Crowding affects structural dynamics and contributes to membrane association of the NS3/4A complex

Area of Science:

• Biophysics
• Virology
• Computational Biology

Background:

• Hepatitis C virus (HCV) protease NS3/4A is crucial for viral replication and a target for antiviral drugs.
• The NS3/4A protease requires the NS4A cofactor, which has flexible, unstructured tails in solution.
• Understanding how the cellular environment affects protease function is vital for developing effective treatments.

Purpose of the Study:

• To investigate the impact of molecular crowding on the internal dynamics and diffusion of HCV NS3/4A protease.
• To elucidate the role of crowding in the structural changes of the NS4A cofactor.
• To explore how crowding influences the NS4A N-terminal tail's interaction with the NS3 protease.

Main Methods:

• Atomistic molecular dynamics simulations of the NS3/4A protease and water molecules.
• Modeling of crowders (polyethylene glycol - PEG) using all-atom and coarse-grained approaches.
• Circular dichroism spectroscopy to experimentally validate structural changes in the NS4A N-terminal tail.

Main Results:

• Crowding, particularly with partially interactive crowders, promotes the formation of a helical structure in the NS4A N-terminal tail.
• Simulations suggest crowding facilitates NS4A N-tail detachment from the protease surface, aiding membrane insertion.
• Crowding plays an active role in inducing functional structural changes in disordered protein fragments.

Conclusions:

• Molecular crowding significantly affects HCV NS3/4A protease dynamics and NS4A cofactor structure.
• Crowding-induced helical formation in NS4A is essential for viral replication machinery assembly.
• These findings highlight the importance of the cellular environment in viral protein function and suggest new therapeutic strategies targeting protein-crowding interactions.