Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg²⁺ Titration Analysis for HIV-1 Ribonuclease H Domain

This article communicates our study to elucidate the molecular determinants of weak Mg²⁺ 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 Mg²⁺ interaction with the protein or interaction of the protein with other solutes that are present in the buffer solution can confound the observed Mg²⁺-titration data. To investigate these indirect effects, we monitored changes in the chemical shifts of backbone amides of RNH by recording NMR ¹H-¹⁵N heteronuclear single-quantum coherence spectra upon titration of Mg²⁺ 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 Mg²⁺ interaction with the active site: (1) weak interaction of Mg²⁺, as a salt, with the substrate-handle region of the protein and (2) overall apparent lower Mg²⁺ affinity in the absence of NaCl compared to that in the presence of 50 mM NaCl. A possible explanation may be that the titrated MgCl₂ 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 Mg²⁺ interaction at the active site of RNH. Taken together, our study provides biophysical insight into the mechanism of weak metal interaction on a protein.