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.