Insertion of a small peptide of six amino acids into the beta7-beta8 loop of the p51 subunit of HIV-1 reverse transcriptase perturbs the heterodimer and affects its activities.
BACKGROUND
HIV-1 RT is a heterodimeric enzyme, comprising of the p66 and p51 subunits. Earlier, we have shown that the beta7-beta8 loop of p51 is a key structural element for RT dimerization (Pandey et al., Biochemistry 40: 9505, 2001). Deletion or alanine substitution of four amino acid residues of this loop in the p51 subunit severely impaired DNA binding and catalytic activities of the enzyme. To further examine the role of this loop in HIV-1 RT, we have increased its size such that the six amino acids loop sequences are repeated in tandem and examined its impact on the dimerization process and catalytic function of the enzyme.


RESULTS
The polymerase and the RNase H activities of HIV-1 RT carrying insertion in the beta7-beta8 loop of both the subunits (p66INS/p51INS) were severely impaired with substantial loss of DNA binding ability. These enzymatic activities were restored when the mutant p66INS subunit was dimerized with the wild type p51. Glycerol gradient sedimentation analysis revealed that the mutant p51INS subunit was unable to form stable dimer either with the wild type p66 or mutant p66INS. Furthermore, the p66INS/p66INS mutant sedimented as a monomeric species, suggesting its inability to form stable homodimer.


CONCLUSIONS
The data presented herein indicates that any perturbation in the beta7-beta8 loop of the p51 subunit of HIV-1 RT affects the dimerization process resulting in substantial loss of DNA binding ability and catalytic function of the enzyme.
Pandey
Pradeep K
PK
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA. pandeypk@umdnj.edu
Kaushik
Neerja
N
Singh
Kamalendra
K
Sharma
Bechan
B
Upadhyay
Alok K
AK
Kumar
Suriender
S
Harris
Dylan
D
Pandey
Virendra N
VN
D000596:Amino Acids / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D019281:Dimerization; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D014461:Ultracentrifugation / Q000379:methods
D000596:Amino Acids; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H