Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• HIV-1 reverse transcriptase (RT) is a key enzyme for viral replication, existing as a p66/p51 heterodimer.
• Single amino acid substitutions, like L289K, can significantly reduce RT dimer affinity and enzymatic activity.

Purpose of the Study:

• To investigate the structural and functional impact of the L289K mutation on HIV-1 RT heterodimer formation.
• To elucidate the role of specific domains within the RT monomer and their interactions in heterodimer assembly.

Main Methods:

• Small-angle X-ray scattering (SAXS) and proton paramagnetic relaxation enhancement (PRE) to study monomer structure.
• Site-specific 19F NMR to probe domain interactions.
• Size exclusion chromatography (SEC) to analyze heterodimer formation.

Main Results:

• The L289K mutation in the p66 monomer (p66_L289K) revealed that the thumb and RNH domains are not rigidly interacting but spatially close.
• Structural modeling predicted that p66_L289K/p51 could form a heterodimer, but p66/p51_L289K could not.
• SEC experiments confirmed that L289K substitution in the p51 subunit, not p66, significantly reduces p66/p51 heterodimer formation.

Conclusions:

• The L289K mutation in the p51 subunit is critical for disrupting p66/p51 heterodimer formation.
• The inter-subunit interaction between RNH and thumb domains is crucial for p66/p51 assembly.
• Understanding these mechanisms can inform the development of novel HIV-1 therapies targeting RT assembly.