Mechanism of NS2B-mediated activation of NS3pro in dengue virus: molecular dynamics simulations and bioassays

Abstract

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.

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.

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.

FIG. 3.

The time dependence of RMSDs from the start structure of the NS2B-NS3pro complex and NS3pro for the C^α 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.

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.

FIG. 5.

Time-dependent atomic distances between C^α 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^α atoms of His51 and Ser135 in the NS2B-NS3pro complex; (B) distance between C^α atoms of His51 and Ser135 in the NS3pro system.

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.

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.

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.

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).

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.