Functional Loop Dynamics and Characterization of the Inactive State of the NS2B-NS3 Dengue Protease due to Allosteric Inhibitor Binding

Abstract

Dengue virus, a flavivirus that causes dengue shock syndrome and dengue hemorrhagic fever, is currently prevalent worldwide. A two-component protease (NS2B-NS3) is essential for maturation, representing an important target for designing antiflavivirus drugs. Previously, consideration has been centered on developing active-site inhibitors of NS2B-NS3pro. However, the flat and charged nature of its active site renders difficulties in developing inhibitors, suggesting an alternative strategy for identifying allosteric inhibitors. The allosterically sensitive site of the dengue protease is located near Ala125, between the 120s loop and 150s loop. Using atomistic molecular dynamics simulations, we have explored the protease's conformational dynamics upon binding of an allosteric inhibitor. Furthermore, characterization of the inherent flexible loops (71−75s loop, 120s loop, and 150s loop) is carried out for allosteric-inhibitor-bound wild-type and mutant A125C variants and a comparison is performed with its unbound state to extract the structural changes describing the inactive state of the protease. Our study reveals that compared to the unliganded system, the inhibitor-bound system shows large structural changes in the 120s loop and 150s loop in contrast to the rigid 71−75s loop. The unliganded system shows a closed-state pocket in contrast to the open state for the wild-type complex that locks the protease into the open and inactive-state conformations. However, the mutant complex fluctuates between open and closed states. Also, we tried to see how mutation and binding of an allosteric inhibitor perturb the connectivity in a protein structure network (PSN) at contact levels. Altogether, our study reveals the mechanism of conformational rearrangements of loops at the molecular level, locking the protein in an inactive conformation, which may be useful for developing allosteric inhibitors.

Computer-aided Affinity Enhancement of a Cross-reactive Antibody against Dengue Virus Envelope Domain III

“…The Molecular Mechanics Poisson Boltzmann or Generalized Born Surface Area (MM-PB(GB)SA has been a widely used method to calculate the binding free energy for protein-protein or protein-ligand systems [42][43][44][45][46][47][48][49][50]. Here, the binding free energy for the wild-type and mutant systems were calculated using the MMPBSA scheme implemented in the python script MMPBSA.py in AMBER18.…”

Loop Dynamics and Conformational Flexibility in Dengue Serine Protease Activity: Noninvasive Perturbation by Solvent Exchange

“…However, to develop an effective drug candidate, it is necessary to understand the intricacy of the protein machinery. 5,6 Key intramolecular loops or residues are known to regulate the activation or inactivation status of several enzymes. 7−13 It is recognized that such important loops/residues have certain kinds of conformations/motions that are absolutely imperative for enzyme kinetics.…”

“…Because of the biological significance of DENV NS2B/NS3 serine protease, continuous effort is being given to develop a suitable drug against it. However, to develop an effective drug candidate, it is necessary to understand the intricacy of the protein machinery. , Key intramolecular loops or residues are known to regulate the activation or inactivation status of several enzymes. − It is recognized that such important loops/residues have certain kinds of conformations/motions that are absolutely imperative for enzyme kinetics. , An example is the WPD (Trp–Pro–Asp) loop of several protein tyrosine phosphatases (PTP). According to the research of Crean et al, WPD along with the neighboring loop dynamics regulates the catalytic activity of the PTPs and responds differently to environmental changes .…”

Novel conformational filter allows unambiguous identification of specific conformational ensembles for the large NS3pro/NS2B Dengue-associated protease; Implications for the appropriate choice of protein conformations in drug discovery

“…If valid, the 'open/closed' hypothesis would require conformational changes to occur upon e.g. activation by substrate or binding to an inhibitor, a concept initially supported by the crystal structure of NS3pro/NS2B in complex with an allosteric inhibitor (PDB code 4M9T) 25 , by molecular dynamics (MD) simulations of NS3pro/NS2B in 'open' conformation whence binding an allosteric inhibitor [26][27][28] and through mutational studies forcing NS2B to assume an 'open' inactive-state conformation 29 . This hypothesis would also imply the simultaneous presence of open and close conformations, with a possible exchange time scale of 10 ms.…”