Structure/function studies of HIV-1(1) reverse transcriptase: dimerization-defective mutant L289K.
Virion-derived HIV-1 reverse transcriptase (RT) has subunits of molecular mass 66 and 51 kDa (p66 and p51, respectively) in an approximately 1:1 ratio. Since enzyme activity appears to depend on dimerization of these subunits, identification of critical regions of primary sequence required for proper dimerization could lead to potential targets for antiviral therapy. A central region of primary sequence contains a leucine hepta-repeat motif from leucine 282 to leucine 310 that has been suggested to be involved in dimerization [Baillon, J. G., Nashed, N. T., Kumar, A., Wilson, S. H., & Jerina, D. M. (1991) New Biol. 3, 1015-1019]. A region including this hepta-repeat was recently shown to be involved in protein-protein interactions required for dimerization [Becerra, S. P., Kumar, A., Lewis, M. S., Widen, S. G., Abbotts, J., Karawya, E. M., Hughes, S. H., Shiloach, J., & Wilson, S. H. (1991) Biochemistry 30, 11708-11719]. To investigate the role of this repeat motif in dimerization, we performed site-directed mutagenesis of these leucine residues from position 282 to position 310. Mutations were introduced into p66 and p51 RT coding sequences, and the individually purified RT subunit polypeptides were compared with wild-type polypeptides for dimerization. Physical characterization of the purified mutant peptides was conducted by circular dichroism analysis. Binding between p66 and p51 was studied by gel filtration, ultracentrifugation, and CD analysis. L289K-p66 was unable to dimerize with itself and wild-type or L289K-p51. The leucine repeat motif in the p66 subunit appears to be critical in formation of the heterodimer.(ABSTRACT TRUNCATED AT 250 WORDS)
Goel
R
R
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Beard
W A
WA
Kumar
A
A
Casas-Finet
J R
JR
Strub
M P
MP
Stahl
S J
SJ
Lewis
M S
MS
Bebenek
K
K
Becerra
S P
SP
Kunkel
T A
TA
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007930:Leucine / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation
D046911:Macromolecular Substances; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D007930:Leucine