STRUCTURE-FUNCTION STUDIES OF HIV-1(1) REVERSE-TRANSCRIPTASE - DIMERIZATION-DEFECTIVE MUTANT L289K
STRUCTURE-FUNCTION STUDIES OF HIV-1(1) REVERSE-TRANSCRIPTASE - DIMERIZATION-DEFECTIVE MUTANT L289K
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DOI：
10.1021/bi00211a009
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发表时间：
1993-12-07
期刊：
影响因子：
2.9
通讯作者：
WILSON, SH
中科院分区：
文献类型：
--
作者：
GOEL, R;BEARD, WA;WILSON, SH
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 in to 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. Conversely, our observation that the wild-type p66 and L289K-p51 can heterodimerize indicates that this leucine residue in p51 is probably not at a hydrophobic protein-protein interface of the heterodimer. These results for subunit dimerization with wild-type and mutant polypeptides were consistent with DNA polymerase activity being dependent on dimerization.
