From the Fels Institute, Temple University,
Philadelphia, Pennsylvania 19140 and the ¶ Department of
Biochemistry and Molecular Biology, University of Kansas Medical
Center, Kansas City, Kansas 66160
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The mosquito-borne dengue viruses are
widespread human pathogens causing dengue fever, dengue hemorrhagic
fever, and dengue shock syndrome, placing 40% of the world's
population at risk with no effective treatment. The viral genome is a
positive strand RNA that encodes a single polyprotein precursor.
Processing of the polyprotein precursor into mature proteins is carried
out by the host signal peptidase and by NS3 serine protease, which requires NS2B as a cofactor. We report here the crystal structure of
the NS3 serine protease domain at 2.1 Å resolution. This structure of
the protease combined with modeling of peptide substrates into the
active site suggests identities of residues involved in substrate recognition as well as providing a structural basis for several mutational effects on enzyme activity. This structure will be useful
for development of specific inhibitors as therapeutics against dengue
and other flaviviral proteases.
Dengue viruses, members of the family Flaviviridae, are
transmitted by mosquitos, Aedes aegypti and Aedes
albopictus, and cause severe and widespread epidemics of diseases
such as dengue fever and dengue hemorrhagic fever/dengue shock syndrome
(for a review, see Ref. 1). The global distribution of dengue virus infections is comparable to that of malaria, with approximately 2.5 billion people at risk, mostly in the southern hemisphere. Nearly 5%
of the close to one million dengue hemorrhagic fever cases each year
are fatal (2). Currently, dengue infections are endemic in all
continents except Europe (for recent reviews, see Refs. 3 and 4).
Management of dengue fever is largely supportive; however, severe
hemorragic manifestation may require blood transfusions (1). No vaccine
is available to protect against dengue virus infections, and thus there
is considerable interest in developing new antiviral therapeutic agents
to combat diseases caused by dengue viruses.
Dengue virus type 2 (Den2),1 the most prevalent
of the four serotypes, contains a
single-stranded RNA of positive polarity with a type I cap structure at
the 5'-end and codes for a single polyprotein precursor (3,391 amino
acid residues for Den2) (5) arranged in the order
NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH. Maturation of the polyprotein precursor occurs both cotranslationally and post-translationally within the endoplasmic reticulum, yielding three structural proteins, C (core), prM (precursor to membrane), and E
(envelope), which are components of the virion, and at least seven
nonstructural (NS) proteins (NS1-NS5). The nonstructural proteins are thought to function in viral replication, although the
components of the viral RNA replicase have not been defined precisely.
Proteolytic processing by at least two different proteases,
one of host and the other of viral origin, liberates the individual viral proteins from the polyprotein precursor. Signal peptidase within
the endoplasmic reticulum cleaves at the C-prM, prM-E, E-NS1, and
NS4A-NS4B junctions (6-8). Processing at the NS1-NS2A junction is
mediated by a host protease within the endoplasmic reticulum which may
be either signal peptidase or another unknown enzyme (9). Maturation of
prM to M is mediated by another host protease located in an acidic
compartment encountered during the exocytic pathway and occurs at a
late step of virion morphogenesis (10). The remaining cleavages in the
polyprotein precursor are mediated by the trypsin-like serine
protease, encoded within the NH2-terminal 180 amino acid
residues of NS3 protein. This protease domain was identified based on
the sequence similarity of NS3 with several viral and cellular serine
proteases (11, 12). Besides the protease domain at the NH2
terminus, the COOH-terminal three-fourths of NS3 contains conserved
motifs found in DEXH family of several viral RNA-stimulated NTPases/RNA
helicases. The RNA helicase activity has been implicated in unwinding
of double-stranded RNA replicative intermediate. NS3 also interacts
with the viral RNA-dependent RNA polymerase (NS5) (13), and
these protein-protein interactions may facilitate the localization of
the viral replicase complex to endoplasmic reticulum membranes where
genome replication occurs (for reviews, see Refs. 14 and 15).
Analysis of polyprotein processing established that the NS3
protease, as a complex with the viral activator protein NS2B (16-18) catalyzes the cleavages at NS2A-NS2B, NS2B-NS3, NS3-NS4A, and NS4B-NS5
sites in the polyprotein which have Lys-Arg, Arg-Arg, Arg-Lys, and
occasionally Gln-Arg at the P2 and P1 positions, followed by a short
chain amino acid Gly, Ala, or Ser at the P1' position (16, 17, 19-23).
Thus, the biologically active viral protease is a heterodimeric complex
of NS2B-NS3. The importance of this protease activity in viral
viability is underscored by the finding that mutations that abolished
protease activity when introduced in the context of an infectious
cDNA clone eliminated virus recovery (16). NS2B has three
hydrophobic regions flanking a conserved hydrophilic domain of
about 40 amino acid residues. This hydrophilic region is necessary and
sufficient for activation of the NS3 protease domain in vivo
(18, 32) and in vitro. Although the hydrophobic regions of
NS2B are dispensable for the protease activity, they are required for
cotranslational membrane insertion of full-length NS2B and its
efficient activation of the NS3 serine protease domain (24).
The covalently linked trypsin-like serine protease domain and
the RNA-stimulated NTPase/RNA helicase domain are also found in
hepatitis C virus (HCV), a new member of Flaviviridae (25, 26),
although HCV does not depend on arthropod vectors for transmission (27). Moreover, unlike the arthropod-borne flaviviral NS3 protease domain, which is activated by the hydrophilic region of NS2B, the
protease domain of HCV is activated by a 19-residue
NH2-terminal hydrophobic region of NS4A protein
(28-33).
Serine proteases are perhaps the best studied (34-40) of the
four classes (serine, aspartic, metallo, and cysteine) of proteases (for a review, see Ref. 41 and the references therein). Pioneering crystallographic, biochemical, and molecular biological studies have
extensively documented the existence of a common catalytic apparatus
(Asp-His-Ser triad) participating in a conserved mechanism of catalysis
among serine proteases (35, 37-39) (for a review, see Ref. 41). The
basic mechanism consists of a charge relay system that transfers the
unfavorable negative charge on the buried carboxyl via the histidine to
the serine. This results in transfer of the Ser O In this study we report the structure of the Den2 protease
domain at 2.1 Å resolution, which provides a structural basis for the
effects of several site-specific mutations on enzyme activity (42).
Modeling of tetrapeptide substrates into the active site indicates that
a number of residues that were identified as potential determinants of
substrate specificity by sequence alignments do make contacts with the
substrate, although there are significant differences from anticipated
interactions. The structure of the Den2 protease reveals a substrate
binding cleft that is small and shallow, except for the S1 and S2
pockets, and a catalytic triad that mimics half of the charge relay
system (43).
The structures of the NS3 protease domain of HCV in the
presence and absence of a synthetic activator region of NS4A have been
reported (44, 45). Comparison of the Den2 protease structure with these
HCV protease structures reveals notable differences; for example, the
structural zinc binding site and the long hydrophobic NH2-terminal loop of the HCV protease are absent in the
Den2 protease. Taken together, this is the first structural report of
an arthropod-borne flavivirus protease, including the differences
between substrate cleavage specificities between the Den2 and HCV
serine proteases (discussed later). As such this structure will be
useful not only for the development of specific inhibitors with
therapeutic potential for treatment of diseases caused by dengue
viruses but will also serve as a model for other serine proteases of
more than 70 members of the arthropod-borne flavivirus family.
Expression and Purification of the Den2 NS3(Protease)
Domain--
5'GGGGTACCGCTGGAGTATTGTGGGAT (underlined
nucleotides 4522-4539 of Den2 genome (5) and
5'-CCCAAGCTTCTTTCGAAAAATGTCATC (underlined complementary
nucleotides 5059-5076) were used for polymerase chain reaction on the
template pTM1-NS2B-3(Pro)-PFH (24). The polymerase chain reaction
product was digested with KpnI and HindIII and
cloned into pQE-30 (Qiagen). This plasmid codes for a hexahistidine tag
fused to the NH2 terminus of the Den2 NS3 protease domain. Escherichia coli strain XL1-Blue MRF' transformed with the
6His-NS3(185aa) plasmid was grown at 37 °C in LB containing 100 µg/ml ampicillin until the OD600 nm reached about 0.5, induced with 1 mM isopropyl
1-thio- Crystallographic Analysis--
Crystals were grown in hanging
drops using a 5-mg/ml solution of the protein. Well solutions contained
200 mM Tris-HCl, pH 7.8, 200 mM LiCl, 4 mM NiCl2, 0.4%
Significant improvement to the electron density map calculated with
weighted SIRAS phases was obtained using DM (50), and a model of
approximately 40% of the protein was built into this map using Bones
and O (51). At this stage similarities to HCV NS3 protease became
apparent, and the rest of the structure was built into SIGMAA (43)
weighted 2Fo-Fc maps calculated using SIRAS phases combined with those
from the partial model. The initial model, consisting of residues
12-178, had an R value of 0.42, and refinement with XPLOR
(52) and model building using O with extension of resolution to 2.1 Å were achieved expeditiously. The slow cooling protocol (53) was used in
refinement and progress was monitored using free R values
(54). The current model includes residues 5-181 of Den2 protease and
64 solvent molecules modeled as water oxygens. The final R
value after restrained individual B factor refinement is 0.186, and the
free R value is 0.228. Root mean square (r.m.s.) deviation
in bond lengths is 0.012 Å, in bond angles 1.2°, and r.m.s. Structure Description--
A side-by-side stereo
representation of the electron density map around the catalytic triad
is shown in Fig. 1. Fig.
2a shows the overall folding
along with the conserved orientation of the catalytic triad.
Comparisons with the structures of HCV protease domain, either as a
heterodimeric complex with the activating NS4A peptide (Protein Data
Base no. 1jxp (56); see also Ref. 45) or in the absence of NS4A
(Protein Data Base no. 1a1q (44)) show similarities expected from
sequence comparisons as well as significant differences. Although both
the amino- and carboxyl-terminal
There are significant differences in the mode of catalysis by
Den2 and HCV proteases. For example, the substrate cleavage specificities of HCV and Den2 proteases are different; HCV protease prefers a Cys residue at the P1 position of the substrates for cleavages at the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites but a Thr
residue at this position for the intramolecular cleavage (NS3-NS4A
site) (28-30, 58, 59) in contrast to a basic residue (Lys or Arg) for
all of the cleavages by Den2 protease (14) (see Table
II). The activating peptide, NS4A,
enhances the efficiency of HCV protease activity in vitro to
a different extent: 100-fold for the cleavage of NS4B-NS5A site but
only 11- and 3-fold for the cleavages of NS4A-NS4B and NS5A-NS5B sites,
respectively (60). However, the Den2 protease requires NS2B for
cleavages of all protease-sensitive sites at least within the
sensitivity of currently available assays. Moreover, the region of NS2B
required for activation of Den2 protease is a hydrophilic domain of 40 residues in contrast to the HCV NS4A activator peptide, which consists
of hydrophobic residues. Thus the mode of activation of Den2 and HCV
protease domains by their respective activator peptides and their
activated states are likely to be different.
Other significant (>2 Å) deviations of Den2 from the fold
of both HCV protease structures occur in strand F1 (Fig. 2,
a and b) where there is a two-residue insertion
in HCV protease relative to Den2 protease, strand D2, the site of a
three-residue insertion in HCV protease, and strand C2 where Den2
protease has a seven-residue insertion relative to HCV protease. Strand
C2, a long 13-residue strand in HCV protease, is split into two shorter
strands of three residues each interrupted by a four-residue coil
segment in Den2 protease. Among the conserved features of all three
protease structures are the small helical turn that carries the
catalytic His residue as well as the helices close to the termini. The
spatial relationship between members of the catalytic triad, His-51,
Asp-75, and Ser-135 is strongly conserved as in other serine proteases,
with Ser-135 being within hydrogen bonding distance (2.7 Å) of N Substrate-Protease Interactions Using Molecular
Modeling--
Sequences around the in vivo cleavage sites
of Den2 protease and seven other flaviviral NS3 proteases (14) are
given in Table II. The substrate binding cleft of the Den2 protease is not very extensive (Figs. 2 and 3,
a and b) and does not appear capable of providing
specific interactions, in the absence of NS2B activating peptide, with
side chains beyond P2 and P2'. This observation is consistent with
heterogeneity of residues beyond these sites seen (14) in flaviviral
proteases (Table II). Most viral proteases are considerably smaller
than their cellular counterparts; several loops that are present in
cellular enzymes that provide specific interactions to P3, P4, and P5
side chains of substrates are absent in the viral proteases (45). It is
possible that heterodimerization with NS2B peptide could generate
additional specific interactions for side chains beyond P2 and P2',
both by altering the protease conformation into an activated state and
by interacting directly with the substrate as has been suggested for
other two-component proteases (45).
Accordingly, molecular modeling was limited to building a
tetrapeptide into the substrate binding cleft. Complexes with
substrates were modeled using well known principles of
serine-protease-substrate interactions (64). Rigid-body transformations
(57) and small manual adjustments were used throughout. Using the
relevant tetrapeptide section of the main chain from a porcine
trypsin-inhibitor complex (65) (Protein Data Base no. 1mct), the
scissile carbonyl was positioned within hydrogen bonding distance of
Table III presents a summary of
results obtained. There are three residues (Ser-131, Tyr-150, and
Ser-163) within the S1 pocket (Fig. 3b), in addition to the
catalytic Ser-135, which are potentially capable of providing specific
stabilizing interactions with the guanidino nitrogen atoms of an Arg
residue at P1. In addition, Leu-115 provides nonspecific van der Waals
interactions. These interactions can also accommodate a Lys side chain
(as found in the NS3-NS4A cleavage site; see Table II) equally well.
All of the residues that form the S1 pocket except Ser-131 and Leu-115 are absolutely conserved (14) among all of the flaviviruses listed in
Table III. Primary specific interaction of the Den2 protease with a
lysyl (NS2A-NS2B site), glutaminyl (NS2B-NS3 site), or an arginyl
(NS3-NS4A and NS4B-NS5 sites) side chain at P2 is provided by Asn-152.
The catalytic His-51 and Asp-75 as well as Gly-151 and Gly-153 make
additional interactions through their main chain atoms. These residues
are also strongly conserved among the listed flaviviruses (Table III).
The O Correlation with Mutational Studies--
Sequence
comparisons (12, 66) identified four sets of residues (labeled as
conserved regions 1-4) which were conserved between flaviviral and
cellular proteases. Conserved region 1 includes strands C1 and D1 (Fig.
2a) along with catalytic His-51; conserved region 2 comprises of strand F1 and catalytic Asp-75; conserved region 3 consists of strand D2 and includes the catalytic Ser-135 together with
several residues expected to contribute to substrate binding; and
conserved region 4 includes strand E2 with additional residues involved
in substrate recognition. Residues Asp-129, Phe-130, Tyr-150, Asn-152,
and Gly-153 were judged to be important for determination of substrate
specificity in Den2 protease as these are analogs of residues 189, 190, 213, 215, and 216 in trypsin (chymotrypsin numbering) which determine
interactions with side chains of the substrate in that enzyme. Asp-129,
in particular, was identified as the residue that lies at the bottom of
the S1 site and provides charge neutralization for the substrate Arg or
Lys side chain (12, 66). A wide ranging site-specific mutagenesis study
reported recently (42) measured change in catalytic activity of the
enzyme resulting from each of a number of mutations in the protease
domain. Residues, mutations of which resulted in significant alteration
of enzyme activity (42), are plotted in Fig. 3c on a C Mutations in Conserved Region 3--
In conserved region
3, Asp-129 and Phe-130 are residues that were expected to form part of
the S1 pocket, with Asp-129 providing charge neutralization of a Lys or
Arg residue at P1, in analogy with trypsin. However, Asp-129 could be
replaced by Glu, Ser, or Ala without significant loss of activity;
substitution by Lys, Arg, or Leu did not abolish activity completely.
Substrate modeling indicates that the side chain carboxylate of Asp-129
is distant (11Å; Fig. 3, b and c) from the S1
pocket and cannot interact with a P1 Arg or Lys side chain without
substantial main chain motion. Phe-130, likewise is too far from the P1
side chain, but both Asp-129 and Phe-130 could provide some van der
Waals contacts for a large side chain at P2', such as a Trp found at
the NS2A-NS2B cleavage site. Ser-135 is an essential residue and cannot
tolerate replacement by Ala; however, a replacement of
Ser-135 by a cysteine also renders the Den2 protease nearly inactive.
Cysteine proteases, in which a Cys plays the role of the active site
serine in serine proteases, have structural requirements that are
similar to those of serine proteases. Mechanistic details of substrate
hydrolysis for the two classes of proteases are also very similar (67). Thus it would be of interest to determine the underlying cause of the
severe loss of activity in the S135C mutant Den2 protease. Gly-133 is
in the loop that carries the catalytic Ser-135 (Fig. 3d) and
is part of a Mutations in Conserved Region 4--
Tyr-150 and Ser-131
(conserved region 3), which were mutated to a number of other residues
(42), are close enough to the basic side chains of either Arg or Lys at
P1 to form a salt bridge or hydrogen bond with small side chain
rotations. In contrast to conclusions from sequence comparisons, it is
Tyr-150 rather than Asp-129 which appears to provide primary charge
stabilization within the S1 pocket. In addition, the side chain of
Ser-163, which is also highly conserved, could stabilize a P1 Arg or
Lys in a different conformation after side chain rotation (Fig.
3c). Tyr-150, in addition, could provide stabilization for a
positively charged side chain through its aromatic electron cloud, a
mode of interaction which has been suggested earlier (68) and
proposed for the other serine proteases (44, 45). Thus replacement of
Tyr-150 by Phe will not reduce activity significantly because although
it would abolish the salt bridge/hydrogen bond to the Tyr-150-OH, it
would not abrogate aromatic stabilization and the potential for
interaction with Ser-131 or Ser-163. However, replacement of Tyr-150
with Ala, Val, or His would be expected to decrease activity because
fewer modes of stabilization of the P1 side chain would be available,
which is consistent with observation (42). It would be interesting to
see the effects of a double or triple mutant of residues Tyr-150,
Ser-131, and Ser-163. Gly-148 is part of the strand E2 packing against
the strand F2 in the COOH-terminal
The S2 pocket of Den2 protease has Asn-152 as one of the
components (Table III) which can provide hydrogen bonding interactions for Lys, Arg, or Gln side chains at P2. A number of carbonyl atoms in
the vicinity provide a electrostatically favorable environment for a
basic P2 side chain. If Asn-152 does indeed provide primary interaction
with the P2 side chain, its replacement with Ala, as was done (42),
would negate this interaction. Its replacement with a Lys would also be
expected to decrease activity because a basic side chain at P2 would be
repelled. A Lys would still be capable of providing an interaction for
a P2 Gln, and perhaps this mutant enzyme will have activity at the
NS2B-NS3 (Table II) junction but not at other sites. It is more
difficult to rationalize the observed decrease in activity of the N152Q
mutant because this would appear to conserve the interaction with a
positively charged substrate. The structure of the heterodimeric
NS2B-NS3 protease, the species that is probably present under the assay conditions of that study (42), might suggest an appropriate rationalization.
Mutations of Other Residues--
It is clear why Den2 protease
loses activity if Gly-153 is replaced (42). Gly-153 is part of the
strand E2 which forms one half of the sheet E2-F2 (Fig. 2a).
It is packed against Ser-163 in the opposite strand and is almost
completely inaccessible to solvent. Substitution of the
Effects of mutations at Val-126 appear to be related to the
packing environment of this residue. Val-126 participates in a hydrophobic cluster that probably defines the conformation of this loop
and which positions Ser-135 appropriately for catalysis. The main chain
beyond Val-126 descends into the active site cleft through a series of
stacked
It is clear from the structure and substrate modeling
experiments that Den2 protease in the absence of interaction with NS2B peptide is not likely to provide specific interactions to substrate side chains beyond P2' on the carboxyl side of the scissile bond and P2
on the amino-terminal side. Such a limited substrate binding cleft is
likely to make the design of specific inhibitors somewhat formidable.
However, the structure of the protease domain provides an excellent
starting point for structural studies of binary complexes with
inhibitors and ternary complexes with inhibitors and activating NS2B
polypeptide. These analyses will provide additional information on both
substrate interactions as well as molecular mechanism of activation. If
NS2B extends the binding site of the protease to provide additional
specific interactions with substrates, that information can be used in
the structure-assisted design of specific inhibitors (69). Moreover, a
heterodimeric NS3 protease complex with NS2B could be an alternate
target aimed at preventing full activation of the enzyme, a strategy
that has been suggested for the HCV protease (45). Based on modeling
calculations presented in this study, interactions that have been
suggested to be important for binding of the substrate are also
conserved among other serine protease domains of arthropod-borne
flaviviruses (some shown in Table III). This observation suggests that
conclusions drawn about substrate binding with the Den2 protease are
also applicable to these other serine proteases. Results reported
here for the Den2 protease are thus of additional significance in
providing a structural basis for design and evaluation of antiviral
therapeutics. This structure-based approach offers considerable promise
toward treatment of a wide range of life-threatening diseases caused
by flaviviruses.
INTRODUCTION
Top
Abstract
Introduction
References
proton to the
histidine, converting the serine into a strong nucleophile for attack
on the peptidyl carbonyl of the substrate. The exquisite selectivity of
proteases for particular substrates is a result of the existence of
specific binding sites (frequently termed pockets) on the enzyme for
amino acid side chains of the substrate(s). The substrate is oriented
by binding of the amino acid side chain of the P1 residue in the S1
pocket (the P and S nomenclature of Schechter and Berger (73) is used; P1 is the substrate residue at the NH2-terminal end, and
P1' is the residue at the COOH-terminal end of the scissile bond), a hydrogen bond between the backbone NH of the P1 residue, and two hydrogen bonds between the carbonyl oxygen of the scissile bond and two
backbone NH groups of the enzyme (oxyanion binding hole). The
reaction proceeds through a tetrahedral transition state with an acyl
enzyme intermediate. Recently, however, serine proteases that lack one
of the three components of the catalytic triad have also been
identified (41). The important role of the predicted catalytic triad
residues (His-51, Asp-75, and Ser-135 in Den2) in the mechanism of
homologous flaviviral NS3 serine proteases was established by
site-directed mutagenesis of these residues which abolished protease activity.
EXPERIMENTAL PROCEDURES
-D-galactopyranoside, and shifted to 30 °C for 4 h. Cells were harvested and resuspended in a lysis buffer (50 mM HEPES, pH 7.5, 0.3 mM NaCl, and 5%
glycerol). Cells were disrupted by either a French press or by
sonication. Bacterial cell lysates were clarified by centrifugation at
27,000 × g for 30 min at 4 °C. The 6His-NS3(185aa)
was distributed in both soluble and pellet fractions, but predominantly
in the latter. The soluble fraction was loaded onto a preequilibrated
Ni2+-agarose affinity resin. The column was then washed
with the lysis buffer (20 × bed volume) and then with the same
buffer containing 40 mM imidazole. The protein was eluted
with the buffer containing 0.5 M imidazole. The pellet
fraction was solubilized in a denaturation buffer (7 M
urea, 0.1 M NaH2PO4, 0.01 M
Tris-HCl, pH 8.0) by incubation for 2 h at 0 °C. The suspension
was clarified by centrifugation at 27,000 × g for 30 min, and the solubilized fraction was loaded onto a preequilibrated
Ni2+-agarose affinity resin. The protein eluate was
refolded by successive dialysis against 50 mM Tris-HCl, pH
7.5, 50 mM NaCl (4 × 1 liter for 14-16 h). The
refolded protein was clarified by centrifugation (27,000 × g for 30 min) and concentrated by ammonium sulfate
precipitation (70% w/v).
-octyl glucoside, and 11% polyethylene glycol 3350. Protein drops (2 µl) mixed with an equal volume of well solution were allowed to equilibrate at 20 °C, and
crystals measuring 0.2 × 0.25 × 0.4 mm were obtained after 3-4 weeks. Space group was determined to be P21 with
a = 48.8, b = 62.4, c = 35.6 Å and
= 96.70 and a solvent content of 50% assuming a
monomer of the protease in the asymmetric unit. A native data set to
2.1 Å was derived from 59,122 measurements made on three crystals (an
average redundancy of 4.6) on a Siemens X1000 area detector system at
room temperature and processed with XDS (46). Data measurement
statistics are presented in Table I. The
structure was determined using a single two-site samarium derivative,
obtained by soaking crystals in a solution containing 2.5 mM samarium acetate solution at pH 7.8 over 2 days. The
strong Bijvoet signal from samarium in conjunction with isomorphous
differences (Table I) was used to obtain an excellent set of SIRAS
phases. All derivative data (80% complete to 2.7 Å ) used in phasing
were measured from one crystal rotated around the crystallographic
b direction to map hkl and h-kl reflections on to the
detector simultaneously and in similar geometry, thus reducing
systematic errors in Bijvoet differences. Systematic errors in both
Bijvoet and isomorphous differences were reduced further by anisotropic
local scaling (47). Positions of the two samarium ions in the
asymmetric unit were determined from the Harker section of a Bijvoet
differences Patterson map and the relative y translation
between them derived from analysis of cross-vectors. The positions were
confirmed from isomorphous differences Patterson maps. Correct
chirality was established by inspection of electron density maps. SIRAS
phasing and refinement were done using MLPHARE (48), part of the
CCP4 package (49) with reflections to 2.7 Å , the limit of usable data
from the derivative. Phasing statistics are given in Table I.
Data measurement, phasing, and refinement statistics
B
between bonded atoms is 3.2 Å2. Analysis using PROCHECK
(55) indicates excellent geometry with no residues in disallowed
regions of the Ramachandran map. Despite requirement for nickel ions in
crystallization, neither the metal ions nor the His tag was visible in
maps. Refinement statistics are presented in Table I.
RESULTS AND DISCUSSION
barrels are six-stranded in both
Den2 and HCV proteases, the strands in the NH2-terminal
domain of Den2 protease are shorter, and the barrel is significantly
more deformed (Fig. 2a). In the carboxyl-terminal domain,
all six strands are more strongly conserved and are of comparable
length. In general, the carboxyl-terminal domain carries most of the
residues that contribute to substrate specificity and is highly
conserved among serine proteases (34), and Den2 protease is not an
exception. Superposition (57) of 68
carbon atoms of the
COOH-terminal domain of Den2 protease (residues 87-167) and either HCV
protease structure (residues 96-186), omitting strands B2 and C2 in
both, yields a r.m.s. deviation of 0.9 Å compared with 0.6 Å for the two structures of HCV protease. Alignment of pairs of structures using
this transformation shows that the conformation of Den2 protease has a
greater resemblance to that of the HCV protease-NS4A complex in this
region. The first 30 residues in the two HCV protease structures have
very different conformations (Fig. 2b). In the absence of
NS4A these residues make
sheet interactions with symmetry-related
molecules. Upon interaction with NS4A peptide, these 30 residues form
two
strands (A0 and A1) and a helix
0. However, the structure of
the Den2 protease in the absence of NS2B peptide more closely resembles
that of the HCV protease-NS4A complex rather than the HCV protease
domain alone (r.m.s. deviation 0.4 Å) (Fig. 2b). A second
major difference between the two HCV structures occurs near helix Ha
and strands D1, E1, and F1. Here again, the conformation of Den2
protease is much closer to that of the HCV protease-NS4A complex (Fig.
2, a and b).
View larger version (47K):
[in a new window]
Fig. 1.
Fit of atomic model to electron density.
A side-by-side stereo view of a 2Fo-Fc map (thin line)
around the catalytic triad contoured at 1.4 is shown with the final
refined model (thick line) superimposed. Phases for the map
were calculated by omitting all of the atoms in the model and refining
the rest of the structure with tight geometric restraints. Several
residues are labeled. The figure was made with CHAIN (72).
View larger version (32K):
[in a new window]
Fig. 2.
Panel a, overall structure of Den2
protease. A C trace of the polypeptide chain (red and
yellow) is shown with individual strands and helices
labeled. The orientation has been chosen to show the catalytic triad
and two water molecules, but the COOH-terminal
barrel can also be
seen. Residues of the catalytic triad are shown as balls and
sticks. Carbon atoms are colored gray, nitrogen
cyan, and oxygen magenta. Sections of the chain
which differ from the homologous HCV enzyme are colored
yellow. Two water molecules that hydrogen bond to His-51 and
Ser-135 and might have significance in catalysis are shown as oversized
purple spheres. Both water molecules are within hydrogen
bonding distance of both N
2 of His-51 and O
of Ser-135. The
upper molecule is 3.0 Å from Ser-135 and 3.1 Å from
His-51; the lower molecule is 2.9 Å from His-51 and 3.3 Å from Ser-135. The figure was made with RIBBONS (70). Panel
b, differences in the NH2-terminal domains of Den2 and
HCV proteases. Superimposed C
traces of HCV protease domain
(yellow) and its complex with NS4A (cyan) and
Den2 protease (magenta) are shown. It can be readily seen
that the overall conformation of the amino-terminal domain of Den2
protease is closer to that of the HCV protease-NS4A complex. Some
structural features are labeled. The figure was made with GRASP
(71).
Sequences around flavivirus serine protease cleavage sites
2
of His-51 (Fig. 2a). Connecting density for this hydrogen
bond was visible in electron density maps. Side chain carboxyl oxygen
atoms of Asp-75 are, however, oriented away from His-51, hydrogen
bonding to main chain N of Trp-50; a hydrogen bonding interaction with His-51 can be generated easily by a small rotation of the side chain,
thus establishing the charge relay system (43). There are also two
water molecules within hydrogen bonding distance of His-51 and Ser-135
which could be relevant to the catalytic mechanism, perhaps by
providing the attacking nucleophile for hydrolysis of the acyl enzyme
intermediate (61, 62). As evidenced by solvent accessibility
calculations (63) using a 1.4 Å probe, the enzyme appears to be in at
least a partially "open" conformation, with the catalytic triad and
the residues in the substrate binding pocket which are accessible to
model substrates. The open conformation suggests that Den2 protease is
likely to have some level of intrinsic activity. Experimental evidence
for this expectation must await development of sensitive in
vitro assays for Den2 protease.
View larger version (110K):
[in a new window]
Fig. 3.
Substrate modeling and structural explanation
of mutational data. Panel a, substrate binding
(top left). A stick model (carbon atoms white,
nitrogen purple, and oxygens red) of the
tetrapeptide RRSW (P2-P2' residues at the NS2A-NS2B cleavage site) is
shown binding in the active site cleft of Den2 protease, represented by
its molecular surface (magenta). The shallowness of the
active site and the side chain of the P1 arginine residue disappearing
into the S1 pocket of the enzyme are readily visible. The figure was
made with GRASP (71). Panel b, illustration of interactions
in the S1 pocket (top right). The contribution of strands E2
and F2 (thin gray ribbon) to the surface shown in
panel a has been removed to provide a clear view of the S1
pocket. Substrate side chains are labeled (P2-P2'), carbon atoms are
colored white, nitrogen purple, and oxygen
red. Enzyme residues near the guanidine moiety of P1
arginine are shown as sticks, colored cyan, and
labeled. Asn-152 is shown in yellow and can be seen
interacting with N of the P2 arginine. Note that protease residues
are shown in their conformations in the native structure to illustrate
the multiple possibilities for interaction, but they are capable of
interaction with the P1 residue by side chain rotations. The figure was
made with GRASP (71). Panel c, mutations causing significant
reduction in enzyme activity (bottom left). Side chains of
residues (stick representation) that caused a reduction of
catalytic activity (42) are plotted on the C
trace (residues beyond
154 are omitted for clarity) of the enzyme (gray). Residues
that are in conserved region 3 are colored cyan, and those
in conserved region 4 colored yellow. Val-126, outside both
conserved regions, is colored magenta. Catalytic His-51 and
Asp-75 (green) are shown for orientation purposes. C
trace of the modeled tetrapeptide (red worm) and the P1 side
chain (carbon gray and nitrogen purple) are also
shown. The figure was made with GRASP (71). Panel d,
hydrophobic cluster stabilizing Ser-135 (bottom right).
Stick representation of the hydrophobic cluster (green)
possibly stabilizing the loop carrying the active site Ser-135
(cyan) is shown. A hydrogen bond between Asp-129 and Ser-127
(magenta) which probably provides additional stabilization
to this loop is also shown. The figure was made with GRASP (71).
nitrogen atoms of Gly-133 (2.71 Å) and Ser-135 (2.8 Å).
Simultaneously the peptide nitrogen was placed at 3.1 Å from the
carbonyl oxygen of Gly-151. Gly-133 and Ser-135 were identified as the
most likely to form the oxyanion hole by comparison with structures of
other serine proteases. Further small translations established hydrogen bonding interactions between the main chain of P1 and P2 residues with appropriate main chain atoms of Gly-153 and Asn-152 to generate the short section of
sheet common to serine protease-inhibitor interactions (64). Side chains were built in their most probable conformations using O to generate the desired sequence at each of the
viral polypeptide junctions (Table II).
1 atom of Asn-152 forms a salt bridge/hydrogen bond with N
of the P2 Arg in the modeled complex (Fig. 3b); alternate
interactions are possible by rearrangement of the side chains of both
the protease and the P2 residue of the substrate to accommodate a Lys
or Gln (Table II). A serine side chain at P1' fits into the S1' pocket
formed by the catalytic His-51 and Ser-135 and residues Gly-35, Ile-36,
and Val-52. Of these Ile-36 interacts only through its main chain atoms
and is not conserved (Table III). A Ser at P2' is also stabilized
exclusively by interaction with main chain atoms of the residues listed
(Table III). A larger side chain at P2', such as a Trp (as in the
NS2A-NS2B cleavage site; see Table II) or an Arg (occurring in an
internal cleavage site within Den2 protease) makes additional van der
Waals interactions with side chain atoms of residues defining the S2' pocket as well as with Asp-129 and Phe-130. It is possible that the
paucity of specific interactions at sites other than P2 and P2' is
caused by the protease being not complexed with NS2B.
Heterodimerization with NS2B could provide additional interactions
beyond the P2 and P2' positions of the substrate side chains directly
as well as indirectly through altered positions of residues in the
protease.
Interacting residues and sequence conservation
trace of the NS3 protease.
turn that positions strand D2 to interact with strand
A2 to form the core
barrel (Fig. 2a). Replacement of
this residue might destabilize the barrel as well as alter the position
of Ser-135 significantly. Other residues in conserved region 3, residues 139-144 (Fig. 4 of Ref. 42), do not seem to play an essential
role in the modeled substrate-enzyme complex. This observation is
consistent with the mutational effects of these residues (42).
barrel (Fig.
2a), and a mutation is likely to cause destabilization of
the barrel, which was indeed observed (42).
hydrogen on
Gly-153 by any other residue will undoubtedly destabilize this sheet
and cause main chain changes in surrounding residues. Modeling studies
indicate that Gly-153 is likely to be one of the residues that form the oxyanion hole, donating its NH to the carbonyl atom of the P1 residue
to stabilize and orient the substrate in the Michaelis complex. Any
changes in its position would, therefore, severely curtail enzyme
activity. In addition, Gly-153 is in the vicinity of Tyr-150 and
Asn-152, both of which are part of the strand E2. Changes in this part
of the main chain will likely alter the positions of Tyr-150 and
Asn-152, further reducing the ability to stabilize the basic P1 side chain.
turns (Fig. 3d). None of these turns is
internally hydrogen bonded, thus deriving their stability from van der
Waals interactions in the vicinity and a hydrogen bond between Asp-129
and Ser-127 side chains (Fig. 3d). The side chains of
Val-126 along with those of Leu-115 from the loop between the strands
B2 and C2 (Fig. 2a) and Ala-160 and Tyr-161 from the strand F2 participate in this cluster (Fig. 3d and Table III).
Replacement of Val-126 with smaller side chains will create a packing
fault, very likely altering the conformation of the loop, making it
more mobile and therefore making it more difficult to position Ser-135 optimally for catalysis. Explanation of severe loss of activity upon
deletion of residues amino-terminal to residue 167 in the NS3
domain2 also follows from the
observation that residues Ala-160 and Ser-163 probably contribute
critical interactions to stabilize the enzyme-substrate complex.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Abdel-Meguid, Babu, Falgout, and Valle for a critical reading of the manuscript. We also thank Dr. Murali for the use of computing facilities at the University of Pennsylvania.
![]() |
FOOTNOTES |
---|
* This work was supported in part by American Cancer Society Grant IRG 204 (to K. M.) and by National Institutes of Health Grant AI-32078 and the Johnson & Johnson Foundation (to R. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1bef) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
§ Present address: CMC, the University of Alabama@Birmingham, 79-THT, MCLM-248, 1918 University Blvd., Birmingham AL, 35294-0005. To whom correspondence should be addressed. Tel.: 205-934-9148; Fax: 205-934-0480; E-mail: murthy{at}onyx.cmc.uab.edu.
Supported in part by a predoctoral fellowship from the Kansas
Health Foundation.
2 S. Clum, S. You, and R. Padmanabhan, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Den2, dengue virus type 2; C, core; prM, precursor to membrane; E, envelope; NS, nonstructural; HCV, hepatitis C virus; r.m.s., root mean square; SIRAS, single isomorphous replacement with anomalous scattering.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|