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Molecular mechanics play an important role in enzyme action and understanding the dynamics of loop motion is key for designing inhibitors of an enzyme, particularly targeting the allosteric sites. For the successful creation of new protease inhibitors targeting the dengue serine protease, our current investigation detailed the intricate structural dynamics of NS2B/ NS3 dengue protease. This enzyme is one of the most essential enzymes in the life cycle of the dengue virus, which is responsible for the activation/processing of viral polyprotein, thus making it a potential target for drug discovery. We showed that the internal dynamics of two regions, fingers 1 and 2 (R24−G39 and L149− A164, respectively) adjacent to the active site triad of this protease, control the enzyme action. Each of these regions is composed of two antiparallel β-strands connected by β-turn/hairpin loops. The correlated bending and rocking motions in the two β-turns on either side of the active site were found to modulate the activity of the enzyme to a large extent. With increasing concentration of cosolvent dimethyl sulfoxide, correlated motions in the finger 2 region get diminished and bending of finger 1 increases, which are also reflected in the loss of enzyme activity. Decreasing temperature and mutations in neighboring nonsubstrate binding residues show similar effects on loop motion and enzyme kinetics. Therefore, in vitro noninvasive perturbation of these motions by the solvent exchange as well as cold stress in combination with in silico molecular dynamics simulations established the importance of the two β-turns in the functioning of dengue virus serotype 2 NS2B/NS3 serine protease.

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