From the Department of Biology and Biochemistry,
University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom and
the ¶ Biochemistry Section, Surgical Neurobiology Branch, NINDS,
National Institutes of Health, Bethesda, Maryland 20982
Received for publication, November 22, 2000, and in revised form, January 3, 2001
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ABSTRACT |
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Eosinophil-derived neurotoxin (EDN), a basic
ribonuclease found in the large specific granules of eosinophils,
belongs to the pancreatic RNase A family. Although its physiological
function is still unclear, it has been shown that EDN is a neurotoxin
capable of inducing the Gordon phenomenon in rabbits. EDN is also a
potent helminthotoxin and can mediate antiviral activity of eosinophils against isolated virions of the respiratory syncytial virus. EDN is a
catalytically efficient RNase sharing similar substrate specificity with pancreatic RNase A with its ribonucleolytic activity being absolutely essential for its neurotoxic, helminthotoxic, and antiviral activities. The crystal structure of recombinant human EDN in the
unliganded form has been determined previously (Mosimann, S. C., Newton, D. L., Youle, R. J., and James, M. N. G. (1996) J. Mol. Biol. 260, 540-552). We have now
determined high resolution (1.8 Å) crystal structures for EDN in
complex with adenosine-3',5'-diphosphate (3',5'-ADP),
adenosine-2',5'-di-phosphate (2',5'-ADP), adenosine-5'-diphosphate (5'-ADP) as well as for a native structure in the presence of sulfate
refined at 1.6 Å. The inhibition constant of these mononucleotides for
EDN has been determined. The structures present the first detailed
picture of differences between EDN and RNase A in substrate recognition
at the ribonucleolytic active site. They also provide a starting point
for the design of tight-binding inhibitors, which may be used to
restrain the RNase activity of EDN.
The eosinophil-derived neurotoxin
(EDN)1 is an eosinophil
protein stored in the matrix of the large secretory granules (1). It is
a small, basic protein (2) that belongs to the pancreatic ribonuclease
A (RNase A; EC 3.1.27.5) superfamily (3) and is also known as RNase-2
or RNase Us. EDN was initially identified by its ability to
induce the Gordon phenomenon (muscle stiffness, ataxia, incoordination,
and spasmodic paralysis) when injected into rabbits (4, 5). Its
neurotoxic effect is achieved through a selective killing of cerebellar
Purkinje cells (6). The protein also displays cytotoxicity against
helminths, single-stranded RNA viruses, and respiratory epithelial
cells; and a role as a host defense protein has been suggested (7).
Damage of host tissues by EDN could contribute to the secondary effects
associated with inflammatory disorders and hypereosinophilic syndromes
(8).
EDN shares 36% amino acid identity with RNase A and 67% identity with
a related eosinophil RNase, eosinophil cationic protein (ECP, also
known as RNase-3) (7). EDN's enzymatic activity is essential for its
neurotoxic, helminthotoxic, and antiviral activities (9-11) and is 3- to 30-fold lower than that of RNase A, depending on the substrate used
(9, 12).
The crystal structure of recombinant EDN in complex with sulfate has
been determined previously at 1.83-Å resolution (13). The topology of
the EDN molecule includes the RNase A fold and core ribonucleolytic
active site architecture (14), which is conserved among all these
molecules, although both ECP (15) and EDN exhibit significant
differences at the peripheral substrate-binding sites (16).
The core of the catalytic site of RNase A consists of subsites
B1, P1, and B2. These subsites
accommodate the phosphate where phosphodiester bond cleavage occurs
(P1) and the nucleotide bases on the 3' and 5' sides of the
scissile bond (B1 and B2, respectively) (17).
In addition, several studies (18-20) have identified additional sites,
including P0 and P2. P0 interacts
with the 5'-phosphate of a nucleotide base bound at B1 and
P2 interacts with the 3'-phosphate of a nucleotide base
bound at B2 (for recent reviews see Refs. 20-22). The
three main catalytic residues of RNase A (His-12, Lys-41, and His-119
of the P1 subsite) are present in EDN as His-15, Lys-38, and His-129. The key B1 residues, Thr-45 and Phe-120 in
RNase A, are also maintained in EDN as Thr-42 and Leu-130, but the
other components of this subsite differ. The B2 subsite is
partially conserved between EDN and RNase A, but subsites
P0 and P2 are not. Although EDN and RNase A
bind only pyrimidines at B1 and prefer purines at
B2, differences in B1 and B2 site
structures give rise to subtle changes in substrate specificity. With
polynucleotide substrates, EDN has a 20-fold preference for cytidine
over uridine; with dinucleotide substrates, EDN has a 2-fold preference
for cytidine at B1 and a 100-fold preference for adenosine
at B2 (9). Two sulfate anions were found in the
EDN·sulfate complex structure (13) occupying two distinct
subsites. One of these is subsite P1, whereas the other is
a site not identified in the structure of RNase A, suggested to
correspond to a new P The biological properties attributed to EDN have been related to its
ribonucleolytic activity (7). The analysis of its substrate specificity
and the identification of the residues involved in substrate
interaction would help in understanding its mechanism of action. In
addition, the identification of nucleotide-based inhibitors may lead to
therapeutic agents for use against the pathological conditions
associated with eosinophil RNases. Here we present the first structures
of recombinant EDN-nucleotide complexes (at 1.8-Å resolution) and make
a detailed comparison with the EDN·sulfate structure at higher
resolution (1.6 Å) than the previous reported structure (13). The
structures of the complexes have revealed a detailed picture of
critical residues involved in the P1 and B2
substrate-binding sites and their flexibility in interaction with
different adenylic nucleotides. The analysis of the sulfate-containing
structure and comparison with the nucleotide complexes also confirm the
presence of a previously suggested P Materials--
Uridylyl-3',5'-adenosine (UpA) was from ICN
Biochemicals. Adenosine-5'-diphosphate (5'-ADP), adenosine
2',5'-diphosphate (2',5'-ADP) and adenosine-3',5'-diphosphate
(3',5'-ADP) were from Sigma Chemical Co.
Protein Purification and Crystallization--
Recombinant EDN
was expressed in Escherichia coli and purified as described
previously (13, 23). Briefly, a synthetic gene for human EDN was cloned
into the pET11c expression vector, and the protein was purified from
inclusion bodies. The recombinant EDN, which contains an additional
methionine residue at the N terminus, has the same specific activity as
the protein purified from the natural host (23) and does not have the
post-translational modifications that are present in EDN isolated from
human body fluids.
Crystals of recombinant EDN were grown using the hanging drop/vapor
diffusion method from drops containing 9 mg/ml protein in 0.1 M sodium cacodylate buffer (pH 6.5), 0.75 M
ammonium sulfate, and 2.5% ethanol. Drops were equilibrated against
reservoirs containing 0.1 M sodium cacodylate buffer (pH
6.5), 1.5 M ammonium sulfate, and 5% ethanol. Single
crystals appeared after 3-4 days at 16 °C.
Diffraction Measurements--
The EDN crystals diffracted to a
minimum Bragg spacing of 1.6 Å on a Synchrotron radiation source at
100 K using a cryoprotectant solution containing 0.1 M
sodium cacodylate buffer, pH 6.5, 1.3 M sodium potassium
tartrate, and 30% 2-methyl-2,4-pentanediol. The systematic absences
and symmetry were consistent with the space group
P212121 with the
following unit cell dimensions: a = 53.4 Å,
b = 57.2 Å, and c = 42.2 Å. There is
one EDN molecule per crystallographic asymmetric unit, and ~50% of
the crystal volume is occupied by solvent. The EDN·2',5'-ADP,
EDN·3',5'-ADP, and EDN·5'-ADP complexes were obtained by soaking
native EDN crystals with 100 mM 2',5'-ADP, 3',5'-ADP, or
5'-ADP for at least 2 days prior to data collection. Because the native
crystals were grown in the presence of ammonium sulfate, sulfate ions
were expected to bind at the active site of EDN as observed previously
(13). Hence, prior to soaking, EDN crystals were transferred to 1-2 ml
of a solution containing 1.5 M sodium potassium tartrate,
5% ethanol, and 0.1 M sodium cacodylate buffer, pH 6.5, for at least 2 days to remove the bound sulfate ions.
Diffraction data for the EDN·sulfate complex were collected to 1.6-Å
resolution at 100 K using the Synchrotron Radiation Source at Daresbury
(UK) on station PX 9.5 using a MAR300 image plate and also at the
Protein Diffraction Beamline at Ellettra-Trieste (Italy) using a MAR345
image plate. Data for the EDN·2',5'-ADP, EDN·3',5'-ADP, and
EDN·5'-ADP complexes were collected (at 1.8 Å) in-house at 100 K
using a MAR300 image plate mounted on an Enraf-Nonius rotating anode
x-ray source with CuK Structure Refinement--
All crystallographic refinement was
carried out using the program X-PLOR 3.851 (26) with the published
structure of EDN·sulfate (13) used as a starting model. The behavior
of the Rfree (27) value was monitored throughout
refinement. Several rounds of refinement, model building, individual
B-factor refinement, and bulk solvent correction as
implemented in X-PLOR 3.851 (28) were performed until the
Rfree value for every model could not be
improved any further. During the final stages of refinement, water
molecules were inserted into the model at positions corresponding to
peaks in the |Fo| Determination of Ki Values--
RNase activity of
EDN was measured by a spectrophotometric method. Assays were carried
out in 0.2 M MES·NaOH, pH 6.5, at 25 °C using
0.5-cm path length cells. UpA was used as a substrate. Substrate and
inhibitor concentrations were determined spectrophotometrically using
the following extinction coefficient: Accession Numbers--
The final atomic coordinates for the four
complexes of EDN (sulfate, 2',5'-ADP, 3',5'-ADP, and 5'-ADP) have been
deposited with the RCSB Protein Data Bank (accession codes 1HI2, 1HI3, 1HI4, and 1HI5, respectively).
Overall Structures--
The structures of EDN detailed here are
very similar to the EDN·sulfate structure reported previously (13).
The r.m.s. difference between C
In all free RNase A structures reported so far, the side-chain of
His-119, which is part of the catalytic triad, was found in two
conformations (A and B with
The r.m.s. differences between the C The Binding of 3',5'-ADP to EDN--
The 3',5'-ADP molecule is
reasonably well defined in the electron density map (Fig.
2A). The conformation of
3',5'-ADP when bound to EDN is very similar to that observed previously
for B2-bound adenosine in the complexes of RNase A with
d(pA)4 (43), d(ApTpApApG) (44), d(CpA) (37, 45), and
2',5'-CpA (45), as well as those frequently observed in free adenylic
nucleotides (46). The glycosyl torsion angle
The inhibitor binds to the P1-B2 region of the
catalytic site of EDN in a manner similar to the binding of the
analogous parts of d(ApTpApApG) (44, 45), d(CpA) (37, 45), and
2',5'-CpA (45) to RNase A (Fig. 2B). The molecular surface
of EDN calculated using the GRASP program (47) shows how the active
site cleft accommodates the 3',5'-ADP nucleotide (Fig.
3A). EDN and 3',5'-ADP engage
in seven hydrogen-bond interactions, and three water molecules form
hydrogen bonds with the inhibitor (Table
III). The 5'-phosphate binds to subsite
P1 (the distance between the phosphorous to sulfur of the
SO
Comparison of the EDN-3',5'-ADP binding mode with the RNase
A·ppA-3'-p complex (38) (PDB code 1AFK) shows equivalent interactions
at the main phosphate site P1 and at the secondary base
site B2 (Fig. 3B). Both of the 5'-phosphate of
3',5'-ADP and the 5'- The Binding of 2',5'-ADP to EDN--
The structure of 2',5'-ADP is
very well defined within the electron density map (Fig. 2C).
The conformation of 2',5'-ADP is typical for protein-bound nucleotides
and the deoxyribose takes up the energetically favored
C2'-endo anti conformation (Table II) (46). The
Although 2',5'-ADP binds to the same P1-B2
region of the EDN active site as 3',5'-ADP (Fig. 2D), there
are striking differences in their interaction with EDN. Interestingly,
in the 2',5'-ADP·EDN complex, a 2'- rather than the 5'-phosphate
occupies the P1 site. Indeed, the 2'-phosphate group forms
a similar extensive set of hydrogen bonds with the side chains of
Gln-14, His-15, and His-129 and with the amide nitrogen of Leu-130
(Table IV) as observed for the
5'-phosphate group in the 3',5'-ADP·EDN complex. However, although
the adenine is almost parallel to the imidazole of His-129, the
five-membered ring does not stack against the imidazole ring as in the
3',5'-ADP complex. Instead, the imidazole ring of His-129 adopts
conformation B (inactive) and packs against the five-membered ring of
adenine in a different orientation (Fig. 2D). There are three water molecules making hydrogen bond interactions with the inhibitor, and one of them mediates interactions between the adenine moiety and the side chain of Asn-70. A shift in orientation of both
Arg-68 and Asp-112 is observed when compared with the sulfate bound EDN
or the 3',5'-ADP complex, although these residues are not directly
interacting with the adenine. The 5'-phosphate is not involved in any
direct interactions with EDN residues and is only involved in a
water-mediated interaction with the The Binding of 5'-ADP to EDN--
The nucleotide is very well
defined in the electron density map (Fig. 2E). The
deoxyribose adopts the C2'-endo anti
energetically favored conformation (46), and the
5'-ADP is bound at the active site of EDN in an extended conformation
with the Inhibition of EDN by Mononucleotides 3',5'-ADP, 2',5'-ADP, and
5'-ADP--
The inhibition constants for these adenylic
mononucleotides have been determined spectrophotometrically. The
Ki values for 3',5'-ADP, 2',5'-ADP, and 5'-ADP are
32 ± 2, 64 ± 4, and 92 ± 7 µM,
respectively. These results are consistent with the crystallographic analysis of EDN complexes described above, i.e. among the
three complexes, 3',5'-ADP binds most avidly to EDN with a maximum
number of contacts with the protein atoms. However, when comparing the determined Ki values with the reported ones in the
literature, differences in pH and ionic strength of the assay mixture
have to be taken into account. Kinetic assays were performed at pH 6.5, the pH used for the crystallization buffer. Previous kinetic characterization of eosinophil RNases indicated that they have a lower
optimum pH for catalytic efficiency in comparison with RNase A. Sorrentino and Libonati (48) reported that of the optimum pH for
catalytic efficiency is 7.5-8.0 for RNase A and 6.5-7.0 for EDN. Thus
a lower pH optimum for the nucleotide interaction should be expected,
because RNase A has an optimum pH of 7.5 for the catalytic constant and
of 5.5 for the substrate affinity constant (49). Therefore, lower
Ki values would be expected for the assayed
mononucleotides if they are analyzed at a pH lower than 6.5. Moreover,
when comparing the reported Ki with previous kinetic
analyses of EDN, it should be considered that the present structural
and kinetic characterization has been performed with the recombinant
protein, whereas the reported kinetic assays used the natural enzyme
(50). Native EDN has several N-glycosylation sites (8) and
is also C-mannosylated at Trp-7 (51). The reported inhibition constants of the same adenylic mononucleotides for RNase A
are in the range of 1 to 8 µM (52, 53), indicating that
these nucleotides have a much higher affinity for RNase A. A
10-fold difference in the Ki values has also been
observed while studying the affinity of dinucleotides for either RNase A or EDN (50), in agreement with kinetic data that show a lower catalytic efficiency for the RNA transphosphorylation reaction with EDN
(9).
EDN·Sulfate Complex--
As reported by Mosimann et
al. (13), two sulfate ions were found in the EDN structure
occupying two distinct subsites (Fig. 4).
The first sulfate, SO All three ligands, 3',5'-ADP, 2',5'-ADP, and 5'-ADP bind to the
catalytic site of EDN, each one in a significantly different manner. In
the EDN·3',5'-ADP complex, the 5'-phosphate binds at the
P1 site engaging in similar interactions with EDN to those observed for phosphate groups bound to RNase A subsite P1.
This was anticipated, because subsite P1 is conserved
between RNase A and EDN. The adenosine binds in an identical manner to
that observed for adenosines bound to the subsite B2 of
RNase A. Subsite B2 is only partially conserved between EDN
and RNase A; residues Asn-67, Gln-69, Asn-71, and Glu-111 in RNase A
are replaced by residues Asn-65, Arg-68, Asn-70, and Asp-112 in EDN.
Atoms N6 and N1 of the adenosine of 3',5'-ADP form hydrogen bonds with Asn-70, and the adenine ring is engaged in stacking interactions with
the imidazole of His-129. All these interactions are very similar to
those observed for the adenosine bound to B2 in the RNase A
complex with d(ApTpApApG) (44), d(CpA) (37), and 2',5'-CpA and
3',5'-d(CpA) (45). However, the replacement of RNase A residue Gln-69
by Arg-68 in EDN restricts EDN from forming an additional hydrogen bond
with adenine as seen in the RNase A·d(ApTpApApG) complex (44). In
addition, the substitution of a Gln residue by Arg seems to force the
adenine ring to occupy a slightly different position. The hydrogen bond
between Arg-68 and Asn-65 observed in the sulfate-bound structure is
not present in the 3',5'-ADP and 2',5'-ADP complexes, where the Arg-68
side chain can shift toward the base. The position of the 3'-phosphate
of 3',5'-ADP indicates a possible location of subsite P2 in
EDN. In RNase A, Lys-7 is the main component of subsite P2
and in EDN it is replaced by Trp-10. The 3'-phosphate group forms a
hydrogen bond with the main-chain nitrogen of the N-terminal residue
Met-0 (an interaction that should not exist in the natural EDN because
Met-0 is an addition due to the expression system) and a water-mediated
interaction with the side chain of Trp-10. In addition, this phosphate
group forms several van der Waals contacts with Trp-10. Therefore, it appears that Trp-10 is the sole component of EDN subsite P2
and contributes only through nonpolar interactions to substrate binding.
The observed binding mode of 2',5'-ADP to EDN was not anticipated on
the basis of the 3',5'-ADP binding to EDN. In the EDN·2',5'-ADP complex, it is the 2'-phosphate that binds to the P1
subsite, whereas the 5'-phosphate points toward the N terminus and is
involved only in water-mediated interactions with the N-terminal
residue Met-0. In addition, there are no interactions between this
phosphate group and Trp-10 (the sole component of subsite
P2), which is 6.9 Å away. The adenosine binds to the
purine binding subsite in a different mode to that observed in previous
RNase A·nucleotide complexes (37-39, 44, 45). The adenine ring is in
a different orientation and does not pack against the imidazole ring of
His-129 due to steric hindrance. In doing so, His-129 adopts
conformation B (as described for RNase A His-119, the inactive
conformation). On the other hand, the 2'-phosphate group of 2',5'-ADP
forms hydrogen bond interactions with EDN at the main phosphate active
site P1 similar to those observed for the 5'-phosphate of
3',5'-ADP. From the present study, we are unable to explain why it is
the 2'-phosphate instead of the 5' that binds to subsite P1
of EDN. Modeling of the 2',5'-ADP molecule onto the 3',5'-ADP structure
revealed that the EDN active site could easily accommodate 2',5'-ADP
with the 5'-phosphate group in P1 and the 2'-phosphate
pointing toward the solvent. This mode of binding might also bring the
adenine moiety to form stacking interactions with His-129. However, the binding mode of 2',5'-ADP to EDN shows the flexibility of the B2 site in EDN, the ability of the P1 site to
accommodate either a 5'- or a 2'-phosphate and the low affinity for
phosphate anions of subsite P2 noted above.
In the EDN·5'-ADP complex it is the EDN is one of a relatively small array of proteins that are
C-mannosylated which involves the attachment of an The EDN sulfate structure has confirmed the position of the two sulfate
molecules at P1 and P
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 phosphate-binding subsite.
1 subsite for EDN
(13). In addition, kinetic results suggest that these nucleotides can
serve as a starting point toward the design of potent inhibitors of
EDN.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
radiation. Raw data images were
indexed, integrated, and corrected for Lorentz and polarization effects
using the program DENZO (24). All data were scaled and merged using the
program SCALEPACK (24). Intensities were then truncated to amplitudes
by the TRUNCATE program (25). Details of data processing statistics are
presented in Table I.
|Fc|
electron density maps with heights greater than 3
and at hydrogen
bond forming distances from appropriate atoms.
2|Fo|
|Fc|
calc maps were also used to
verify the presence of the peaks. Water molecules with a temperature
factor higher than 65 Å2 were excluded from subsequent
refinement steps. In the case of EDN·ligand complexes, the ligand
molecule was included during the final stages of refinement. The
details of refinement are given in Table
I. The program PROCHECK (29) was used to
assess the quality of the final structure. Analysis of the Ramachandran (
) plot (30) showed that all residues lie in the allowed regions.
Crystallographic statistics
261 = 23,500 M
1 cm
1 for UpA (31) and
259 = 15,400 M
1
cm
1 for 5'-ADP, 2',5'-ADP, and 3',5'-ADP (32). The
activity was measured by following the initial reaction velocities
using the difference molar absorbance coefficient
286 = 570 M
1 cm
1 for the
transphosphorylation reaction of UpA (33). Ki was
determined by the Dixon method (34) using three different substrate
concentrations from 0.15 to 0.4 mM of UpA, and eight inhibitor concentrations from 5 to 400 µM;
1/vo, where vo is the initial velocity, was plotted against the inhibitor concentration [I]
(data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
atoms in the two
sulfate-bound structures is 0.15 Å. The corresponding values for the
EDN·3',5'-ADP, EDN·2',5'-ADP, and EDN·5'-ADP complexes are 0.31, 0.29, and 0.28 Å, respectively. In the present EDN·sulfate structure, the additional methionine (Met-0) residue from the recombinant protein was observed.
1 = 150° and
1 =
60°, respectively) (35, 36). Conformation A is
compatible with nucleotide binding (37-39) whereas low pH or the
presence of sulfate/phosphate in the active site favors conformation B
(40-42). The side chain of the corresponding EDN residue, His-129, is
in conformation B in the EDN·sulfate (
1 =
67°),
EDN·2',5'-ADP (
1 =
65°), and EDN·5'-ADP (
1 =
74°) complex structures, whereas in the
EDN·3',5'-ADP (
1 = 152°) complex structure it is in
conformation A.
atoms of the
structures of the present 1.6-Å resolution EDN·sulfate complex and
those of the 3',5'-ADP, 2',5'-ADP, and 5'-ADP complexes are 0.29, 0.27, and 0.27 Å, respectively. The corresponding values within the complexes, 3',5'-ADP against 2',5'-ADP and 5'-ADP are 0.27 and 0.31 Å,
respectively, whereas the r.m.s. difference between the 2',5'-ADP and
the 5'-ADP complexes is 0.18 Å. The differences between the four
protein structures are very small, concentrated in the loop regions,
and seemingly unrelated to the presence of the different inhibitors.
The EDN·sulfate structure, determined at 1.6-Å resolution, contains
152 water molecules. The structures of the 3',5'-ADP, 2',5'-ADP, and
5'-ADP complexes are at a slightly lower resolution (1.8 Å) and
contain 123, 110, and 132 water molecules, respectively. The residues
at the active site are oriented similarly in all four complexes, with
the exception of His 129 (noted above), and there are no significant
conformational changes due to inhibitor binding. A numbering scheme
and torsion angle definitions are shown in Fig.
1 using 5'-diphosphoadenosine
3'-phosphate (ppA- 3'-p) as a reference molecule (38) for the three
adenylic mononucleotides.
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Fig. 1.
General numbering scheme and torsion angle
assignment for ppA-3'-p (38, 46).
adopts the
anti-conformation, whereas the ribose is at the
C2'-endo conformation. The
torsion angle is
in the unusual sp range (Table
II), but its value (
29°) is very
close to the highly favorable
sc region (
30° to
90°) (46) and the 5'-phosphate group is oriented toward the
adenosine. The
torsion angle, dictated by the orientation of the
3'-phosphate group, is in the +ac region as commonly found
in both free and protein-bound nucleotides (46).
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Fig. 2.
A, C, and E,
diagrams of the 1.8-Å sigmaA 2|Fo| |Fc| electron density map of 3',5'-ADP,
2',5'-ADP, and 5'-ADP, respectively. Electron density maps were
calculated using the standard protocol as implemented in X-PLOR 3.851 (60) from the EDN model before incorporating the coordinates of each
inhibitor, are contoured at the 1.0
level, and the refined structure
of the inhibitor is shown. B, D, and
F, diagrams showing the interactions of 3',5'-ADP,
2',5'-ADP, and 5'-ADP with EDN, respectively. EDN residues are drawn as
ball-and-stick models, water molecules appear as gray
spheres, and the nucleotide molecules are shown in dark
gray. Hydrogen bonds are indicated by dashed
lines.
Torsion angles for 3',5'-ADP, 2',5'-ADP, and 5'-ADP when bound to EDN
-amino group of Met-0 at the N terminus and participates in a
water-mediated interaction with the side chain of Trp-10. There are
also numerous van der Waals contacts between the inhibitor and Trp-7,
Cys-62, Arg-68, Asn-70, Ala-110, Val-128, and His-129. With the
exception of residue His-129, a shift in Asp-112 side-chain orientation
and a slight movement of Arg-68 (~1.0 Å) from its position in the
EDN·sulfate complex toward the inhibitor, there are no other
significant conformational changes in the catalytic site of EDN upon
3',5'-ADP binding.
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Fig. 3.
A, the molecular surface of EDN
calculated using the program GRASP (47). The 3',5'-ADP molecule
is shown in red as a ball-and-stick model. The
EDN residues interacting with 3',5'-ADP are shown in yellow.
B, superimposed structures of EDN-3',5'-ADP and RNase
A·ppA-3'-p (38). EDN and RNase A residues are shown in
green and light gray, respectively. The
nucleotides 3',5'-ADP and ppA-3'-p are drawn in dark green
and black, respectively. Hydrogen bonds are indicated by
dashed lines. Diagram was drawn with BOBSCRIPT (61).
Hydrogen bond interactions of 3',5'-ADP
-phosphate of ppA-3'-p are located at
P1. His-119 in RNase A and His-129 in EDN are both in the
same plane and stack against the five-membered ring of adenine.
Although the binding of adenine in both EDN-3',5'-ADP and RNase
A-ppA-3'-p is almost coplanar (as observed in RNase A complexes with
substrate analogs d(CpA) (37) and d(ApTpApA) (44)), the six- and
five-membered rings are reversed. Finally, the 3'-phosphates of
ppA-3'-p and 3',5'-ADP are located at the N-terminal ends of RNase A
and EDN, respectively.
torsion angle around the C4'-C5' bond of the adenosine is in the
+sc range, and the
torsion angle has a value of 158°.
Both these angles are in the range frequently observed in protein-bound
nucleotides (46).
-amino group of Met-0 at the N
terminus (Fig. 2D). The binding of 2',5'-ADP does not
trigger any conformational changes at the active site of the EDN
molecule and, apart from residues Gln-14, His-15, His-129, and Leu-130,
2',5'-ADP does not interact directly with any other parts of EDN.
Hydrogen bond interactions of 2',5'-ADP
torsion angle is
in the +sc range. The
and
pp torsion angles around the
phosphoester bond are in the +sc and +ac
range, respectively. These values are in the accepted range
for both free and protein-bound nucleotides (Table II) (46).
- rather than the
-phosphate group at subsite P1 and the adenosine located in a region away from the
B2 subsite close to but not exactly at B1 (Fig.
2F). The
-phosphate group engages in a hydrogen-bonding
network with the peptide nitrogen of Leu-130 and the side-chain atoms
of Gln-14, His-15, and His-129 (found in conformation B). The
-phosphate forms a hydrogen bond with the
-amino group of Lys-38,
whereas the 2'-hydroxyl group of the ribose makes two hydrogen bonds
with the main-chain atoms of Gln-40 (Table
V). Furthermore, EDN and 5'-ADP
participate in an extended water-mediated hydrogen bond network
involving seven water molecules, and the residues Trp-7, Gln-14,
Arg-36, Asn-39, Gln-40, Val-128, His-129, Leu-130, and Asp-131. In
addition, 5'-ADP also has van der Waals interactions with His-82.
Hydrogen bond interactions of 5'-ADP
1 on the basis
of its proximity to the P
1 phosphate of (Tp)4 upon superposition of the EDN·sulfate structure onto the RNase A-(Tp)4 complex (54). In both structures the
SO
1
and the N
2 atoms of the side chain of Arg-36, and with the
main-chain atoms of Asn-39 and Gln-40; there is also an interaction
with a conserved water molecule (Fig. 4D and Table VI). The
only interaction described in the previous structure (13), which is not
observed in the present structure, is the involvement of the side chain
of Gln-40 with SO
View larger version (30K):
[in a new window]
Fig. 4.
A and C, diagrams of the
1.8-Å sigmaA 2|Fo| |Fc| electron density map calculated using the
standard protocol as implemented in X-PLOR 3.851 (58) from the EDN
model before incorporating the coordinates of sulfates A and B,
respectively. The map is contoured at the 1.0
level. The EDN
residues are shown in light gray. B and
D, diagrams showing the interactions of sulfate ions A and B
with EDN, respectively. EDN residues are drawn as ball-and-stick
models, water molecules appear as dark gray spheres,
and the sulfate molecules are shown in black. Hydrogen bonds
are indicated by dashed lines. Diagrams were drawn with
BOBSCRIPT (61).
Hydrogen bond interactions of sulfate anions
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphate rather than the
-phosphate that occupies the P1 subsite while the
adenosine binds close to the EDN pyrimidine binding subsite
B1. The
-phosphate engages in interactions similar to
those made by the phosphate groups and sulfate ion located at
P1 as seen in the other two EDN complexes described above.
In addition, the
-phosphate group forms a hydrogen bond with Lys-38.
This mode of binding for the pyrophosphate group has been observed
previously in RNase A complexes. In previous structural studies of
RNase A with three potent nucleotide inhibitors that had a
5'-pyrophosphate group (38, 39), the
- instead of the
-phosphate
was found to bind to subsite P1. That mode of binding drove
the adenosine to adopt the syn instead of the
anti conformation with the six- instead of the five-membered ring of the adenine stacking against the imidazole ring of His-119. However, in EDN this does not occur. Instead, the binding of the
-phosphate group in the P1 position forces the adenosine
out of B2 to a new location. Thus in this complex the
binding of the adenosine to EDN is stabilized through two hydrogen
bonds made by the ribose with Gln-40 and through van der Waals
interactions of the adenine ring with Lys-38, Gln-40, and His-82.
-mannosyl residue
via a C-C link to the indole moiety of the first tryptophan in the
recognition sequence of Trp-X-X-Trp (55, 56). The
site of mannosylation is Trp-7, and the modification is absent in the recombinant protein (enzymatically active) used in this study (23).
There is much interest in this novel post-biosynthetic modification but
its role in structure and/or activity is not yet known. In the
recombinant EDN·nucleotide complexes presented here, Trp-7 makes van
der Waals interactions with the nucleotide in both 3',5'-ADP and 5'-ADP
complexes at the active site of the protein. Based on these structural
observations, we predict that these interactions will also be present
in the natural protein. It is quite likely that the mannosyl residue(s)
is(are) positioned on the opposite face of the active site and
may not have a direct role on inhibitor binding to the protein. This
hypothesis can only be tested through structural study of one or more
of these inhibitors with the C-mannosylated protein.
1 sites. The presence of
an additional anchoring site at P
1 was also proposed for
ECP (a close homolog of EDN) both by structural analysis (15) and
kinetic studies (16). Superpositions of the structures of the
EDN·3',5'-ADP, EDN·2',5'-ADP, EDN·5'-ADP, and EDN·sul-fate complexes (Fig. 5) reveal that the
positions and the orientations of all three phosphate groups and
sulfate A at the P1 subsite are almost identical. All these
groups participate in a similar hydrogen bond pattern with EDN and seem
to be the anchoring point for each inhibitor. The binding of the
adenosine is optimal only in the case of 3',5'-ADP, because this is the
only inhibitor where the adenosine ring forms hydrogen bonds with EDN
and binds in the purine binding subsite. The positions of the adenosine
moiety in the other two complexes seem to be dictated by the binding of
the phosphate in P1. To optimize their interactions, each
ligand adopts a different conformation, indicating that diversity in ligand binding can be achieved with subtle modifications to the parent
ligand molecule mainly through the flexible side chain of His-129.
Examples where analogous inhibitors adopt different binding mode/s are
well documented (57). For example, 2-deoxy-D-glucose 6-phosphate and D-glucose 6-phosphate, which differ only in
one hydroxyl group, bind in a totally different manner to glycogen phosphorylase (58).
View larger version (24K):
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Fig. 5.
Stereo views of the superimposed structures
of 3',5'-ADP (cyan), 2',5'-ADP
(orange), 5'-ADP (green), and
sulfates (red) when bound to EDN.
The analysis of EDN·ligand complexes and the comparison with the new sulfate bound EDN at higher resolution (1.6 Å) allowed the identification of some of the key residues implicated in EDN substrate binding. The residues implicated in the main phosphate active site P1 have been confirmed, and we have been able to analyze the B2 site environment and its flexibility for different adenylic mononucleotides. Furthermore, we have confirmed that Trp-10 is the sole component of subsite P2. Although the mononucleotides interact with the enzyme in quite different orientations, some common features are observed. In all three complexes, one of the phosphates is invariably located at P1, even when the adenine does not directly interact with the protein (e.g. 5'-ADP complex) and adopts a completely different orientation. We can conclude that the interactions at P1 are the main driving force for all the observed nucleotide binding analyzed. Interactions at P1 are conserved for the three adenylic nucleotide complexes and the sulfate complex, as is the case in the well documented RNase A·nucleotide complexes. On the other hand, the position of the adenine base is considerably different for each complex. A position of the adenine analogous to the substrate interaction is only feasible in the 3',5'-ADP complex. The comparative analysis of EDN complexes and the sulfate-bound structure have allowed the identification of the residues directly involved in the ligand interaction. We can therefore conclude that adenine binding in EDN mainly involves ribonucleolytic active site residues Asn-70 and His-129.
The EDN·inhibitor complexes presented here suggest ways for further
rational design of tight binding inhibitors of this enzyme against
pathological conditions associated with eosinophil RNases. It also
highlights that subtle alterations in the chemical structure of an
inhibitor can generate significant changes upon binding to the
protein. Thus the process of rational design may not follow a
predictable course. However, the observations presented here emphasize
the importance of crystal structure analysis intertwined with modeling
studies toward achievement of significant enhancement in potency in the
inhibitor design process. Finally, the binding mode of nucleotides with
EDN should also prove useful for the design of inhibitors of other
biologically active RNase superfamily enzymes.
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ACKNOWLEDGEMENTS |
---|
We thank the staff at the Synchrotron Radiation Source, Daresbury (UK) and Sincrotrone Trieste (Italy) for their help during diffraction data collection and Michael James and Steven Mosimann for the atomic coordinates of EDN at 1.8-Å resolution. We also thank members of the Structural Biology Group for the constructive criticisms of the manuscript.
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FOOTNOTES |
---|
* This work was supported by a Medical Research Council (UK) Programme grant (9540039) and by a Wellcome Trust (UK) Equipment grant (055505) (to K. R. A.).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 1HI2, 1HI3, 1HI4, and 1HI5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Current address: Institute of Biological Research and Biotechnology, The National Hellenic Research Foundation, 48 Vas Constantinou Ave., Athens 11635, Greece.
** To whom correspondence should be addressed: Tel.: 44-1225-826-238; Fax: 44-1225-826-779; E-mail: K.R.Acharya@bath.ac.uk.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M010585200
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ABBREVIATIONS |
---|
The abbreviations used are: EDN, eosinophil-derived neurotoxin; RNase A, bovine pancreatic ribonuclease A; ECP, eosinophil cationic protein; UpA, uridylyl-3',5'-adenosine; 3', 5'-ADP, adenosine-3',5'-diphosphate; 2', 5'-ADP, adenosine-2',5'-diphosphate; 5'-ADP, adenosine-5'-diphosphate; d(CpA), deoxycytidyl-3',5'-deoxyadenosine; 2', 5'-CpA, cytidylyl-2',5'-adenosine; d(Up), 2'-deoxyuridine-3'-phosphate; ppA-3'-p, 5'-diphosphoadenosine 3'-phosphate; r.m.s., root mean square.
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