Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg 2+ Titration Analysis for HIV-1 Ribonuclease H Domain

• Published in: The journal of physical chemistry. B 2016-12, Vol.120 (49), p.12420-12431
• Main Authors: Karki, Ichhuk, Christen, Martin T, Spiriti, Justin, Slack, Ryan L, Oda, Masayuki, Kanaori, Kenji, Zuckerman, Daniel M, Ishima, Rieko
• Format: Article
• Language: English
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/acs.jpcb.6b08323

This article communicates our study to elucidate the molecular determinants of weak Mg 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 interaction with the protein or interaction of the protein with other solutes that are present in the buffer solution can confound the observed Mg -titration data. To investigate these indirect effects, we monitored changes in the chemical shifts of backbone amides of RNH by recording NMR H- N heteronuclear single-quantum coherence spectra upon titration of Mg 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 interaction with the active site: (1) weak interaction of Mg , as a salt, with the substrate-handle region of the protein and (2) overall apparent lower Mg 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 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 interaction at the active site of RNH. Taken together, our study provides biophysical insight into the mechanism of weak metal interaction on a protein.