18th International HIV Drug Resistance Workshop Basic Principles & Clinical Implications June 9–13 2009, Fort Myers, Florida, USA

HIGH-RESOLUTION STRUCTURES OF HIV-1 RT/RNA:DNA TERNARY COMPLEXES WITH TENOFOVIR DIPHOSPHATE AND dATP

Antivir Ther 2009; 14 Suppl 1:A19 (abstract no. 17)

K Das^1,2, R Bandwar^1,2, J Bauman^1,2, S Tuske^1,2, SH Hughes³ and E Arnold^1,2
1Center for Advanced Biotechnology and Medicine (CABM), Rutgers University, Piscataway, NJ, USA; ²Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA; ³HIV Drug Resistance Program, NCI-Frederick, Frederick, MD, USA

BACKGROUND: The polymerase of HIV RT can synthesize DNA from either a DNA or an RNA template. However, if RT copies an RNA template, the resulting RNA/DNA hybrid is a substrate for the second enzymatic activity of RT, RNase H. Structural differences between RNA:DNA versus DNA:DNA substrates and their interactions with RT are crucial for understanding the enzymatic activities of RT.

METHODS: We determined the crystal structures of D498N mutant RT/RNA:DNA (27:21-mer) in complexes with the NRTI drug TFV diphosphate (TFV-DP) or with the normal substrate dATP. Each structure was determined at 2.6 Å resolution. The D498N RNase H active site mutation was used to block potential RNase H cleavage.

RESULTS AND DISCUSSION: Both structures were obtained in a new crystal form that diffracted X-rays to 2.6 Å resolution, the highest reported for any RT-nucleic acid structure. Significantly tighter packing of RT molecules in the crystal lattice enhanced the resolution. The high-quality electron density maps help precisely position the nucleic acid, TFV-DP and dATP. The RT-bound RNA:DNA template–primer has structural differences from the corresponding RT-bound DNA:DNA duplex. The canonical base pairing of the RNA:DNA duplex is disrupted in a region between the two active sites. The 2´-OH group of the RNA-template overhang forms a hydrogen bond with the main chain carbonyl of G152. The binding of TFV-DP and dATP at the polymerase active site in the RT/RNA:DNA is similar to their binding to RT/ DNA:DNA complexes.

2009-06-09
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