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 Citations (Scopus)

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

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Peptides
Proteins
Assays
Monomers
Human immunodeficiency virus 1 reverse transcriptase
RNA-Directed DNA Polymerase
Ultracentrifugation
Immunoprecipitation
Protein Binding
Virion
Dimers
Free energy
Gel Chromatography
Gels
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Protein-Protein Interactions of HIV-1 Reverse Transcriptase : Implication of Central and C-Terminal Regions in Subunit Binding. / Becerra, S. Patricia; Kumar, Amalendra; Widen, Steven; Abbotts, John; Karawya, Essam M.; Wilson, Samuel H.; Lewis, Marc S.; Hughes, Stephen H.; Shiloach, Joseph.

In: Biochemistry, Vol. 30, No. 50, 01.12.1991, p. 11707-11719.

Research output: Contribution to journalArticle

Becerra, SP, Kumar, A, Widen, S, Abbotts, J, Karawya, EM, Wilson, SH, Lewis, MS, Hughes, SH & Shiloach, J 1991, 'Protein-Protein Interactions of HIV-1 Reverse Transcriptase: Implication of Central and C-Terminal Regions in Subunit Binding', Biochemistry, vol. 30, no. 50, pp. 11707-11719. https://doi.org/10.1021/bi00114a015
Becerra, S. Patricia ; Kumar, Amalendra ; Widen, Steven ; Abbotts, John ; Karawya, Essam M. ; Wilson, Samuel H. ; Lewis, Marc S. ; Hughes, Stephen H. ; Shiloach, Joseph. / Protein-Protein Interactions of HIV-1 Reverse Transcriptase : Implication of Central and C-Terminal Regions in Subunit Binding. In: Biochemistry. 1991 ; Vol. 30, No. 50. pp. 11707-11719.
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AU - Abbotts, John

AU - Karawya, Essam M.

AU - Wilson, Samuel H.

AU - Lewis, Marc S.

AU - Hughes, Stephen H.

AU - Shiloach, Joseph

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