Related Experiment Video
Updated: Oct 4, 2025

Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods
Published on: December 21, 2019
Characterization of an Allosteric Pocket in Zika Virus NS2B-NS3 Protease
Naiá Porã Santos1, Lucianna Helene Santos1, Mariana Torquato Quezado de Magalhães1
1Biochemistry and Immunology Department, Biological Sciences Institute, Federal University of Minas Gerais (UFMG), Avenida Antônio Carlos 6627, Belo Horizonte, Minas Gerais 31270-901, Brazil.
Abstract:
The NS2B-NS3 protease from Zika virus (ZIKV NS2B-NS3pro) cleaves the viral polyprotein, being essential for its replication and a therapeutic target. Inhibitors that target the active site of ZIKV NS2B-NS3pro have been developed, but they tend to have unfavorable pharmacokinetic properties due to their highly positive charge. Thus, the characterization of allosteric sites in this protease provides new strategies for inhibitor development. Here, we characterized a new allosteric pocket in ZIKV NS2B-NS3pro, analogous to the one previously described for the dengue virus protease. Molecular dynamics simulations indicate the presence of cavities around the residue Ala125, sampling protein conformations in which they are connected to the active site. This link between the residue Ala125 and the active site residues was reinforced by correlation network analysis. To experimentally verify the existence of this allosteric mechanism, we expressed and purified the Ala125Cys mutant of ZIKV NS2B-NS3pro and demonstrated that this variant is inhibited by the thiol-containing chemical probes 5,5'-dithiobis-(2-nitrobenzoic acid) and aldrithiol, which do not affect the activity of the wild-type protein. Inhibition of the mutant protein is reversed by the addition of strong reducing agents, supporting the involvement of Cys125 in covalent bond formation and enzyme inhibition. Together, our results provide experimental evidence for an allosteric pocket in ZIKV NS2B-NS3pro, in the region around Ala125, and computational insights on the structural connection between this region and the enzyme active site.
More Related Videos
Related Concept Videos
Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Ligand Binding and Linkage
Induced-fit Model
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical...
Cooperative Allosteric Transitions
Enzymes
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
Conserved Binding Sites
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...

