Cysteine Disulfide Traps Reveal Distinct Conformational Ensembles in Dengue Virus NS2B-NS3 Protease

Abstract

The dengue virus protease (NS2B-NS3pro) plays a critical role in the dengue viral life cycle, making it an attractive drug target for dengue-related pathologies, including dengue hemorrhagic fever. A number of studies indicate that NS2B-NS3pro undergoes a transition between two widely different conformational states: an “open” (inactive) conformation and a “closed” (active) conformation. For the past several years, the equilibrium between these states and the resting conformation of NS2B-NS3pro have been debated, although a strong consensus is emerging. To investigate the importance of such conformational states, we developed versions of NS2B-NS3pro that allow us to trap the enzyme in various distinct conformations. Our data from these variants suggest that the enzymatic activity appears to be dependent on the movement of NS2B and may rely on the flexibility of the protease core. Locking the enzyme into the “closed” conformation dramatically increased activity, strongly suggesting that the “closed” conformation is the active conformation. The observed resting state of the enzyme depends largely on the construct used to express the NS2B-NS3pro complex. In an “unlinked” construct, in which the NS2B and NS3 regions exist as independent, co-expressed polypeptides, the enzyme rests predominantly in a “closed”, active conformation. In contrast, in a “linked” construct, in which NS2B and NS3 are attached by a nine-amino acid linker, NS2B-NS3pro adopts a more relaxed, alternative conformation. Nevertheless, even the unlinked construct samples both the “closed” and other alternative conformations. Given our findings, and the more realistic resemblance of NS2B-NS3pro to the native enzyme, these data strongly suggest that studies should focus on the “unlinked” constructs moving forward. Additionally, the results from these studies provide a more detailed understanding of the various poses of the dengue virus NS2B-NS3 protease and should help guide future drug discovery efforts aimed at this enzyme.

A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques

“…This fast cleavage validated the amino acid sequences of our linkers as well as the application of "unlinked" versions of both recombinant DENV-2 and ZIKV proteases, in which the NS2B and NS3 regions were produced as independent polypeptides. This choice was based on our own demonstration that the "unlinked" version of DENV-2 protease rests predominantly in a "closed," active conformation, in contrast with the less active, relaxed conformation, adopted by the frequently used "linked" construct, in which NS2B and NS3 polypeptides are attached by a nine-amino acid linker (31).…”

Proteases from dengue, West Nile and Zika viruses as drug targets

“…There is an ongoing debate in the literature whether the linked or unlinked protease constructs are more suitable for drug discovery campaigns (Kang et al 2017;Nitsche 2018;Nitsche et al 2014). The linked constructs tend to be more stable, whereas the unlinked constructs are thought to resemble a state that is closer to the native one (de la Cruz et al 2014;Hill et al 2018;Li et al 2017a). Recent studies with Zika virus protease suggest that the covalent linker may prevent the formation of the catalytically active complex to a certain degree (Li et al 2017a), whereas no significant differences for inhibitor binding could be observed between linked and unlinked Zika virus protease constructs (Nitsche et al 2019).…”

“…After initial challenges with heterogeneity in sample preparations, most reports concluded that both, the dengue and West Nile virus proteases, predominately adopt the closed conformation in solution, regardless of the construct (linked or unlinked) or the presence of an inhibitor (de la Cruz et al 2014Cruz et al , 2011Kim et al 2013;Su et al 2009). Observed conformational flexibilities may relate to exchange phenomena between open and closed states, the extent of which can differ between constructs (Hill et al 2018). In the case of Zika virus protease, NMR experiments indicated sample heterogeneity promoted by the Gly 4 SerGly 4 linker and ligand-binding events (Li et al 2017a;Mahawaththa et al 2017).…”

Structure and Dynamics of Zika Virus Protease and Its Insights into Inhibitor Design

“…An outbreak occurred during 2015-2016 and over 2 million people were affected [4][5][6][7]. Effort has been made to develop antivirals and vaccines to combat the virus while no specific medical treatment is available [8][9][10][11].…”