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

Thomas Schmidt, Charles D. Schwieters, and G. Marius Clore
PNAS September 3, 2019 116 (36) 17809-17816; first published August 5, 2019 https://doi.org/10.1073/pnas.1911086116

  1. Contributed by G. Marius Clore, July 9, 2019 (sent for review June 28, 2019; reviewed by David S. Cafiso, David J. Weber, and Peter E. Wright)

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Significance

HIV-1 reverse transcriptase, the enzyme that catalyzes the conversion of viral RNA into DNA, is initially released from the Gag-Pol polyprotein as a p66 homodimer precursor. Further cleavage by HIV-1 protease removes the RNase H domain of only a single subunit to yield the mature p66/p51 heterodimer. We have determined the spatial subunit organization within the p66 homodimer from a large number of distances between spin labels obtained by EPR. We show that the structural subunit asymmetry of the p66/p51 heterodimer is preserved in the p66 homodimer and that only one of the RNase H domains is accessible to HIV-1 protease.

Abstract

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.

Footnotes

  • ↵1To whom correspondence may be addressed. Email: mariusc{at}mail.nih.gov.

  • Author contributions: T.S., C.D.S., and G.M.C. designed research, performed research, analyzed data, and wrote the paper.

  • Reviewers: D.S.C., University of Virginia; D.J.W., University of Maryland, Baltimore; and P.E.W., The Scripps Research Institute.

  • The authors declare no conflict of interest.

  • See Commentary on page 17614.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1911086116/-/DCSupplemental.

Published under the PNAS license.

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Spatial domain organization in the HIV-1 reverse transcriptase p66 homodimer precursor probed by double electron-electron resonance EPR
Thomas Schmidt, Charles D. Schwieters, G. Marius Clore
Proceedings of the National Academy of Sciences Sep 2019, 116 (36) 17809-17816; DOI: 10.1073/pnas.1911086116
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