Human immunodeficiency virus‐1 reverse transcriptase heterodimer stability

Abstract

Structural and biochemical evidence strongly supports a heterodimeric (p66p5 1) active form for human immunodeficiency virus-1 reverse transcriptase (RT). Heterodimer stability was examined by sedimentation analysis as a function of temperature and ionic strength. Using NONLIN regression software, monomer-dimer-trimer and monomer-dimer-tetramer association models gave the best fit to the analytical ultracentrifuge sedimentation equilibrium data. The heterodimer is the predominant form of RT at 5 "C, with a dimerization K , value of 5.2 X IO5 M-' for both models. KO values of 2.1 X IO5 and 3.8 X IO5 M-' were obtained for the respective association models at 20 "C. RT in 50 and 100 mM Tris, pH 7.0, completely dissociates at 37 "C and behaves as an ideal monomeric species. The dissociation of RT as a function of increasing temperature was also observed by measuring the decrease in sedimentation velocity ( s ,~~) .If the stabilization of the heterodimer was due primarily to hydrophobic interactions we would anticipate an increase in the association from 21 "C to 37 "C. The opposite temperature dependence for the association of RT suggests that electrostatic and hydrogen bond interactions play an important role in stabilizing heterodimers. To examine the effect of ionic strength on p66p51 association we determined the changes in s , , ,~~ as a function of NaCl concentration. There is a sharp decrease in s , ,~~ between 0.10 and 0.5 M NaCI, leading to apparent complete dissociation. The above results support a major role for electrostatic interactions in the stabilization of the RT heterodimer.Keywords: association constants; electrostatic interactions; heterodimer stability; immunodeficiency virus-1 ; reverse transcriptase; sedimentation equilibrium; sedimentation velocity The human immunodeficiency virus (HIV) reverse transcriptase (RT) is the enzyme responsible for the conversion of the viral genome from a single-stranded RNA to double-stranded DNA, which is then integrated into the cellular genome by a viral-coded integrase. RT has been a major target for the treatment of acquired immunodeficiency syndrome (AIDS). The native enzyme has DNA polymerase and RNase H activities and exists as a heterodimer containing polypeptides of approximately 66 and 5 1 kDa (DiMarzo-Veronese et al., 1986; Lightfoote et al., 1986). The p66 subunit contains both the DNA polymerase and RNase H functional domains, whereas the smaller polypeptide, p51, lacks the carboxyl-terminal RNase H domain (Hansen et al., 1988). RT and the viral DNA integration protein are synthesized as part of the gag-pol precursor polyprotein (for review see, *This paper is dedicated to the memory of Dean L. Richard.

Human immunodeficiency virus type 1 reverse transcriptase: role of Tyr115 in deoxynucleotide binding and misinsertion fidelity of DNA synthesis.

“…The 51 kDa subunit was obtained with an extension of six histidines located at its N-terminal end. The formation of heterodimers was facilitated by changing the ionic strength and temperature conditions while processing the bacterial extracts to favour the association of the RT subunits (Lebowitz et al, 1994). The 66 kDa subunit was constitutively expressed by cultures harbouring plasmid p66(RT) (Hizi et al, 1988), and contained two extra amino acids at the N-terminal end (Met-Val-) which were not found in the viral RT obtained from purified HIV (Di Marzo Veronese et al, 1986).…”

Characterization of Selected Strains of Pneumococcal Surface Protein A

“…Band centrifugation was also employed (33). Uncorrected s values were corrected to s w,20 using the standard formula (34 The percentage of the secondary structure elements such as ␣-helix and random coil and the coiled-coil structure were calculated based on the sequence data using the PHD program (26,27) and Matcher software (28), respectively.…”

Characterization and Structural Analysis of Novel Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Involved in the Regulation of Resistance to Nonnucleoside Inhibitors

“…All calculations were assessed by a Linux cluster of five Intel Xeon dual processors at 3.2 GHz with 2 Gb of RAM. In order to consider the protein-protein contribution term at the p66-p51 interface, we used the GROMACS utility g_energy, and we selected the Coul-SR protocol to analyze electrostatic interactions in the short range (28).…”