Structure of HIV-1 reverse transcriptase/d4TTP complex: Novel DNA cross-linking site and pH-dependent conformational...

Area of Science:

• Structural Biology
• Virology
• Biochemistry

Background:

• Stavudine (d4T) was an early HIV treatment, acting as a chain-terminating nucleoside analog.
• Understanding the interaction of d4T triphosphate (d4TTP) with HIV-1 reverse transcriptase (RT) is crucial for antiviral drug development.

Purpose of the Study:

• To determine the X-ray structures of d4TTP bound to HIV-1 RT using a novel cross-linking strategy.
• To investigate the pH-dependent structural dynamics of the HIV-1 RT catalytic complex.

Main Methods:

• Crystallization and X-ray diffraction of cross-linked HIV-1 RT/dsDNA/d4TTP complexes.
• Structural analysis of complexes at various pH values (7 to 9.5).
• Disulfide cross-linking strategy targeting the fingers subdomain of RT.

Main Results:

• The first structure of d4TTP in a catalytic complex with HIV-1 RT was determined.
• pH-dependent motions (~0.5 Å) of the fingers subdomain were observed, affecting the dNTP-binding pocket.
• Cross-linking strategies at different sites (I63C and Q258C) showed minimal perturbation of RT/DNA structures.

Conclusions:

• The observed pH-dependent finger motion likely explains the pH-activity profile of HIV-1 RT.
• Despite d4T's clinical limitations, its chemical modification may inspire new polymerase-targeting nucleoside analogs.