Enzymatic activities are usually investigated in dilute solutions even though these reactions occur in crowded cellular environments. We describe how crowding affects the activity, internal dynamics and diffusion of the hepatitis C virus (HCV) protease — NS3/4A. This enzyme is crucial for viral replication and is used as the therapeutic target.<br/><br/>We applied atomistic molecular dynamics simulations with two types of crowders that are typically used in experiments: the polyethylene glycol (PEG) and polysaccharide (Ficoll). We also developed a coarse-grained bead-shell model of a crowder to separate the effects arising from the excluded volume and physical interactions of macromolecules (Ostrowska N. et al., Biophys. J., 20:3795, 2021). The bead-shell crowder model can be used in the simulations with explicit water molecules. We found that the translational and rotational diffusion of the protease, crowders and peptide substrates was reduced to a different extent in the simulations with the crowding agents. PEG polymers exhibited both hydrophobic and polar interactions with the protease whereas Ficoll interactions with the protease were scarce. Simulations helped explain our fluorescence-based experiments, which have shown that Ficoll crowders increased and PEG decreased the reaction velocity of the NS3/4A-catalysed peptide cleavage (Popielec A. et al. Biochimie, 176:169, 2020). In addition, the presence of Ficoll increased the inhibition constant of telaprevir, the anti-HCV drug. The NS3/4A protease has unstructured tails that have to anchor close to the human endoplasmic reticulum membrane. Both molecular dynamics simulations and circular dichroism spectroscopy experiments suggest that crowding helps the NS3/4A disordered tails fold into a helix, which possibly contributes to NS3/4A membrane anchoring.

Crowders affect diffusion and dynamics of the hepatitis C viral protease

ACS Spring 2022

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