The β1′−β2′ Motif of the RNase H Domain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Responsible for Conferring Open Conformation to the p66 Subunit by Displacing the Connection Domain from the Polymerase Cleft

The heterodimeric human immunodeficiency virus type 1 reverse transcriptase is composed of p66 and p51 subunits. While in the psi subunit, the connection domain is tucked in the polymerase cleft; it is effectively displaced from the cleft of the catalytically active p66 subunit. How is the connection domain relocated from the polymerase cleft of p66? Does the RNase H domain have any role in this process? To answer this question, we extended the C-terminal region of p51 by stepwise addition of N-terminal motifs of RNase H domain to generate p54, p57, p60, and p63 derivatives. We found all of the C-terminal extended derivatives of p51 assume open conformation, bind to the template-primer, and catalyze the polymerase reaction. Glycerol gradient ultracentrifugation analysis showed that only p54 sedimented as a monomer, while other derivatives were in a homodimeric conformation. We proposed a model to explain the monomeric conformation of catalytically active p54 derivative carrying additional 21-residues long beta 1'-beta 2' motif from the RNase H domain. Our results indicate that the beta 1'-beta 2' motif of the RNase H domain may be responsible for displacing the connection domain from the polymerase cleft of putative monomeric p66. The unstable elongated p66 molecule may then readily dimerize with p51 to assume a stable dimeric conformation.