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

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

Research output: Contribution to journalArticlepeer-review

63 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

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