Formation of a quaternary complex of HIV-1 reverse transcriptase with a nucleotide-competing inhibitor and its ATP enhancer.
J Biol Chem
; 288(24): 17336-46, 2013 Jun 14.
Article
em En
| MEDLINE
| ID: mdl-23598281
ABSTRACT
Nucleotide-competing reverse transcriptase inhibitors were shown to bind reversibly to the nucleotide-binding site of the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). Here, we show that the presence of ATP can enhance the inhibitory effects of the prototype compound INDOPY-1. We employed a combination of cell-free and cell-based assays to shed light on the underlying molecular mechanism. Binding studies and site-specific footprinting experiments demonstrate the existence of a stable quaternary complex with HIV-1 RT, its nucleic acid substrate, INDOPY-1, and ATP. The complex is frozen in the post-translocational state that usually accommodates the incoming nucleotide substrate. Structure-activity relationship studies show that both the base and the phosphate moieties of ATP are elements that play important roles in enhancing the inhibitory effects of INDOPY-1. In vitro susceptibility measurements with mutant viruses containing amino acid substitutions K70G, V75T, L228R, and K219R in the putative ATP binding pocket revealed unexpectedly a hypersusceptible phenotype with respect to INDOPY-1. The same mutational cluster was previously shown to reduce susceptibility to the pyrophosphate analog phosphonoformic acid. However, in the absence of INDOPY-1, ATP can bind and act as a pyrophosphate donor under conditions that favor formation of the pre-translocated RT complex. We therefore conclude that the mutant enzyme facilitates simultaneous binding of INDOPY-1 and ATP to the post-translocated complex. Based on these data, we propose a model in which the bound ATP traps the inhibitor, which, in turn, compromises its dissociation.
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Base de dados:
MEDLINE
Assunto principal:
Piridonas
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Trifosfato de Adenosina
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HIV-1
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Fármacos Anti-HIV
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Transcriptase Reversa do HIV
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Indóis
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Nitrilas
Limite:
Humans
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article