
Phe in P1) when the accessibility score of the corresponding site is >0.50. For example, Ndl1* can cleave Ndl at Arg-1385 and can
separate Ndl2 from Ndl1. Ndl1* can also cleave at Arg-1094, yielding a
fragment that may correspond to the non-diffusible 38-kDa fragment
reported by LeMosy et al. (30). Ea* and Snk* are predicted
to cleave the prodomain of Gd at Arg-187 and can generate the 50-kDa
fragment described by LeMosy et al. (3). Furthermore, Ea*
can cleave the prodomain of Snk at Arg-100, thereby producing the
50-kDa fragment observed when Ea* and Snk are coexpressed (3).
Relative binding free energies G (in kcal/mol) of
25-residue peptide-protease complexes calculated from three-dimensional
models
G = 0.0) to facilitate comparison with other
enzyme-target complexes.
IVGGSHTSALQWPF1158
and the two Gd fragments
201ESLHVAIGEPK
SSDGITSPVFVDDD225 (cleavage 30 residues upstream of the standard activation site) and
126FMTQIQLEHIR
KLSFIPDKKSSLLL150
(C2/factor B-type cleavage site 83 residues upstream of the
standard activation site) were docked on the active site of Ndl1*.
Several single mutations have been made in ndl, and their
associated phenotypes have been reported previously (33). We introduced
these mutations in the three-dimensional model of Ndl1* and optimized
the structure by 500 cycle-conjugated gradient minimization on the
residues within 6 Å from the site of mutation. Mutations are displayed in Fig. 3 where residues are visible at the protein surface in its
front view. The mutant C1114S (C1S) loses the disulfide
bond with Cys-1252 (Cys-122) that connects the A and B
chains, but it is processed and secreted normally and retains partial
activity. The A chain is usually inconsequential to function in serine
proteases (34). The mutants G1280S (G140S) and G1282R
(G142R) affect the position of the highly conserved
Trp-1281 (Trp-141) that lines part of the S1' specificity
site. Either mutation may change the backbone and side chain
orientation of Trp-141 with resulting poor substrate
binding and loss of protease activity as seen experimentally (33). The
same argument holds for the mutant V1278M (V138M) whose
bulkier side chain is expected to perturb Trp-141. The
mutant G1334R (G197R) has a protrusion into the S2' pocket
that may cause steric hindrance with the substrate backbone around the
scissile bond, thereby explaining the loss of activity seen
experimentally (33). The mutant H1355L (H215L) perturbs the
hydrophobic core next to the active site (Fig. 3A). The His
residue at this position replaces the highly conserved Trp seen in
almost all of the serine proteases. The hydrophobic residue Ile-207 of
Gd or Val-1140 of Ndl can make contacts with Leu-1355
(Leu-215). However, non-conservative replacements of
residue 215 in thrombin cause a drastic drop in activity
(35), which may explain the total loss of activity of the Ndl1* mutant
(33). The mutant A1360T (A221T) perturbs the backbone of
the 220-loop responsible for Na+ binding (36).
Mutations at the same position in thrombin result in the loss of
Na+ binding and decreased protease activity (36), which
again explains the results seen for the Ndl1* mutant (33).
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Fig. 3.
Three-dimensional homology models of
protease-substrates Ndl1*-Gd-(201-225) (A),
Gd*-Snk-(173-197) (B), Snk*-Ea-(116-141)
(C), and Ea*-Spz-(210-234) (D).
The models of catalytic domains are shown as solvent-accessible surface
areas. Peptide substrates are displayed as sticks (green for
carbons, blue for nitrogens, red for oxygens, and
yellow for sulfurs). Surface of enzyme residues making
contact (<6Å) with the corresponding substrates are
colored in orange. Mutated residues described in
the text are colored in magenta. Some mutated
residues are not visible on the particular displayed view or because
they are not accessible to solvent.
FVDDDEDDVLEHQF234 was
docked onto the active site of Gd*. A potential activation site with
chymotrypsin-like specificity is
128TQIQLEHIRKL
SFIPDKKSSLLLDP152 located
near the C2/factor B-type cleavage site (Fig. 1). Gd* carries two
insertions in the 60 and 149-loops relative to
chymotrypsin, a feature also observed in thrombin (34) where the
insertions contribute to the narrow substrate specificity. The
60-loop in Gd* covers the substrate residues at P1 and P1'.
The 149-loop is quite flexible, judging from the various
conformations obtained in the fifty best models, and interacts loosely
with substrate residues at P3'-P8' (Fig. 3B). Mutant
alleles of gd have been identified and grouped in three
complementation groups (37). Mutations in the catalytic domain G466D
(G193D), G469E (G196D), and G484D
(G211D) are all in the same group complementing with the
group with mutations in the propeptide domain. G466D places the acidic
side chain at the bottom of the S4' site but does not disturb the
aromatic cluster formed by Phe-275, Trp-388, and Phe-390. The
hypomorphic effect of this mutation (gd6) is
moderate (37). On the other hand, G469E introduces a charged side chain
into a hydrophobic cluster formed by Ile-265 (Ile-33), Phe-294 (Phe-59), Val-306 (Val-64), Val-328
(Val-90), and Ile-331 (Ile-88). The
unfavorable steric hindrance is partially compensated by backbone
torsions in the structure core in the vicinity of the catalytic Ser-468
(Ser-195) and His-292 (His-57). A similar effect is observed for the mutant G484D where the charged side chain
perturbs the hydrophobic cluster formed by Tyr-377
(Tyr-130), Leu-472 (Leu-201), Phe-445
(Phe-181), Tyr-513 (Tyr-228), and Ala-514 (Ala-229) with resulting rearrangement of the backbone
structure of the S3-S4 sites and of the 186-loop and
220-loop shaping the Na+ binding site. G469E
(gd10 allele) and G484D
(gd7 allele) dramatically compromise the
activity of Gd and yield completely dorsalized embryos.
IVGGPTRHG-LFPH197 was
docked onto the active site of Gd* with the P1 residue Leu-183 into the
chymotrypsin-like S1 pocket in contact with Ser-468
(Ser-189), Ile-463 (Ile-190), and Ile-467
(Ile-194) (contacts detailed in Fig. 2B and
structure detailed in Fig. 3B). The small side chain of
Ala-488 (Ala-215) in Gd* opens a cavity bordered by Leu-342
(Leu-97), and Tyr-344 (Tyr-99) that
accommodates the P6 residue Val-178 of Snk. Of the other Snk residues,
Val-181 at P3 contacts Ala-489 (A216) and Leu-490
(Leu-217), and Phe-195 at P11' stacks favorably against Trp-388 (Trp-141), Phe-390 (Phe-143), and
Phe-275 (Phe-34), whereas Leu-194 makes close contacts with
Leu-401 (L149d) and Phe-390 (Phe-143).
IYGGMKTKIDEFPW141 was
docked on Snk* (contacts detailed in Fig. 2C and structure
detailed in Fig. 3C). Snk* carries Gly-370
(Gly-189) in the S1 pocket, consistent with either trypsin
or chymotrypsin activity. Residue His-371 (His-190) makes
the S1 pocket more prone to interact with hydrophilic rather than
hydrophobic side chains. Ea residue Arg-127 fills the S1 pocket of
Snk*, making two H-bonds with the backbone carbonyls of His-371
(His-190) and Phe-402 (Phe-218).
VGGSDERFL-CRSIR234
(FBgn0003495, SP-48607) was docked onto the Ea* active site (contacts
detailed in Fig. 2D and structure detailed in Fig.
3D) with Arg-220 at P1 bound to Asp-332
(Asp-189). Several naturally occurring mutations of Ea*
have been identified that lead to dominant or recessive phenotypes of
dorsoventral differentiation (24). Dominant alleles are A325V
(A183V), P373S (P225S), R335C
(R192C), G336S (G193S), G371R
(G223R), G283S (G142S), V360 M
(V213M), and G131E (G19E). Recessive alleles
are G339R (G196R), G363E (G216E), S172L
(S56L), and C324Y (C182Y). We introduced these
mutations in Ea* and optimized the three-dimensional models by 500 cycle-conjugated gradient minimization of residues within 6 Å from the
site of mutation. The mutant A325V (A183V) carries a bigger
side chain in a densely packed region. We colored the positions of the
mutated residues over the Ea* surface in Fig.
3D. The mutant P373S (P225S) perturbs the
backbone of the 220-loop that is crucial for
Na+ binding and substrate recognition (36, 38). The
substitution may promote weak Na+ binding and enhanced
catalytic activity, thereby explaining the gain-of-function phenotype
observed experimentally (24). The mutant R335C (R192C)
lacks one ion-pair interaction with the bound Spz. The mutant G336S
(G193S) introduces a side chain into the S1' pocket that
may lead to an incorrect orientation of the scissile bond and loss of
catalytic activity. The mutant G371R (G223R) perturbs the
220-loop backbone. The mutant G283S (G142S) may
displace Trp-141 nearby by constraining the backbone of the
S' sites. The mutant V360M (V213M) reduces the
accessibility of the S1 pocket and impairs the binding of substrates
carrying Arg or Lys at P1. Other Ea* mutants that are expected to
compromise substrate binding are G131E (G19E) that places
an acidic side chain in a hydrophobic environment, G339R
(G196R) and G363E (G216E) that occlude the S1
pocket, S172L (S56L) that places a bulkier side chain in a densely packed region, and C324Y (C182Y) that removes the
disulfide bond and stability of the hydrophobic core next to the active site.
Rescue of ea- or gd-null embryos by wild-type and mutant mRNA
injections
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Fig. 4.
Representative gastrulation patterns of
injected embryos. Recipient embryos in A-F are of the
genotype ea4/ea5022rx1,
whereas those in G and H are
gd7/gd7. All of the
embryos are oriented with their anterior ends to the left
and dorsal surface up. Injection of Ea wild type (WT)
(A) results in a wild-type gastrulation pattern in which
cells on the ventral side invaginate to form the ventral furrow,
posterior cells migrate anteriorly on the dorsal side
(arrowhead), and a headfold is visible only faintly along
the lateral surface (asterisk). The Ea S195A
(B), Ea E70A (C), and Ea
E70K (data not shown) mutants are completely inactive in
dorsoventral patterning, giving a complete dorsalization in which there
is no ventral furrow or headfold, cells at the posterior do not
migrate, and multiple symmetric folds appear along the
anterior-posterior axis of the embryo. The Ea P225I mutant
gives a weak partial rescue (D) in which the posterior cells
migrate forward, but no ventral furrow forms and there are still
multiple infoldings along the embryo anterior-posterior axis. Ea
P225S (E) and Ea P225Y
(F) mutants cause an intermediate ventralization of the
embryo with the headfold prominent on the dorsal side
(asterisk) and some anteriorward displacement of posterior
cells along the dorsal side. Ea P225S typically gave more
anteriorward movement of these cells than did Ea P225Y,
consistent with a milder ventralization (supported by analysis of
cuticle elements in Fig. 5), but these could not be readily
distinguished in scoring gastrulation so they were grouped together in
Table II. A more extreme ventralization could be seen with the
injection of Gd wild type (WT) (G), Gd
Y225P (H), or Gd Y225A (data not
shown) in which there is a very prominent ventral furrow, no anterior
displacement of posterior cells, and only a small headfold visible on
the dorsal side of the embryo.
View larger version (154K):
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Fig. 5.
Representative cuticle patterns of injected
embryos. Recipient embryos in A-E are of the genotype
ea4/ea5022rx1, whereas those
in F and G are
gd7/gd7. When evident,
embryos are oriented approximately with their anterior ends to the
left and dorsal surface up. The external cuticle develops
late in embryogenesis, but its elements reflect the earlier patterning
along the dorsoventral axis. A, injection of Ea wild type
(WT) results in a hatching embryo with bare dorsal cuticle,
laterally derived filzkörper (arrowhead) and head
skeleton (asterisk), and rows of ventral denticles
(inverted V). B, an uninjected embryo does not
develop either lateral or ventral structures and is considered
completely dorsalized. The Ea S195A, Ea E70A,
and Ea E70K mutants showed a similar phenotype, but their
weak cuticles did not usually survive additional processing required
for injected embryos. C, Ea P225S typically
produced mildly ventralized embryos that retained filzkörper and
a partial head skeleton, but their ventral denticles extend more
dorsally than WT (this embryo was injected with 0.5 mg/ml mRNA).
D, Ea P225I produced moderately dorsalized
embryos with the rescue of filzkörper but rarely head skeleton
and never ventral denticles. E, Ea P225Y
typically produced moderately ventralized embryos lacking lateral
filzkörper or head skeletons and having moderate expansion of
disorganized ventral denticles around the embryo circumference (this
embryo was injected with 0.5 mg/ml mRNA). F, a
moderately to strongly ventralized embryo injected with Gd
Y225P mRNA (0.06 mg/ml), showing strong expansion of
ventral denticles. At high doses (0.6 mg/ml), Gd WT (G) and
the Gd Tyr-225 mutants often gave rise to embryos with
completely circumferential, disorganized ventral denticles.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Tyr replacement. However, the P225Y
substitution in tissue plasminogen activator is not sufficient to
introduce Na+ binding and actually results in reduced
catalytic activity (44). The introduction of Na+ binding in
this protease requires substitution of a large number of residues in
addition to
Pro-225.4
Therefore, it is remarkable that the P225Y substitution in Ea* has such
a profound effect on its catalytic activity, consistent with a gain of
function that likely results from Na+ binding. This
observation motivates the analysis of this protease in terms of kinetic
and direct structural studies and offers new and important insights
into ongoing efforts to engineer Na+ binding and enhanced
catalytic activity in serine proteases of medical and biotechnological relevance.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Carl Hashimoto for offering decisive guidance at early stages of this project. E. K. L. is grateful to Lora LeMosy for computing assistance and to Fu-Shin Yu and Ke-Ping Xu for the use of their phase-contrast photomicroscope in Fig. 5.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Research Grants HL49413 and HL58141 (to E. D. C.) and NS36570 (to J. B. S.).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 on-line version of this article (available at
http://www.jbc.org) contains supplemental Table III and Fig. 6.
E. D. C. dedicates this article to Professor Eraldo Antonini on the occasion of the 20th anniversary of his untimely death on March 19, 1983.
To whom correspondence should be addressed. Dept. of
Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., Box 8231, St. Louis, MO 63110. Tel.: 314-362-4185; Fax: 314-747-5354; E-mail:
enrico@biochem.wustl.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M211820200
2 Underlined numbers refer to positions aligned with chymotrypsin(ogen). Non-underlined positions refer to the corresponding zymogen precursor sequence.
3 E. LeMosy, unpublished data.
4 E. Di Cera, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Ea, Easter zymogen; Chy, chymotrypsin; Ea*, activated Easter; Gd, Gastrulation Defective zymogen; Gd*, activated Gastrulation Defective; Ndl, Nudel zymogen; Ndl*, activated Nudel; Snk, Snake zymogen; Snk*, activated Snake; Spz, Spätzle; Thr, thrombin; Try, trypsin; PDB, Protein Data Bank; FB, Flybase; SP, Swiss Protein.
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REFERENCES |
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