Protein-Protein Interactions of HIV-1 Reverse Transcriptase: Implication of Central and C-Terminal Regions in Subunit Binding

S. Patricia Becerra, Amalendra Kumar, Steven Widen, John Abbotts, Essam M. Karawya, Samuel H. Wilson, Marc S. Lewis, Stephen H. Hughes, Joseph Shiloach

Research output: Contribution to journalArticle

61 Scopus citations

Abstract

Human immunodeficiency virus 1 reverse transcriptase (RT) purified from virions is composed of a ~51000 Mr polypeptide and a ~66000 Mr polypeptide that are thought to be in heterodimer structure (Chandra et al., 1986; Hansen et al., 1988; Starnes & Cheng, 1989) and are identical except for a 15 000 Mr C-terminal truncation in the smaller species (Di Marzo-Veronese et al., 1986). We prepared individual bacterial-recombinant RTs as the ~66000 Mr polypeptide (p66) or as the ~51000 Mr polypeptide (p51) and then conducted various in vitro protein-protein binding experiments. Analytical ultracentrifugation studies in 0.25 M NaCl at pH 6.5 revealed that p66 was in monomer-dimer equilibrium with KA of 5.1 × 104 M-1. p51 failed to dimerize and behaved as a monomer under these conditions. Mixing of the p66 and p51 polypeptides resulted in a 1:1 heterodimer with KA of 4.9 × 105 M-1. These results on formation of the p66/p66 homodimer and p66/p51 heterodimer were confirmed by gel filtration analysis using FPLC Superose-12 columns. Binding between p66 and individual p66 segment polypeptides also was observed using an immunoprecipitation assay. Binding between p51 and p66 in this assay was resistant to the presence of ~ 1 M NaCl, suggesting that the binding free energy has a large hydrophobic component. C-Terminal truncation of p66 to yield a 29-kDa polypeptide eliminated binding to p66, and N-terminal truncation of p66 to yield a 15-kDa peptide also eliminated binding to p66. The results indicate that purified individual RT peptides p51 and p66 are capable of binding to form a 1:1 heterodimer and suggest that the central region of p66 is required for this subunit binding; the C-terminal region (15 000 Mr) of p66 appears to be required also, as p51 alone did not dimerize.

Original languageEnglish (US)
Pages (from-to)11707-11719
Number of pages13
JournalBiochemistry
Volume30
Issue number50
DOIs
StatePublished - Dec 1 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Protein-Protein Interactions of HIV-1 Reverse Transcriptase: Implication of Central and C-Terminal Regions in Subunit Binding'. Together they form a unique fingerprint.

  • Cite this

    Becerra, S. P., Kumar, A., Widen, S., Abbotts, J., Karawya, E. M., Wilson, S. H., Lewis, M. S., Hughes, S. H., & Shiloach, J. (1991). Protein-Protein Interactions of HIV-1 Reverse Transcriptase: Implication of Central and C-Terminal Regions in Subunit Binding. Biochemistry, 30(50), 11707-11719. https://doi.org/10.1021/bi00114a015