Integrative structural biology studies of HIV-1 reverse transcriptase binding to a high-affinity DNA aptamer.

Autor:	Tuske S; Center for Advanced Biotechnology and Medicine, And Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA., Zheng J; Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, 33458, USA., Olson ED; Department of Chemistry and Biochemistry, Center for RNA Biology, And Center for Retrovirus Research, The Ohio State University, Columbus, OH, 43210, USA., Ruiz FX; Center for Advanced Biotechnology and Medicine, And Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA., Pascal BD; Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, 33458, USA., Hoang A; Center for Advanced Biotechnology and Medicine, And Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA., Bauman JD; Center for Advanced Biotechnology and Medicine, And Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA., Das K; Center for Advanced Biotechnology and Medicine, And Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA., DeStefano JJ; Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park, MD, 20740, USA., Musier-Forsyth K; Department of Chemistry and Biochemistry, Center for RNA Biology, And Center for Retrovirus Research, The Ohio State University, Columbus, OH, 43210, USA., Griffin PR; Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, 33458, USA., Arnold E; Center for Advanced Biotechnology and Medicine, And Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA.
Jazyk:	angličtina
Zdroj:	Current research in structural biology [Curr Res Struct Biol] 2020; Vol. 2, pp. 116-129. Date of Electronic Publication: 2020 Jun 30.
DOI:	10.1016/j.crstbi.2020.06.002
Abstrakt:	The high-resolution crystal structure of HIV-1 reverse transcriptase (RT) bound to a 38-mer DNA hairpin aptamer with low pM affinity was previously described. The high-affinity binding aptamer contained 2'-O-methyl modifications and a seven base-pair GC-rich tract and the structure of the RT-aptamer complex revealed specific contacts between RT and the template strand of the aptamer. Similar to all crystal structures of RT bound to nucleic acid template-primers, the aptamer bound RT with a bend in the duplex DNA. To understand the structural basis for the ultra-high-affinity aptamer binding, an integrative structural biology approach was used. Hydrogen-deuterium exchange coupled to liquid chromatography-mass spectrometry (HDX-MS) was used to examine the structural dynamics of RT alone and in the presence of the DNA aptamer. RT was selectively labeled with 15 N to unambiguously identify peptides from each subunit. HDX of unliganded RT shows a mostly stable core. The p66 fingers and thumb subdomains, and the RNase H domain are relatively dynamic. HDX indicates that both the aptamer and a scrambled version significantly stabilize regions of RT that are dynamic in the absence of DNA. No substantial differences in RT dynamics are observed between aptamer and scrambled aptamer binding, despite a large difference in binding affinity. Small-angle X-ray scattering and circular dichroism spectroscopy were used to investigate the aptamer conformation in solution and revealed a pre-bent DNA that possesses both A- and B-form helical character. Both the 2'-O-methyl modifications and the GC tract appear to contribute to an energetically favorable conformation for binding to RT that contributes to the aptamer's ultra-high affinity for RT. The X-ray structure of RT with an RNA/DNA version of the aptamer at 2.8 Å resolution revealed a potential role of the hairpin positioning in affinity. Together, the data suggest that both the 2'-O-methyl modifications and the GC tract contribute to an energetically favorable conformation for high-affinity binding to RT. Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Databáze:	MEDLINE
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