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Revisiting monomeric HIV-1 protease. Characterization and redesign for improved properties
John M Louis1, Rieko Ishima, Issa Nesheiwat
1Laboratory of Chemical Physics, National Institute Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. jmlouis@helix.nih.gov
The Journal of Biological Chemistry
|December 7, 2002
Summary
Researchers engineered monomeric HIV-1 protease by linking N- and C-terminal regions with disulfide bonds. This novel monomeric form offers new possibilities for developing HIV-1 protease inhibitors targeting the dimerization interface.
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- The C-terminal interface residues (96-99) of HIV-1 protease are crucial for dimerization.
- N-terminal residues and Arg(87) contribute to dimer stability.
- Intramonomer interactions, like Asp(29)-Arg(87), significantly impact dimerization.
Purpose of the Study:
- To investigate the role of intramonomer interactions in HIV-1 protease dimerization.
- To design and create stable monomeric HIV-1 protease variants.
- To explore novel therapeutic strategies by targeting monomeric protease.
Main Methods:
- Site-directed mutagenesis to create specific amino acid substitutions (e.g., T26A, Q2C/L97C).
- Introduction of cysteine residues to form intramolecular disulfide bonds linking N- and C-termini.
- Biochemical assays to assess protein folding, dimerization, and aggregation.
Main Results:
- Mutants like T26A destabilized the dimer, leading to monomeric forms prone to aggregation.
- Intramolecular disulfide bonds successfully linked N- and C-terminal regions, yielding stable monomeric variants.
- The Q2C/L97C variant demonstrated a native-like single subunit fold.
Conclusions:
- Stable monomeric HIV-1 protease can be engineered using intramolecular disulfide bonds.
- Monomeric HIV-1 protease offers a new target for drug discovery, distinct from the active dimer.
- This approach expands the potential for developing novel antiviral inhibitors against HIV-1 protease.