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Journal of Virology, January 2009, p. 1060-1070, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01325-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mechanism of NS2B-Mediated Activation of NS3pro in Dengue Virus: Molecular Dynamics Simulations and Bioassays

Zhili Zuo,¹ Oi Wah Liew,¹ Gang Chen,¹ Pek Ching Jenny Chong,¹ Siew Hui Lee,¹ Kaixian Chen,² Hualiang Jiang,² Chum Mok Puah,^1* and Weiliang Zhu^2*

Centre for Biomedical & Life Sciences, Singapore Polytechnic, 500 Dover Road, Singapore 139651,¹ Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People's Republic of China²

Received 25 June 2008/ Accepted 20 October 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The NS2B cofactor is critical for proteolytic activation of the flavivirus NS3 protease. To elucidate the mechanism involved in NS2B-mediated activation of NS3 protease, molecular dynamic simulation, principal component analysis, molecular docking, mutagenesis, and bioassay studies were carried out on both the dengue virus NS3pro and NS2B-NS3pro systems. The results revealed that the NS2B-NS3pro complex is more rigid than NS3pro alone due to its robust hydrogen bond and hydrophobic interaction networks within the complex. These potent networks lead to remodeling of the secondary and tertiary structures of the protease that facilitates cleavage sequence recognition and binding of substrates. The cofactor is also essential for proper domain motion that contributes to substrate binding. Hence, the NS2B cofactor plays a dual role in enzyme activation by facilitating the refolding of the NS3pro domain as well as being directly involved in substrate binding/interactions. Kinetic analyses indicated for the first time that Glu92 and Asp50 in NS2B and Gln27, Gln35, and Arg54 in NS3pro may provide secondary interaction points for substrate binding. These new insights on the mechanistic contributions of the NS2B cofactor to NS3 activation may be utilized to refine current computer-based search strategies to raise the quality of candidate molecules identified as potent inhibitors against flaviviruses.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The spectrum of clinical manifestations caused by the dengue virus, namely, dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, are increasing in severity and present major threats to global health. In the tropics and subtropical regions, the fatality rate ranges between 1 and 10% of more than 1 million cases of dengue hemorrhagic fever every year. With exacerbation of disease infection rates aided by global warming, growing urbanization, and rapid expansion of international trade and travel, the virus is now endemic in more than 100 countries and threatens more than a quarter of the world's population. Currently, there is no vaccine or effective therapeutic agent available to protect against or cure acute dengue viral infections.

Dengue viruses are members of the Flaviviridae family. They are small, enveloped positive-sense RNA viruses transmitted by Aedes aegypti and Aedes albopictus mosquitoes (6). Dengue virus type 2 (DEN2), the most prevalent of the four serotypes, contains a single-stranded RNA and encodes a large single polyprotein precursor of 3,391 amino acid residues which consists of three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (16). Processing of the polyprotein precursor to release mature viral proteins is mediated co- and posttranslationally by host proteases and the virus-encoded two-component protease NS2B-NS3pro (8). Thus, the essential role of NS2B-NS3pro for viral replication makes it an attractive target for the development of effective antiviral drug inhibitors with therapeutic applications against dengue hemorrhagic fever and dengue shock syndrome.

NS3 protease is a trypsin-like serine protease shown to harbor a classic serine protease catalytic triad comprised of residues His51, Asp75, and Ser135 (30). The N-terminal one-third of the dengue virus NS3 protease (NS3pro) is required for protease activity, and the C-terminal two-thirds are associated with the enzymatic functions of a nucleoside triphosphatase and RNA helicase (20). The activating NS2B cofactor is a prerequisite for catalytic activity of the NS3 protease as demonstrated by in vitro studies with synthetic peptide substrates and natural polyprotein precursors (11). Hence, the two-component NS2B-NS3pro protease represents a structurally more relevant target than NS3pro alone for functional studies and drug discovery research. However, the mechanism by which NS2B contributes to high activation of NS3pro remains poorly understood. Elucidation of the prerequisite role of NS2B will pave the way for discovering and designing new drugs against dengue diseases.

To understand why NS2B-NS3pro is much more active than NS3pro alone and to elucidate the mechanistic role of the NS2B cofactor in NS3pro activation at the atomic level, molecular dynamics (MD) simulations, principal component analysis (PCA), and molecular docking studies were performed. For the first time, computational studies revealed the contributions of NS2B to NS3pro catalytic activity, namely, (i) enhancement of the structural stability of NS3pro and adoption of a functional three-dimensional (3D) conformation, (ii) formation of favorable domain motion to facilitate substrate binding, (iii) formation of additional ligand binding sites on NS3pro, and (iv) the key residues on the NS2B cofactor, viz. Asp50 and Glu92, interacting directly with substrate. The combined effects of these four contributory factors render NS2B-NS3pro a better target for substrate binding. To validate these observations, further in vitro studies were carried out. The recombinant forms of the NS2B-NS3pro complex and three other variants containing amino acid substitutions at the putative interaction sites were expressed and purified from Escherichia coli. The proteolytic activities of the four enzymes on an octapeptide fluorogenic substrate were assayed. The kinetic evidence from K_m, K_cat, and K_cat/K_m ratios provided some support for the observations based on MD simulation and docking calculations.

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Preparation of substrates and enzyme. N--Benzoyl-DL-arginine-p-nitroanilide (BAPNA), a well-known small chromogenic substrate, and eight newly designed octapeptides were prepared as ligands used in our docking study. The structural information for the nine ligands is listed in Table 1. The eight octapeptides were designed based on known NS3pro recognition cleavage sequences present on the polyprotein precursor of dengue virus type 2 (2, 23, 24, 26-29). Particular care was taken to ensure that each octapeptide substrate contained the minimal determinants for cleavage sequence recognition, namely, dibasic residues in the P1 and P2 positions and a preferred small polar amino acid in the P1' position. The positions of P1, P2, and P1', etc., were predicted as usually used (15). Each ligand comprises four residues on the prime (C-terminal to the scissile bond) and nonprime (N-terminal to the scissile bond) sides of the cleavage site. Molecular docking is based on these ligands with an attached N-terminal butoxycarbonyl (BOC) and C-terminal morpholineethanesulfonic acid (MES) to reflect the analogous structures that are usually used in biological assays. In this way, the most efficiently processed substrate could be predicted for use in bioassays to verify our model.

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TABLE 1. Structures of the nine studied substrates and their estimated docking scores for binding to the proteases

High-quality crystal structures of NS3pro (Protein Data Bank [PDB] entry 1DF9) and NS2B-NS3pro (PDB entry 2FOM) (10) were extracted from the PDB. Protein atom types and energy potentials were assigned according to the Amber 4.0 force field with the Kollman all-atom charges encoded in SYBYL 7.3 (Tripos Associates Inc., St. Louis, MO).

MD simulations. The two models (NS3pro and NS2B-NS3pro complex) were solvated with the simple point charge water model within a rectangular periodic box and neutralized. The systems were maintained at a constant temperature of 300 K and a reference pressure of 10⁵ Pa using the Berendsen thermostat method (3). The LINCS algorithm (14) was used to constrain all bonds involving hydrogen atoms. Electrostatic interactions between charged groups at a distance of less than 9 Å were calculated explicitly; long-range electrostatic interactions were calculated using the particle-mesh Ewald method (9) with a grid width of 1.2 Å and a fourth-order spline interpolation. A cutoff distance of 14 Å was applied for the Lennard-Jones interactions. The MD simulations were performed using the GROMACS package version 3.3 (4) with the GROMOS96 force field (31). Finally, 10-ns MD simulations were performed on both systems.

Principal component analysis. PCA was carried out in order to identify the most significant fluctuation modes of the proteins. The central hypothesis of this method is that only the motions along the eigenvectors with large eigenvalues are important for describing the functionally significant motions in the protein (1, 18). PCA is based on the calculation of diagonalization of the covariance matrix C, whose elements are defined as follows: C_i = (x_i – x_i)(x_j – x_j) (i, j = 1, 2, 3, ..., 3N) (1), where x_i is a Cartesian coordinate of the ith C atom, N is the number of the C atoms considered, and x_i represents the time average over all the configurations obtained in the simulation. The eigenvectors of the covariance matrix, V_k, obtained by solving V_k^TCV_k = _k, where _k is the kth eigenvalue of the covariance matrix C, denote a set of 3N-dimensional directions, or principal modes, along which the fluctuations observed in the simulation are uncoupled with respect to each other (i.e., C_ij = 0 if i j), and can be analyzed separately. Domain motion analyses based on the PCA results were performed using the DYNDOM program (12).

Docking studies. There is a strict requirement for Arg or Lys residues in the P1 and P2 positions and a preference for small polar amino acids, predominantly glycine, alanine, or serine, in the P1' position for cleavage activity (21, 32). These should be regarded as critical residues for activity of or cleavage sequence recognition by the NS3 protease. These conserved residues in the substrate imply that they are likely to adopt similar conformations upon binding to the same subsite of the NS3 protease. Studies based on the crystal structure of NS3pro complexed with a Bowman-Birk inhibitor (1DF9) suggested that the P1-arginine "sits" in the S1 binding pocket. Thus, in keeping with the structural and spatial constraints defined by this crystal structure, the backbone of the P1 substructure was initially restricted to the S1 subsite for calculations at the early stage of molecular docking.

Two powerful and widely used docking algorithms, GOLD 3.0 (genetic optimization for ligand docking) and AutoDock 3.0.3, were used to perform the docking studies and provide cross-validation. The default parameters in GOLD 3.0 were used, except that a maximum number of 300,000 operations and a population of 200 individuals were imposed. The GoldScore fitness function and ChemScore fitness function were used sequentially, and the best-fitting binding mode was identified. The binding energy was calculated with AutoDock 3.0.3 based on the best-fit model from GOLD docking. The initial number of individuals in the population was 150. The maximum number of generations, energy evaluations, and docking runs were set to 1.6 x 10⁶, 5.0 x 10⁵, and 10, respectively. The AutoDock was performed as described in previous work (33).

Construction of NS2B-Gly-NS3pro181 and its variants. Taking into consideration the modeled binding characteristics of the substrates, our data identified the residues Glu92 and Asp50 in the NS2B cofactor and residues Gln27, Gln35, and Arg54 in NS3pro to be important for the substrate binding via hydrogen bond interactions. It is noteworthy that sequence alignment shows that these residues are conserved in all four dengue virus serotypes with the exception of Glu92. Interestingly, of 27 diverse members of the flavivirus family, Asp50 and Glu92 in NS2B are conserved in 6 and 17 members, respectively. Our molecular docking results show that elimination of the predicted hydrogen bond interactions would have some negative impact on enzyme activity. Hence, three variants were designed with the following substitutions with short side chain residues (Ala or Gly): (i) NS2B(D50A/E92A)-Gly-NS3pro181, with two alanine substitutions in the NS2B domain; (ii) NS2B-Gly-NS3pro181(Q35G), with one glycine substitution in the NS3 domain; (iii) NS2B-Gly-NS3pro181(Q27G/R54G), with two glycine substitutions in the NS3 domain.

The DEN2 NS2B-NS3 protease expressed for functional assays comprised the 47-residue hydrophilic core region of NS2B linked to the N-terminal 181 amino acids of NS3 via a nine-residue flexible G₄-S-G₄ linker. The DNA specifying this protease, designated NS2B-Gly-NS3pro181, was incorporated with an N-terminal His₆ tag and artificially synthesized according to E. coli codon preference for insertion into pBluescript SK as a 5' EcoRI-3' XhoI fragment. Three further variants of NS2B-Gly-NS3pro181 were also constructed, one comprising a double mutation in the NS2B fragment [NS2B(D50A/E92A)-Gly-NS3pro181], one with a single mutation in the NS3 fragment [NS2B-Gly-NS3pro181(Q35G)], and the third construct with a double mutation in the NS3 fragment [NS2B-Gly-NS3pro181(Q27G/R54G)]. A schematic representation of the NS2B-Gly-NS3pro181 construct and variants are shown in Fig. 1.

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FIG. 1. Schematic representation of the NS2B-Gly-NS3pro181 construct (a) and variants (b, c, and d), showing the NS2B fragment joined to the NS3 fragment via nucleotides encoding the G4-S-G4 linker. The sites with codon changes required to introduce the target substitutions in the NS2B and NS3 fragments are indicated by triangles. The cognate residues that are substituted are shown below the triangles. Key restriction enzyme sites used for the generation of the constructs are also indicated.

To generate the variant containing D50A and E92A substitutions on the NS2B fragment, two oligonucleotides spanning the NcoI-EarI and EarI-NarI sites were synthesized with the target mutations. These oligonucleotides were then fused in frame via the EarI site and used to replace the NcoI-Nar I fragment of NS2B-Gly-NS3pro181. To generate the variants containing either the single substitution of Q35G or the double substitutions of Q27G and R54G on the NS3 fragment, two oligonucleotides spanning the NarI-BglII sites were synthesized with the appropriate target mutations and used to replace the NarI-BglII fragment of NS2B-Gly-NS3pro181. Authenticity of all synthetic coding sequences and the reading frame was verified by DNA sequencing. A directional insertion of NS2B-Gly-NS3pro181 and its variants were finally cloned into the 5' NcoI and 3' XhoI sites of the expression vector pET19b (Novagen). The amino acid sequence of the protein expressed from pET19b/NS2B-Gly-NS3pro181 is shown in Fig. 2.

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FIG. 2. Amino acid sequence of NS2B-Gly-NS3pro181. Residues 6 to 11 specifying the N-terminal His6 tag are underlined. Residues 18 to 64 indicated in bold typeface comprise the 47-amino-acid hydrophilic core sequence derived from the DEN2 NS2B sequence (GenBank accession no. U872412). Residues 65 to 73 comprise the G4-S-G4 flexible linker (italicized and underlined). The amino acids of the NS3 sequence (PDB entry 1BEF) are highlighted in gray. The locations of the amino acid mutations to generate the variants are indicated by triangles and the substituted residue is shown below the triangle.

Expression and purification of functional viral proteases. The four pET19b vectors generated for expression of NS2B-Gly-NS3pro181 and its variants were transformed into competent BL21(DE3) cells. The bacterial cultures were grown and induced with isopropyl-β-D-thiogalactopyranoside (IPTG) under conditions described by Liew et al. (22) except that the culture temperature following induction was maintained at 25°C. The cells were harvested by centrifugation (5,000 x g for 10 min) at 5 h postinduction and lysed with BugBuster HT protein extraction reagent (Novagen). The soluble protein fractions were collected by centrifugation at 8,000 x g for 20 min at 4°C and purified in a single chromatographic step using nickel as the metal ion in immobilized metal affinity chromatography according to the methods described by Liew et al. (22). The viral proteases were eluted in elution buffer (1 M imidazole, 50 mM NaCl, 20 mM Tris-HCl, pH 7.9) and dialyzed against 5 mM Tris-Cl, pH 8.0, using 12-kDa molecular weight cutoff tubing. Recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by Western blotting using in-house rabbit polyclonal antibodies against NS3pro. The immunodetected viral protease bands revealed by horseradish peroxidase chromogenic substrates on Immobilon-P membranes were quantified by densitometric scanning (GS-800 imaging densitometer; Bio-Rad, Hercules, CA) using the volume function of the Quantity One quantitation software (Bio-Rad).

Protease assays. The activities of NS2B-Gly-NS3pro181 and its variants were measured by cleavage of the fluorogenic substrate Dabcyl-KQRRGRIE-Edans (Sigma-Aldrich), in 200-µl reaction mixtures. Dabcyl-KQRRGRIE-Edans was designed based on the sequence of the AQRRGRIG cleavage site. This substrate was selected because data from docking studies showed BOC-AQRRGRIG-MES to be the most active ligand. The substrate was incubated in 96-well plates with 100 nM recombinant protease at 37°C in a buffer containing 50 mM Tris-Cl, pH 9.0, 0.1% Triton X-100, and 30% glycerol. The increase in fluorescence intensity was monitored continuously using an Infinite M200 microplate reader (Tecan) at an excitation wavelength of 340 nm and emission wavelength at 480 nm. The initial velocity was determined from the linear portion of the progress curve, and the values of the kinetic parameters K_m, K_cat, and K_cat/K_m were calculated from weighted nonlinear regression of initial velocities as a function of eight substrate concentrations [S] from 1 µM to 80 µM assuming Michaelis-Menten kinetics, where = V_max [S]/([S] + K_m). All calculations were performed using GraphPad Prism software. Each data point was obtained from three independent experiments and is reported as the mean ± the standard error of the mean.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
RMSD and interaction network. To check whether our MD simulations were satisfactorily performed, the root mean square deviation (RMSD) of the C atoms with reference to the initial structures over the 10-ns simulation time were determined and are shown in Fig. 3A and B. The RMSD of the NS2B-NS3pro complex system tends to be convergent after 1.0 ns, while the RMSD in the system of NS3pro converges at 6.0 ns. This, together with total energy, temperature, mass density, and volume during the simulations (data not shown), means that the NS2B-NS3pro system reaches equilibrium with respect to its initial structure faster than the NS3pro system, which indicates that the former is more stable. The RMS fluctuations (RMSFs) of individual residues obtained by averaging atomic fluctuations are shown in Fig. 3C and D. Overall, NS3pro alone has a larger residue fluctuation than the NS3 chain in NS2B-NS3pro. The number of hydrogen bonds and hydrophobic interaction pairs within both structures was compared using LIGPLOT, and they are listed in Table 2. Remarkably, the NS3 chain in the dual-component complex with 27 residues fewer than NS3pro alone in the sequence has 30 more hydrogen bonds and 54 more hydrophobic interaction pairs than that in NS3pro alone, and there are 29 hydrogen bonds and 59 hydrophobic interaction pairs between the chain of NS2B and NS3 in the complex. Therefore, NS2B not only interacts with NS3 via the hydrogen bonds and hydrophobic interaction, but also it changes the interaction network in NS3.

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FIG. 3. The time dependence of RMSDs from the start structure of the NS2B-NS3pro complex and NS3pro for the C atom in the 10-ns MD simulations and residue fluctuations calculated by averaging atomic fluctuations (RMSFs) in 10-ns MD simulations. The residues of the catalytic triad (His51, Asp75, and Ser135) are indicated by the gray bar. (A) Time evolution of RMSD values in the NS2B-NS3pro model; (B) time evolution of RMSD values in the NS3pro model; (C) residue fluctuations in the NS2B-NS3pro MD simulation, with the NS2B residues highlighted in red; (D) residue fluctuation in the NS3pro MD simulation.

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TABLE 2. Hydrogen bonds and hydrophobic interaction pairs in the structures of NS3pro (1DF9 from PDB) and the NS2B-NS3pro complex (2FOM from PDB)

Domain motion of both systems. The effect of NS2B on the motions of the NS3pro conformation was studied by PCA calculations based on the MD simulation results for the NS2B-NS3pro complex and NS3pro, and the corresponding motion modes were identified using the program DYNDOM (13). The first five principal components (PCs) of C atoms account for approximately 50% and 60% of the total sample variance for the NS2B-NS3pro complex and NS3pro systems, respectively. Therefore, these five principal components were used to identify motion modes. Notably, only the fifth mode of the NS2B-NS3pro complex (Fig. 4A) and the third mode of NS3pro (Fig. 4B) were identified to be related to domain motion, therefore representing the most significant fluctuation mode for comparing the stability of the two proteins.

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FIG. 4. Principal component mode of motion. The colors of the arrows denote the particular moving domain (right-hand rule). The fixed domains are shown in blue, the residues involved in interdomain bending are green, the moving domains are red, and the rest are gray. The binding clefts are indicated by the red circle and are enlarged on the right. (A) The fifth motion mode of the NS2B-NS3pro complex; (B) the third motion mode of NS3pro.

To further substantiate the influence of NS2B on the motion of NS3, the distance between C atoms of His51 and Ser135, which corresponds to the width of the active site entrance, were monitored at the point when both MD simulations become relatively stable after 6 ns. In the NS2B-NS3 protease complex (Fig. 5A), the distance vibrates from 7.8 to 9.2 Å regularly with an average of 8.4 Å. However, the distance in NS3pro narrows to as little as about 6.2 to 7.8 Å, with an average of 6.8 Å (Fig. 5A).

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FIG. 5. Time-dependent atomic distances between C atoms of the key residues on the two sides of the binding cleft after 6-ns MD simulations. The average values are indicated by the straight line. (A) Distance between C atoms of His51 and Ser135 in the NS2B-NS3pro complex; (B) distance between C atoms of His51 and Ser135 in the NS3pro system.

The contribution of NS2B cofactor to substrate binding. The molecular surfaces of both structures were generated by SYBYL 7.3 and compared (Fig. 6). Two subpockets were found around the active site of NS3pro (1DF9), namely S1 and S1' (Fig. 6B), and five new potential subpockets in addition to S1 and S1' were discovered around the active site of NS2B-NS3pro (2FOM), viz., S2, S2', S3, S3', and S4' (Fig. 6A).

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FIG. 6. Molecular surface map of binding pockets (highlighted by red circles) generated by the MOLCAD module in SYBYL 7.3 and colored according to their electrostatic properties. The color scale, ranging from negative to positive, is shown between panels A and B. (A) Potential binding pockets in NS2B-NS3pro; (B) potential binding pockets in NS3pro. The subpockets interacting with the P1 and P2 residues, respectively, are designated S1, S2, and so forth, while those interacting with the P1' and P2' residues, respectively, are designated S1', S2', and so forth.

A molecular docking study was performed to further investigate the effects of structural differences between NS3pro and NS2B-NS3pro on ligand binding by using GOLD 3.0 (Darwinian genetic algorithm) (17) and AutoDock 3.0.3 (25). Table 1 summarizes the binding scores for all nine ligands. Figure 7 depicts the interaction pattern between ligands and the proteases, illustrated by the well-known small-molecule ligand BAPNA and the most active dibasic peptide ligand, BOC-AQRRGRIG-MES, binding to NS2B-NS3pro and NS3pro, respectively. We can see from these figures that only the side chain of BAPNA interacts with the S1 pocket of NS2B-NS3pro while BAPNA wholly binds into the S1 pocket in NS3pro, suggesting that this small ligand binds more strongly to NS3pro than the protease complex.

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FIG. 7. The docking prediction of BAPNA and substrate (BOC-AQRRGRIG-MES) bound to the catalytic site of NS2B-NS3pro and NS3pro. The binding subpocket is indicated by the red circle. (A) Interaction of BAPNA in the binding groove of NS2B-NS3pro; (B) interaction of BAPNA in the binding groove of NS3pro. To give a better view, the molecular surface is set at half-transparency and some key residues are shown. (C) Binding of BOC-AQRRGRIG-MES to the surface of NS2B-NS3pro; (D) binding of BOC-AQRRGRIG-MES to the surface of NS3pro.

The details of 3D and theoretical molecular interactions of the most powerful ligands (BOC-AQRRGRIG-MES) with NS2B-NS3pro are summarized in Fig. 8. In the S1 pocket, the negatively charged oxygen from the side chains of Tyr50, the main chain of Leu128, Asp129, and Ser131 are all directed toward the inside of the pocket. Also, in the case of the S2 pocket, the oxygens from the side chain of Asp75 and main chain of Val72 and Lys73 can interact directly with the positively charged groups on the P2 residue. The same situation can be seen for the S3 pocket. The predicted fleet hole of the S1' and S3' pockets and the polar atoms (oxygen and nitrogen atoms from main chain residues) interacting directly with substrates corroborates the catalytic preference of the enzyme complex for small and polar amino acids in the P1' and P3' positions (21). The pockets of S2' and S4' are also important for the binding of substrates. Gln35 and Glu92 (NS2B) in the S2' pocket may provide a hydrogen bond interaction with a positive side chain of the substrate. MD simulation suggests that the side chains of residues Asp50 and Arg54 from NS2B can swing flexibly to the S4' pocket. Thus, the side chains of these residues, when suitably located toward the S4' pocket, together with Gln27 may influence substrate binding through highly specific and directional hydrogen bond interactions in the S4' pocket.

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FIG. 8. Details of the molecular interactions between substrate (BOC-AQRRGRIG-MES) with NS2B-NS3pro. (A) 3D representation of the enzyme-substrate interaction (blue, substrate; magenta, NS3pro moiety; yellow, NS2B moiety). The dashed lines represent hydrogen bonds and important interaction points between substrate and residues in the binding site of NS2B-NS3pro. The schematic structures were prepared using the PyMol programs (DeLano Scientific, San Carlos, CA). (B) Summary of theoretical molecular interactions; some important residues identified from docking and MD simulation studies are highlighted in bold.

Expression and purification of NS2B-Gly-NS3pro181 and its variants. Previous studies showed that the hydrophilic core region of NS2B was more or less functionally equivalent to that of full-length NS2B and that the presence of this central core domain significantly improved the recovery of soluble recombinant NS3pro in E. coli (19). Hence, the expression constructs for the production of wild-type and mutated DEN2 NS2B-NS3pro in this work comprised the 47-residue NS2B cofactor domain linked to NS3pro181 via a nonapeptide linker.

The theoretical molecular size of the wild-type viral protease and its mutant derivatives is 26.9 kDa. SDS-PAGE analysis showed migration of these recombinant proteins to approximately the 30-kDa position (Fig. 9A). Western blotting of the purified proteases (Fig. 9B) showed no autoproteolysis at the NS2B-NS3 junction, since the canonical NS2B-NS3 recognition site was not incorporated in the polypeptide sequence. In harmony with observations from other studies (5, 19), our recombinant proteases accumulated to levels representing about 30% of total bacterial protein, with >90% being partitioned to the insoluble fraction. Nevertheless, sufficient soluble proteases were obtained for the in vitro assays in previous studies and this present work.

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FIG. 9. Analysis of NS2B-Gly-NS3pro181 and its variants expressed in E. coli strain BL21(DE3). A. Time course accumulation of NS2B-Gly-NS3pro181 (indicated by arrows) in the soluble and insoluble cellular fractions at time points (in hours) after induction with IPTG. Proteins were separated by 16% SDS-PAGE and stained with Coomassie blue. Similar accumulation patterns were also observed for the enzyme variants (data not shown). B. Western blot of purified soluble NS2B-Gly-NS3pro181 and its variants immunodetected with polyclonal antibodies raised against NS3pro and visualized by using 3,3'-diaminobenzidine tetrahydrochloride substrate development. Autoproteolysis between the NS2B-NS3 junction was not observed, but additional products migrating above the 25-kDa marker position were immunodetected. Similar bands were also observed by Leung et al. (18), who attributed these to translation at internal sites downstream of the start codon. Lane 1, His6-tagged NS3pro; lane 2, NS2B-Gly-NS3pro181; lane 3, NS2B(D50A E92A)-Gly-NS3pro181; lane 4, NS2B-Gly-NS3pro181(Q35G); lane 5, NS2B-Gly-NS3pro(Q27G R54G). MW, protein molecular weight marker (Precision Plus protein standards; Bio-Rad).

Characterization of enzymatic activity. In vitro assays were performed to measure the kinetic parameters for processing the substrate, Dabcyl-KQRRGRIE-Edans (Sigma-Aldrich). Evaluation of the consequences of mutations on the NS2B moiety (D50A E92A enzyme variant) in comparison to substitutions on the NS3 sequence (single Q35G and double Q27G R54G mutants) was carried out. The rate of substrate hydrolysis and the Michaelis-Menten equilibrium constants are listed in Table 3. The rank order of the catalytic rate K_cat for these enzymes is wild type > D50A E92A > Q35G > Q27G R54G. As expected, the double mutant of NS2B-Gly-NS3pro181 (Q27G R54G) exhibits the lowest proteolytic activity. On the other hand, the binding affinities (K_m) of the substrate for the various enzymes vary in the order of D50A E92A > Q27G R54G Q35G > wild type, leading to the following rank order of enzyme efficiency (K_cat/K_m): wild type > D50A E92A > Q35G > Q27G R54G. The K_m values of the mutated enzymes are increased marginally, which means the binding affinities of the substrate for them are slightly decreased. Interestingly, double mutations on the NS2B moiety had the greatest negative impact on binding affinity. Replacement of Asp50 and Glu92 with alanine residues in the NS2B cofactor domain resulted in a 1.7-fold decrease in substrate affinity as well as reaction rate, translating to an overall 1.6-fold decline in catalytic efficiency compared to the wild type. We attribute this to less interaction provided to substrates by the NS2B cofactor after mutation.

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TABLE 3. Kinetic parameters for the hydrolysis of peptide substrate by the wild-type NS2B-Gly-NS3pro181 and its mutant derivatives, NS2B(D50A E92A)-Gly-NS3pro181, NS2B-Gly-NS3pro181(Q35G), and NS2B-Gly- NS3pro181(Q27G R54G)

As expected, double Q27G R54G mutations in the NS3 region diminished ligand binding to a greater extent than single point mutations, indicating that the predicted weak intermittent interaction from Arg54 is plausible. In stark contrast, mutations on the NS3 moiety resulted in greater adverse impacts on catalytic activity than mutations on the NS2B cofactor. In addition, the single Q35G substitution showed a more detrimental effect on enzyme activity than the double Q27G R54G mutant. This can be explained by the fact that Gln35 is located in the S2' subpocket, which is closer to the catalytic triad than Gln27 and Arg54 in the S4' subpocket.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The RMSD of MD simulations converge after 1.0 ns and 6.0 ns, respectively, which suggests that the simulations were carried out satisfactorily and 10-ns MD simulations should be sufficient for elucidating the basic dynamics of the NS2B-NS3pro complex and NS3pro. RMSD of the NS2B-NS3pro complex also indicates that it is more stable than NS3pro itself. The RMSF spanning across all residues of the NS3pro system is higher than those of the NS2B-NS3pro complex. Compared to the NS3pro system, smaller fluctuations were observed for the critical residues (His51, Asp75, and Ser135) (Fig. 3C and D) around the active site of NS2B-NS3pro. This difference between the RMSF of the two systems strongly suggests that NS3pro itself is more flexible than the NS2B-NS3pro complex system.

The model of NS2B-NS3pro places the binding cleft of the catalytic triad between the fixed domain and moving domain located on the two β-barrels. The hinge residues involved in the domain motion comprise the residues representing the active site of NS2B-NS3pro. This strongly suggests dynamic domain motion, with regular opening and closing of the cleft that translates into changes in the shape and spatial volume of the binding site, thereby facilitating substrate entry, binding, unbinding, and exit from the catalytic pocket of the complex. Indeed, the distance evolution along simulation time between the C atoms of His51 and Ser135 in the NS2B-NS3pro complex fluctuates regularly (Fig. 5A). In contrast, the binding cleft in the NS3pro system is located in the fixed domain indicated in the figure and notably remote from the moving domain (Fig. 4B). Therefore, the domain motion changes neither the volume nor geometry of the binding site. The distance between C atoms of His51 and Ser135 in NS3pro (Fig. 5B) was obviously shorter on average than that in the NS2B-NS3pro complex during the MD simulation. This gives direct evidence that the catalytic pockets between the two systems are substantially different and confirms the influence of NS2B on binding site conformation. We can conclude that the NS2B cofactor not only enhances the structural stability of the NS3 domain but also contributes to proper domain motion that facilitates substrate accessibility and promotes catalytic activity.

There are a greater number of hydrogen bonds and hydrophobic interaction pairs in the 3D structure of the NS2B-NS3pro complex than in the NS3pro structure (Table 2). Furthermore, 29 hydrogen bonds and 59 hydrophobic interaction pairs formed between the chain of NS2B and NS3 in the complex structure. Accordingly, the presence of NS2B not only affects the principal component mode of motion of the protease but also confers greater rigidity to NS3 in the NS2B-NS3pro complex. Therefore, the NS2B cofactor contributes significantly and is essential to the 3D structure and biological function of NS3pro.

In addition to the greater structural rigidity accorded by the hydrogen bond and hydrophobic interaction networks in NS2B-NS3pro, new additional binding sites not previously observed in the NS3pro structure were discovered on the molecular surface of the protease complex. Only two subpockets around the active site of NS3pro were observed, while five more potential subpockets were found on the surface of the complex structure (Fig. 6). Therefore, the NS2B cofactor acts like a hand, clutching the NS3 protease with the "thumb-like" segment inserted between two random loops in domain I and the other "fingers" holding on to domain II (Fig. 10). The strong clasping action of the "hand" reorientates the random loops into β-turns in both domains I and II. In addition, the structural geometry of the newly modeled domain of NS3 in the NS2B-NS3pro complex is clearly distinct from that of NS3pro alone. The significant changes in secondary and tertiary structures induced by NS2B finally lead to the generation of five additional subpockets around the active site of NS2B-NS3pro (Fig. 6A). This predicted topology is in agreement with the observed strong hydrogen bond and hydrophobic networks in the complex deemed to play important roles in modeling and stabilizing the structure. We surmise that the extra binding sites in the complex induced by the presence of the NS2B cofactor provide a further basis for the improved catalytic activity of NS2B-NS3pro. The following molecular docking calculations support this deduction.

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FIG. 10. Cartoon presentation of the crystal structure of NS3pro in the absence and presence of the NS2B cofactor (blue, domain I; red, domain II; green dashed line, demarcation of domain I and domain II). (A) Structure of NS3pro protease (PDB entry 1DF9); (B) structure of the NS2B-NS3pro protease complex (PDB entry 2FOM). The NS2B segment is indicated in yellow.

Biochemical data revealed that small-ligand BAPNA is more active against NS3pro than NS2B-NS3pro (32), and our predicted binding scores (AScore, GoldScore, and Gold-ChemScore) corroborate well with experimental evidence (Table 1). The S1 pocket in NS3pro is deeper than the analogous site in NS2B-NS3pro, and the residues of Glu101, Ser131, and Asp129 in the deep hole of S1 in NS3pro exhibit multipoint interactions via hydrogen bonding with BAPNA (Fig. 7B), which explains the better fit of BAPNA in NS3pro compared with NS2B-NS3pro (Fig. 7A). Furthermore, all the octapeptides have stronger predicted binding affinity to NS2B-NS3pro than to NS3pro (AScore) (Table 1), suggesting that NS2B-NS3pro adopts a catalytically more relevant target conformation than NS3pro. Again, this is in good agreement with the earlier findings that the formation of additional interaction sites allows all the side chains of natural and longer substrates to bind efficiently to NS2B-NS3pro (32). It is notable that NS2B residues Glu92, Leu51, and Asp50 lie in the S2' and S4' pockets of the NS2B-NS3pro complex. Evidently, the NS2B cofactor not only facilitates the folding of the NS3 protease into a more active and rigid structure but also contributes residues that interact with substrates. This is also consistent with the above findings on the positive influence on NS3pro protease activity in the presence of NS2B attributed to formation of a strong hydrogen bond and hydrophobic networks, extra binding sites, and proper motion modes.

Just as expected, a small but distinct decline in the kinetic properties of the variants is shown in the experiments and is sufficient to substantiate our modeling predictions, since these mutations do not abolish primary critical residues involved in the binding and catalytic properties of the protease. The kinetic fitness of the three enzyme variants compared with the wild type provides some indication that the residues of Asp50 and Glu92 on NS2B and Gln27, Gln35, and Arg54 on NS3pro may interact directly with the substrate during the proteolytic process. Interestingly, the studies of Chambers et al. (7) on yellow fever virus NS2B-NS3pro indicated that charged-to-alanine mutations in the NS2B region have less deleterious effects on cleavage activity compared to the protease domain. Mutations on NS2B that had the greatest effect on cleavage activity were located at the charged cluster near the N-terminal conserved cofactor region at positions 47 and 49. Our observations were that single or double mutations on the DEN2 NS3 protease domain (Q35G and Q27G R54G) resulted in greater reduction in catalytic efficiency than double mutations on NS2B (D50A E92A). This result compares well with the observations with the yellow fever virus protease. In addition, the charged cluster at positions 47 and 49 is located near our predicted Asp50 residue on the DEN2 NS2B moiety.

Our predictions based on molecular simulation and calculation studies are congruent with the key observations from bioactivity assays: (i) mutations on NS2B have some influence on substrate binding; (ii) mutations on NS3 lead to minor reductions in both substrate binding and catalytic activity; (iii) the Q27G R54G mutations affecting the S4' subpocket have less effect on overall catalytic efficiency than the Q35G substitution on the S2' subpocket positioned nearer to the catalytic binding pocket. The effects of the mutations on substrate binding and catalytic efficiency of the NS2B/NS3 proteases are weak. However, this is to be expected, as our computational analyses predict the contributions of these residues as secondary interaction sites. Nonetheless, we cannot exclude other possible inadvertent effects that the mutations could have on the kinetic fitness of the variant enzymes. Other than the intended abolishment of hydrogen bond interactions with ligands, substitutions with the smaller residues, alanine or glycine with short side chains, might also have some influence on the spatial volume and shape of the secondary binding pockets. Hence, the overall effects on enzyme activity by disruption of hydrogen bond and hydrophobic interactions may be modulated by other inadvertent changes to the secondary binding pockets.

In summary, molecular docking showed that the binding energy of substrates to NS2B-NS3pro is higher than that to NS3pro. MD simulation showed that the NS2B-NS3pro complex system is significantly different from and more rigid than NS3pro alone. Furthermore, PCA revealed a regular domain motion generated in the presence of the cofactor. This dynamic motion should be essential to substrate accessibility, suggesting that NS2B is important for protease stability and inhibitor recognition. The residues Glu92 and Asp50 in the NS2B cofactor and residues Gln27, Gln35, and Arg54 in NS3pro are directly involved in interaction between the ligand and the binding pockets on the enzyme. Kinetic assessment of the proteolytic activity of wild-type and mutated NS2B-NS3pro enzymes provides some support that the residues identified by modeling are involved in substrate binding. Computational and biofunctional evidence presented here points to a possible dual role for NS2B cofactor-mediated activation of NS3pro: (i) to facilitate the folding of the enzyme complex to a catalytically active conformational state and (ii) to provide direct but weak secondary interaction sites for substrate binding. Although the computational predictions along with a modest set of kinetic data of the NS2B-NS3pro system in this study might not be fully representative of the global architecture of the DEN2 NS3 viral protease, our comparative studies by computational and experimental approaches offer a set of relatively reliable information for explaining the conformational and functional changes that accompany the interaction of the NS2B cofactor with the catalytic protease domain. The information expands current understanding of structural reorientation of the NS3pro enzyme in the presence of the NS2B moiety and the mechanistic role of this cofactor in influencing binding interactions with ligands and substrate processing. It also provides a useful clue for discovering new drugs against flaviviruses.

 
We gratefully acknowledge financial support from Singapore Polytechnic and the Singapore Totalisator Board Project (grant no. 11-27801-36-M103), the 863 Program of China (grant no. 2006AA02Z336), the international collaboration project of China (grant no. 2007DFB30370), and the 973 program of China (grant no. 2004CB518901).

 
* Corresponding author. Mailing address for C. M. Puah: Centre for Biomedical & Life Sciences, Singapore Polytechnic, 500 Dover Road, Singapore 139651. Phone: (65) 6772 1896. Fax: (65) 6870 8004. E-mail: puah{at}sp.edu.sg. Mailing address for W. Zhu: Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People's Republic of China. Phone: 86-21-50805020. Fax: 86-21-50807088. E-mail: wlzhu{at}mail.shcnc.ac.cn

Published ahead of print on 29 October 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Virology, January 2009, p. 1060-1070, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01325-08
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