Spatial domain organization in the HIV-1 reverse transcriptase p66 homodimer precursor probed by double electron-electron resonance EPR

Proc Natl Acad Sci U S A. 2019 Sep 3;116(36):17809-17816. doi: 10.1073/pnas.1911086116. Epub 2019 Aug 5.


HIV type I (HIV-1) reverse transcriptase (RT) catalyzes the conversion of viral RNA into DNA, initiating the chain of events leading to integration of proviral DNA into the host genome. RT is expressed as a single polypeptide chain within the Gag-Pol polyprotein, and either prior to or following excision by HIV-1 protease forms a 66 kDa chain (p66) homodimer precursor. Further proteolytic attack by HIV-1 protease cleaves the ribonuclease H (RNase H) domain of a single subunit to yield the mature p66/p51 heterodimer. Here, we probe the spatial domain organization within the p66 homodimer using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure a large number of intra- and intersubunit distances between spin labels attached to surface-engineered cysteines. The DEER-derived distances are fully consistent with the structural subunit asymmetry found in the mature p66/p51 heterodimer in which catalytic activity resides in the p66 subunit, while the p51 subunit purely serves as a structural scaffold. Furthermore, the p66 homodimer precursor undergoes a conformational change involving the thumb, palm, and finger domains in one of the subunits (corresponding to the p66 subunit in the mature p66/p51 heterodimer) from a closed to a partially open state upon addition of a nonnucleoside inhibitor. The relative orientation of the domains was modeled by simulated annealing driven by the DEER-derived distances. Finally, the RNase H domain that is cleaved to generate p51 in the mature p66/p51 heterodimer is present in 2 major conformers. One conformer is fully solvent accessible thereby accounting for the observation that only a single subunit of the p66 homodimer precursor is susceptible to HIV-1 protease.

Keywords: DEER-derived distances; HIV-1 reverse transcriptase; asymmetric homodimer; p66 homodimer precursor; site-directed spin labeling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Electron Spin Resonance Spectroscopy* / methods
  • HIV Reverse Transcriptase / chemistry*
  • HIV Reverse Transcriptase / metabolism
  • Humans
  • Models, Molecular
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Domains and Motifs*
  • Protein Multimerization*
  • Staining and Labeling
  • Structure-Activity Relationship


  • reverse transcriptase, Human immunodeficiency virus 1
  • HIV Reverse Transcriptase