title: Examining the role of the HIV-1 reverse transcriptase p51 subunit in positioning and hydrolysis of RNA/DNA hybrids.
abstract: Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has illuminated novel and important contacts between structural elements at the C terminus of the noncatalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. In particular, a short peptide spanning residues Phe-416-Pro-421 was shown to interact with the DNA strand, cross the minor groove of the helix, and then form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate. At the base of the adjoining α-helix M', Tyr-427 forms a hydrogen bond with Asn-348, the latter of which, when mutated to Ile, is implicated in resistance to both nucleoside and non-nucleoside RT inhibitors. Based on our structural data, we analyzed the role of the p51 C terminus by evaluating selectively mutated p66/p51 heterodimers carrying (i) p51 truncations that encroach on α-M', (ii) alterations that interrupt the Asn-348-Tyr-427 interaction, and (iii) alanine substitutions throughout the region Phe-416-Pro-421. Collectively, our data support the notion that the p51 C terminus makes an important contribution toward hybrid binding and orienting the RNA strand for catalysis at the RNase H active site.
authors.affiliation: RT Biochemistry Section, HIV Drug Resistance Program, Center for Cancer Research, NCI, National Institutes of Health, Frederick Maryland 21702, USA.
authors.forename: Suhman
authors.initials: S
authors.lastname: Chung
authors.forename: Jennifer T
authors.initials: JT
authors.lastname: Miller
authors.forename: Mikalai
authors.initials: M
authors.lastname: Lapkouski
authors.forename: Lan
authors.initials: L
authors.lastname: Tian
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D020125:Mutation, Missense; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D012367:RNA, Viral; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
keywords: Enzyme Mechanisms; Enzyme Structure; HIV; Protein-Nucleic Acid Interaction; Reverse Transcription