Metal and ligand binding to the HIV-RNase H active site are remotely monitored by Ile556

Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Nucleic Acids Research (Impact Factor: 9.11). 08/2012; 40(20). DOI: 10.1093/nar/gks791
Source: PubMed


HIV-1 reverse transcriptase (RT) contains a C-terminal ribonuclease H (RH) domain on its p66 subunit that can be expressed
as a stable, although inactive protein. Recent studies of several RH enzymes demonstrate that substrate binding plays a major
role in the creation of the active site. In the absence of substrate, the C-terminal helix E of the RT RNase H domain is dynamic,
characterized by severe exchange broadening of its backbone amide resonances, so that the solution characterization of this
region of the protein has been limited. Nuclear magnetic resonance studies of 13C-labeled RH as a function of experimental conditions reveal that the δ1 methyl resonance of Ile556, located in a short, random
coil segment following helix E, experiences a large 13C shift corresponding to a conformational change of Ile556 that results from packing of helix E against the central β-sheet.
This shift provides a useful basis for monitoring the effects of various ligands on active site formation. Additionally, we
report that the RNase H complexes formed with one or both divalent ions can be individually observed and characterized using
diamagnetic Zn2+ as a substitute for Mg2+. Ordering of helix E results specifically from the interaction with the lower affinity binding to the A divalent ion site.

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