Relationship between enzyme activity and dimeric structure of recombinant HIV-1 reverse transcriptase.
The multifunctional enzyme human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer composed of a 66-kDa (p66) subunit and a p66-derived 51-kDa (p51) subunit. p66/p51 HIV-1 RT contains 1 functional DNA polymerase and 1 ribonuclease H (RNase H) active site, which both reside in the p66 subunit at spatially distinct regions. In this study, we have investigated the relationship between the heterodimeric structure of HIV-1 RT and its enzymatic properties by introducing mutations at RT codon W401 that inhibit the formation of p66/p51 heterodimers. We demonstrate a striking correlation between abrogation of both HIV-1 RT dimerization and DNA polymerase activity. In contrast, the p66 monomers exhibited only moderately slowed catalytic rates of DNA polymerase-dependent and DNA polymerase-independent RNase H cleavage activity compared with the wild-type (WT) enzyme. Furthermore, no major changes in the unique cleavage patterns were observed between the WT and mutant enzymes for the different substrates used in the RNase H cleavage assays. Based on these results, and on our current understanding of HIV-1 RT structure, we propose that the p66 monomer can adopt an open tertiary conformation that is similar to that observed for the subunit in the heterodimeric enzyme. We also propose that the formation of intersubunit interactions in HIV-1 RT regulates the establishment of a functional DNA polymerase active site.
Tachedjian
G
G
Molecular Interactions Group, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia.
Radzio
J
J
Sluis-Cremer
N
N
D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
D021122:Protein Subunits; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H