Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg2+ Titration Analysis for HIV-1 Ribonuclease H Domain
Open accessAbstract
This article communicates our study to elucidate the molecular determinants of weak Mg 2+ interaction with the ribonuclease H (RNH) domain of HIV-1 reverse transcriptase in solution. As the interaction is weak (a ligand-dissociation constant >1 mM), nonspecific Mg 2+ interaction with the protein or interaction of the protein with other solutes that are present in the buffer solution can confound the observed Mg 2+ -titration data. To investigate these indirect effects, we monitored changes in the chemical shifts of backbone amides of RNH by recording NMR 1 H– 15 N heteronuclear single-quantum coherence spectra upon titration of Mg 2+ into an RNH solution. We performed the titration under three different conditions: (1) in the absence of NaCl, (2) in the presence of 50 mM NaCl, and (3) at a constant 160 mM Cl – concentration. Careful analysis of these three sets of titration data, along with molecular dynamics simulation data of RNH with Na + and Cl – ions, demonstrates two characteristic phenomena distinct from the specific Mg 2+ interaction with the active site: (1) weak interaction of Mg 2+, as a salt, with the substrate-handle region of the protein and (2) overall apparent lower Mg 2+ affinity in the absence of NaCl compared to that in the presence of 50 mM NaCl. A possible explanation may be that the titrated MgCl 2 is consumed as a salt and interacts with RNH in the absence of NaCl. In addition, our data suggest that Na + increases the kinetic rate of the specific Mg 2+ interaction at the active site of RNH. Taken together, our study provides biophysical insight into the mechanism of weak metal interaction on a protein.
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Crystal structures of reverse transcriptase with N-hydroxy naphthyridinone inhibitors demonstrated direct active-site binding and showed that H539 sterically prevents the inhibitors from flipping to the opposite orientation.
“Crystal structures of RT with N-hydroxy naphthyridinone inhibitors also experimentally demonstrated direct RNH active-site interaction by the inhibitors and showed that the position of H539 sterically prohibits the naphthyridinones from flipping to an opposite orientation [78] (Figure 5b).”
From Retroviral RNase H: structure, mechanism, and inhibition (2021) · cites this paper as [78]
The Introduction and Conclusions discuss Mg²⁺ binding to the HIV-1 RNase H domain, not inhibitor-bound crystal structures. Searches for N-hydroxy naphthyridinones, H539, and inhibitor orientation found no passage addressing the claimed binding or steric restriction; the crystal structures listed in the References do not establish this claim. (LLM, conf=0.76)
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- Authors
- Karki, I., Christen, M. T., Spiriti, J., Slack, R. L., Oda, M., Kanaori, K., Zuckerman, D. M., & Ishima, R.
- Affiliations
- Kyoto Prefectural University (JP)
- Corresponding author
- Ishima, Rieko
- ORCID
- Venue
- The Journal of Physical Chemistry B
- Publisher
- American Chemical Society (ACS)
- Published
- 2016-12-05
- Volume
- 120
- Issue
- 49
- Pages
- 12420-12431
- ISSN
- 1520-5207 (ISSN-L)
- 1520-6106
- PMC
- PMC5850897
- OpenAlex
- W2559970489
- Semantic Scholar
- 635daa2948c313bcc6d06984630f6c31753258f1
- Cited by
- 5
- References
- 65
- Citations by year
- 2021: 1 · 2020: 1 · 2019: 2 · 2018: 1
- FWCI
- 0.50
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- The Journal of Physical Chemistry B (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of Allergy and Infectious Diseases (AI R01 100890)
- National Institute of General Medical Sciences (GM R01 105401)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced NMR Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Health Sciences › Medicine › Infectious Diseases › HIV/AIDS drug development and treatment
- Keywords
- HIV-1 reverse transcriptase
- MeSH terms
- Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Binding Sites, Cations, Divalent, Cloning, Molecular, Escherichia coli, Genetic Vectors, Kinetics, Magnesium, Models, Molecular, Protein Binding, Quantum Theory, Recombinant Proteins, Substrate Specificity, HIV-1, Gene Expression, Ribonuclease H, Nuclear Magnetic Resonance, Biomolecular, Catalytic Domain, HIV Reverse Transcriptase, Protein Interaction Domains and Motifs, Molecular Dynamics Simulation
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