Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent.
We have devised a high-resolution protein footprinting methodology to dissect HIV-1 reverse transcriptase (RT) contacts to the viral RNA:tRNA complex. The experimental strategy included modification of surface-exposed lysines in RT and RT-viral RNA:tRNA complexes by the primary amine selective reagent NHS-biotin, SDSPAGE separation of p66 and p51 polypeptides, in gel proteolysis, and comparative mass spectrometric analysis of peptide fragments. The lysines modified in free RT but protected from biotinylation in the nucleoprotein complex were readily revealed by this approach. Results of a control experiment examining the RT-DNA:DNA complex were in excellent agreement with the crystal structure data on the identical complex. Probing the RT-viral RNA:tRNA complex revealed that a majority of protein contacts are located in the primer-template binding cleft in common with the RT-DNA:DNA and RT-RNA:DNA species. However, our footprinting data indicate that the p66 fingers subdomain makes additional contacts to the viral RNA:tRNA specific for this complex and not detected with DNA:DNA. The protein footprinting method described herein has a generic application for high-resolution solution structural studies of multiprotein-nucleic acid contacts.
Kvaratskhelia
Mamuka
M
HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702, USA.
Miller
Jennifer T
JT
Budihas
Scott R
SR
Pannell
Lewis K
LK
Le Grice
Stuart F J
SF
D000595:Amino Acid Sequence; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D019921:Biotinylation; D004247:DNA / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D008239:Lysine / Q000737:chemistry; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D011485:Protein Binding; D011487:Protein Conformation; D023302:Protein Footprinting / Q000379:methods*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D012357:RNA, Transfer, Lys / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013388:Succinimides / Q000494:pharmacology*
D007202:Indicators and Reagents; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; D001710:Biotin; D004247:DNA; D054303:HIV Reverse Transcriptase; D008239:Lysine