Nucleobase carbonyl groups are poor Mg²⁺inner-sphere binders but excellent monovalent ion binders—a critical PDB survey

Abstract

Precise knowledge of Mg 2+ inner-sphere binding site properties is vital for understanding the structure and function of nucleic acid systems. Unfortunately, the PDB, which represents the main source of Mg 2+ binding sites, contains a substantial number of assignment issues that blur our understanding of the functions of these ions. Here, following a previous study devoted to Mg 2+ binding to nucleobase nitrogens, we surveyed nucleic acid X-ray structures from the PDB with resolutions ≤2.9 Å to classify the Mg 2+ inner-sphere binding patterns to nucleotide carbonyl, ribose hydroxyl, cyclic ether, and phosphodiester oxygen atoms. From this classification, we derived a set of "prior-knowledge" nucleobase Mg 2+ binding sites. We report that crystallographic examples of trustworthy nucleobase Mg 2+ binding sites are fewer than expected since many of those are associated with misidentified Na + or K +. We also emphasize that binding of Na + and K + to nucleic acids is much more frequent than anticipated. Overall, we provide evidence derived from X-ray structures that nucleobases are poor inner-sphere binders for Mg 2+ but good binders for monovalent ions. Based on strict stereochemical criteria, we propose an extended set of guidelines designed to help in the assignment and validation of ions directly contacting nucleobase and ribose atoms. These guidelines should help in the interpretation of X-ray and cryo-EM solvent density maps. When borderline Mg 2+ stereochemistry is observed, alternative placement of Na + , K + , or Ca 2+ must be considered. We also critically examine the use of lanthanides (Yb 3+ , Tb 3+) as Mg 2+ substitutes in crystallography experiments.

The multiple flavors of GoU pairs in RNA

“…The displacement of that water molecule allows for a tighter packing with another base pair or to the insertion of a protein atom in a protein complex The displacement of the U also creates a binding site frequently occupied by a hydrated potassium ion with binding via hydration water molecules to O4(U), O6(G), and N7(G) in the major groove (Figure ) …”

“…18 • The displacement of the U also creates a binding site frequently occupied by a hydrated potassium ion with binding via hydration water molecules to O4(U), O6(G), and N7(G) in the major groove ( Figure 2). 19,20 With those usual GoU pairs, it is the departure from standard Watson-Crick pairs that is exploited for structural and functional purposes. In the complexes formed, the geometry of GoU pairs does not change conformation; at the very most, one or two water molecules exchange against a ligand in the complex.…”

Deflating the RNA Mg2+bubble. Stereochemistry to the rescue!

“…The dynamical differences between bound K + and Mg 2+ are a direct consequence of the ion stereochemistry that has been repeatedly described 6,7,[11][12][13] . In short, the main distinctive character of these ions, besides their charge, resides in their idiosyncratic coordination distances (Mg 2+ : ≈2.1 Å; K + : ≈2.8 Å; Fig.…”

“…This accomplishment involved the use of the dedicated I23 in-vacuum long-wavelength beamline at the Diamond Light Source facility 2 to detect the anomalous signal of this biologically essential cation [3][4][5] . It allowed RY 1 to revisit the ribosome ionic structure proposing that electron density patterns, previously attributed to Mg 2+ , can now safely be associated with K + when coordination distances and geometries command it, as demonstrated earlier through the use of more candid stereochemically based methods 6,7 . Precisely, the authors focused on the role of K + at the decoding center, showing that a monovalent ion acts as a structural link between the protein uS12, the mRNA codon and the rRNA.…”

“…These binding modes are recurrently observed in potassium channels, nucleic acid quadruplexes and other RNA systems 4,6,15 . K + and Mg 2+ have also distinct binding preferences 6,7 . The primary ligands for monovalent ions in nucleic acids are nucleobase carbonyl groups 7 .…”