Intrinsic Flexibility of West Nile Virus Protease: In Solution Characterization

West Nile virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection can cause severe neurological disease and fatalities in humans. Efforts are ongoing to develop antiviral drugs that inhibit the WNV protease, a viral enzyme required for polyprotein processing. Unfortunately, little is known about the solution structure of recombinant WNV protease (NS2B-NS3pro) used for antiviral drug discovery and development, although X-ray crystal structures and NMR studies have provided valuable insights about the interactions between NS2B-NS3pro and peptide-based inhibitors. To determine the solution structure and dynamics of the substrate-free WNV NS2B-NS3pro and understand the conformational changes due to substrate binding, we performed Circular dichroism spectroscopy, fluorescence experiments, small angle X-ray scattering (BioSAXS) and Fourier transform infrared spectroscopy (FT-IR) experiments. Our results suggested that in absence of a substrate analogous, the NS3pro domain adopted a well-folded compact tertiary structure similar to the observed crystallographic structures. However, while the NS2B cofactor maintained its secondary structure folding elements, it adopted multiple conformations distinct from previously described open and closed structures. In presence of a substrate analogous, WNV NS2B-NS3pro acquires a more compact folding state, which can be represented by the closed state described by X-ray crystallography data, suggesting that the substrate analogue acts as a folding inductor. This detailed description of the folding state and intrinsic flexibility of WNV NS2B-NS3pro can aid structure-guided discovery and optimization of WNV protease inhibitors as well as improve understanding of WNV polyprotein post-translation processing.

Name thesis:degree_name

Molecular Biophysics Educational Track (Doctoral)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Structural Biology

Grantor

The University of Texas Medical Branch at Galveston

Author dc:creator

• Garces fernandez, Andrea

Advisor dc:contributor.advisor

• Choi, Kyung

Committee members dc:contributor.committeemember

• Watowich, Stanley
• Braun, Werner
• Freiberg, Alexander
• López-Lastra, Marcelo
• Olano, Juan

• West Nile Virus
• protease
• NS2B-NS3pro
• BioSAXS
• FT-IR
• conformational heterogeneity
• drug design

Handle dc:identifier.uri

http://hdl.handle.net/2152.3/860

OAI identifier oai:identifier

oai:utmb-ir.tdl.org:2152.3/860