The motif D loop of human immunodeficiency virus type 1 reverse transcriptase is critical for nucleoside 5'-triphosphate selectivity.
Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the "palm" domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment from Taq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3'-deoxy 3'-azidothymidine 5'-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.
Canard
B
B
Centre National de la Recherche Scientifique, Ecole Supérieure d'Ingénieurs de Luminy, Architecture et Fonction des Macromolécules Biologiques, 163 avenue de Luminy, 13288 Marseille cedex 9, France.
Chowdhury
K
K
Sarfati
R
R
Doublié
S
S
Richardson
C C
CC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012984:Software; D013379:Substrate Specificity; D013912:Threonine
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012265:Ribonucleotides; D013912:Threonine; D054303:HIV Reverse Transcriptase; D008239:Lysine