HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis.
Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions. Limited proteolysis of the homodimer with alpha-chymotrypsin also resulted in cleavage to a stable Mr 66,000/51,000 mixture, and proteolysis with trypsin resulted in the transient formation of some Mr 51,000 polypeptide. These results are consistent with the reverse transcriptase molecule having a protease-sensitive linker region following a structured domain of Mr 51,000. Further digestion with trypsin resulted in cleavage of the Mr 51,000 polypeptide after residue 223, yielding peptides of apparent Mr 29,000 and 30,000. A minor peptide of Mr 40,000 was also produced by cleavage of the Mr 66,000 polypeptide after residue 223. About half the original Mr 66,000 polypeptides remained resistant to proteolysis and existed in complex with the above peptides in solution. During both chymotrypsin and trypsin digestion there was an increase in the reverse transcriptase activity caused by a doubling of Vmax with little change in Km for dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)
Lowe
D M
DM
Department of Molecular Sciences, Wellcome Research Laboratories, Beckenham, Kent, U.K.
Aitken
A
A
Bradley
C
C
Darby
G K
GK
Larder
B A
BA
Powell
K L
KL
Purifoy
D J
DJ
Tisdale
M
M
Stammers
D K
DK
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002918:Chymotrypsin / Q000378:metabolism; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011994:Recombinant Proteins; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin