title: Thermodynamics of HIV-1 reverse transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors.
abstract: HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a reinterpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.
authors.affiliation: Architecture et Réactivité des ARN, CNRS/Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bec
authors.forename: Benoit
authors.initials: B
authors.lastname: Meyer
authors.forename: Marie-Aline
authors.initials: MA
authors.lastname: Gerard
authors.forename: Jessica
authors.initials: J
authors.lastname: Steger
authors.forename: Katja
authors.initials: K
authors.lastname: Fauster
authors.forename: Philippe
authors.initials: P
authors.lastname: Wolff
authors.forename: Dominique
authors.initials: D
authors.lastname: Burnouf
authors.forename: Ronald
authors.initials: R
authors.lastname: Micura
authors.forename: Philippe
authors.initials: P
authors.lastname: Dumas
authors.forename: Eric
authors.initials: E
authors.lastname: Ennifar
mesh_terms: D002151:Calorimetry; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D058105:Polymerization; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D009711:Nucleotides; D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase