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

• Published in: Biochemistry (Easton) 2017-07, Vol.56 (27), p.3434-3442
• Main Authors: Pandey, Ashutosh K, Dixit, Updesh, Kholodovych, Vlad, Comollo, Thomas W, Pandey, Virendra N
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Dimerization; Enzyme Stability; HIV Reverse Transcriptase - chemistry; HIV Reverse Transcriptase - genetics; HIV Reverse Transcriptase - metabolism; HIV-1 - enzymology; Hydrophobic and Hydrophilic Interactions; Kinetics; Models, Molecular; Molecular Docking Simulation; Molecular Weight; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Conformation; Protein Conformation, beta-Strand; Protein Folding; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Ribonuclease H, Human Immunodeficiency Virus - chemistry; Ribonuclease H, Human Immunodeficiency Virus - genetics; Ribonuclease H, Human Immunodeficiency Virus - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.7b00005

The heterodimeric human immunodeficiency virus type 1 reverse transcriptase is composed of p66 and p51 subunits. While in the p51 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 β1′−β2′ motif from the RNase H domain. Our results indicate that the β1′-β2′ 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.