Mg2+ ions: do they bind to nucleobase nitrogens?

Abstract

Given the many roles proposed for Mg²⁺ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg²⁺ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg²⁺ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg²⁺ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na⁺, K⁺, NH₄⁺ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg²⁺ ions do not bind to N7 atoms. Without much of a surprise, Mn²⁺, Zn²⁺ and Cd²⁺, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn²⁺ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg²⁺ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments.

Figure 1.

The Mg²⁺ first hydration shell is strictly defined as deduced from high-resolution crystal structures. (A) d(Mg²⁺…Ow) histogram derived from the CSD (version 5.37, update February 2016; R-factors ≤ 5%). No disordered, error containing, polymeric or powder structures were included. Standard deviations for the average Mg²⁺…Ow coordination distances are given in parenthesis. The water exclusion zone and the second Mg²⁺ coordination shell are marked by a grey and a light blue rectangle, respectively. (B) An ultra high-resolution Mg[H₂O]₆²⁺ CSD x-ray structure examplifyes the strict octahedral water arrangement around Mg²⁺(125).

Figure 2.

Mg²⁺ coordination to purine N7 atoms derived from PDB structures. (A) d(Mg²⁺…N7) histogram (derived from the PDB; May 2016; resolution ≤ 3.0 Å). Ions with B-factors ≤ 1.0 Å² and ≥ 79 Å² were excluded. The different ion binding zones are colored according to Figures 1 and 2B. (B) Scheme showing the different ion binding zones in front of the purine N7 atom (the oxygen and nitrogen atoms able to associate with a cation are shown in red and blue, respectively). The d(Mⁿ⁺…N6/O6) expected distance range is also indicated. Note that these cutoff distances are indicative and simply suggest that the potential ion assignments close to the mentioned limits should be considered with greater care.

Figure 3.

Unrealistic binding of Mg²⁺ to adenine N7 atoms. Mg²⁺ to N6 coordination distances are shown in cyan. The red cross is used to mark misidentified Mg²⁺ ions. (A) Ill-placed Mg²⁺ according to electron density patterns and coordination distances. (B) The Mg²⁺ hydration sphere is incomplete with erratic coordination distances. Fixing the hydration sphere of this ion with a proper hexacoordinated geometry would result in clashes similar to those shown in Supplementary Figure S5.

Figure 4.

Mg²⁺ close to guanine N7 atoms in PDB structures. The cyan question mark, red cross and green mark are used to identify: (i) sites where either Na⁺ or Mg²⁺ match the electron density, (ii) a misidentified and (iii) a correctly placed ion. (A) The d(Mg²⁺…Ow) coordination distances, shown in magenta, were irrealistically modeled to 2.18 Å. A larger Na⁺ could equally fit into this density pattern. (B) Mg²⁺ is distant from the electron density center, leading to underestimated d(Mg²⁺…N7) and d(Mg²⁺…O6) distances. (C) Incomplete and poorly defined Mg²⁺ coordination shell. The coordination distances suggest the presence of Na⁺ or water but not Mg²⁺. (D) A reliable but rare pentahydrated coordination pattern with separate densities for water and Mg²⁺. Similar patterns are reported in a set of high-resolution Z-DNA crystal structure (PDB codes: 1VRO, 292D, 2DCG, 2ELG, 336D).

Figure 5.

Mⁿ⁺ bound to a double N7 motif. (A) Schematical representation of this ion binding pattern with two guanines. Guanine-adenine combinations were also identified (Table 2). (B) 2D diagram showing Mg²⁺ distances with respect to each of the bound N7 atoms. The Mg²⁺ dots are colored according to their distance to the closest N7 atom (see Figure 2). The colored rectangular boxes frame the ions with respect to both coordination distances. (C) A Mg²⁺ placed close to site IV in an E. coli structure (Table 2). Though the coordination distance is correct, this ion assignment is ambiguous since ion and water B-factors < 1.0 Å² are not consistent with those of the guanines. These facts hint to the presence of a more electron dense ion such as Zn²⁺. (D) Mg²⁺ placed close to site IV in a T. thermophilus structure. This ion assignement is ambiguous since, although the coordination distances are in agreement with those of Mg²⁺, the F_o−F_c density (in orange) points to the presence of a more electron dense ion, possibly Zn²⁺.

Figure 6.

Mⁿ⁺ bound to N7/O6 atoms of RpG steps. (A) Schematical representation of this ion binding pattern. (B) Mg²⁺ binding as reported in a group I intron structure. (C) Probable Na⁺ binding observed in a group I intron structure of slightly better resolution.

Figure 7.

Mg²⁺ bound to N7 and anionic oxygens. (A) Overlap of 51 Mg²⁺ found in helix 11 of large ribosomal subunits with d(Mg²⁺…N7/OP) ≤ 2.4 Å. Loop configuration is taken from a H. marismortui structure (PDB code: 4V9F; resolution 2.4 Å). All structures were superimposed on the adenine base. (B) Mg²⁺ bound to a (G)N7 and a phosphate group in B-DNA (crystal contact); separate density peaks for ion and water allow for more reliable ion identification. (C) Mg²⁺ bound to a (G)N7 and a glutamate carboxyl group in a RNA/protein complex.

Figure 8.

Na⁺ coordination. (A) Pentahydrated Na⁺ bound to (G10)N7 in a hammerhead ribozyme structure. (B) Based on coordination distances in the 2.3–2.5 Å range, this pentahydrated Mg²⁺ is probably a Na⁺. Note that some water molecules display isolated density blobs even at a 2.4 Å resolution.

Figure 9.

Polyamine misattributions. (A) Mg²⁺ ions with inapropriate coordination distances are close to N7 atoms in a human SRP helix 6 structure. (B) A spermine molecule—spermine is mentioned in the crystallization conditions—has tentativelly been fitted into the electron density in place of the original Mg²⁺ and water molecules. (C) A misplaced symmetry related polyamine lined up on the major groove of a Z-DNA G=C pair. Note the coordination pattern of the hydrated –NH₃⁺ head that fits a pentahydrated Mg²⁺ (see Figure 4D).

Figure 10.

Missassigned Mg²⁺ ions close to imine N1/N3 atoms. (A) This figure illustrates the pitfalls of placing ions into poorly defined density patterns. See, for example, the unrealistic Mg²⁺ to arginine contact. (B) The tetrahedral coordination inferred from the solvent density at the N3 site and d(Mg²⁺…N/O) in the 2.4–3.0 Å range suggest the presence of a water molecule and excludes that of Mg²⁺.

Figure 11.

d(Mg²⁺…Ow) histogram for nucleic acid crystal structures (PDB; May 2016; resolution ≤ 3.0 Å) that emphasize the systematic use of crystallographic restraints around 2.07 and 2.18 Å. The d(Na⁺…Ow) histogram peaks around 2.4 Å and no peaks associated with crystallographic restraints are apparent (see insert; resolution ≤ 2.0 Å). Supplementary Figure S9 displays d(Mn²⁺/Na⁺…Ow) histograms where peaks due to the use of restraints are also absent (resolution ≤ 3.0 Å).