Metal-binding sites in nucleic acids can have a range of thermodynamic and kinetic properties.
Based on theory and experiment, Draper and Misra6have emphasized three types of association of cations with nucleic acids that provide useful starting categories (seeFigure 5):
(a) ‘‘territorial’’ binding, a mobile charged layer of hydrated cations associated with the nucleic acid polyanion;
(b) nonspecific ‘‘site-binding,’’ association of hydrated cations with pockets of negative elec- trostatic potential created by the nucleic acid structure; and
(c) specific ‘‘site-binding,’’ in which at least one cation aqua ligand is replaced by a ligand from the nucleic acid.
The bulk of this chapter will describe metal sites in category (c), which are specific metal sites that have been localized in X-ray or spectroscopic studies of nucleic acid structures. In order to put these sites into perspective within the complexities of a nucleic acid polyanion, this section provides a brief description of all three categories.
8.29.3.1 Nucleic Acids are Polyelectrolytes: Counterion Condensation Theory
A formal theoretical model for the association of cations with polyelectrolytes is provided by counterion condensation theory. In its ideal form this theory assumes a linear polyanionic chain consisting of regularly spaced charges and of infinite length, in a medium of constant dielectric constant.
Three important properties of the ions involved in counterion condensation are the following.
First, the cations are considered to maintain full hydration and to be ‘‘territorially’’or diffusely bound, meaning that they are mobile, not localized on the nucleic acid. Second, the effective concentration of cations in this mobile layer becomes independent of the bulk at a relatively low threshold concentration. Third, because of charge–charge repulsion, the effective compensation of negative charge on the nucleic acid by the cation counterions is modeled to be slightly less than 1:1. Evidence for these properties has been derived for DNA oligomers using NMR spectroscopy, among other techniques.2 A final and critical point concerning metal interactions with nucleic acids is that the apparent affinity of a metal ion for a site will depend on the counterion atmosphere. For example, the apparent affinity of a divalent ion for a specific site will decrease with increasing concentrations of background monovalent ions. Moreover, the entropic cost of displacing the counterion atmosphere must be considered when metals bind to specific sites in RNA and DNA. These factors lead to relatively low apparent affinities of specific metal ion sites in oligonucleotides in comparison with metal sites found in proteins or small-molecule inorganic complexes.
The assumption that is made in formal counterion condensation theory of a linear, infinitely long oligonucleotide chain breaks down for shorter oligonucleotides and complex RNA structures with irregular shapes. The properties of ion atmospheres for such structures have more recently been modeled using Poisson–Boltzmann-type electrostatic models.15
Figure 5 Three general types of cation interactions with nucleic acid oligonucleotides (after Misra and Draper6).
8.29.3.2 Nonspecific Site-binding
Metal sites in nucleic acids are sometimes predicted from measurements of functional properties, such as stability of the folded structure, that depend on the presence of specific classes of cations.
The classic example of such a site is found in folding studies of transfer RNA (tRNA), whose tertiary structure is stabilized by addition of divalent cations such as MgII, CaII, or MnII (see Figure 6).15,16 The stabilities of RNA and DNA duplexes are always increased by addition of monovalent and divalent cations. Tertiary structure, however, generally creates regions with close association of phosphates and other groups that may accommodate an ion of a particular size and charge properties. Such metal sites may be categorized as ‘‘site-bound’’ or ‘‘localized’’ on the nucleic acid. ‘‘Localized’’ ions may provide sufficient population to be observable in X-ray
B.
A.
C.
OMG34 O2P OMG34 NT
2.25 A21 O1P
2.30
1.98 G20 O2P M11
M5 M4
M7 M6
M9
M3 M2 M8
M1
M10
(a)
3′ 5′
Figure 6 Structure and metal sites of yeast tRNAPhe. (a) The secondary structure (A), ribbon diagram of folded RNA (B), and two specific metal sites (C) (reproduced by permission of Cambridge University Press from Shiand Moore16, vol. 6, pp 1091–1109). (b) Metal site M2 at the intersection of the D and T C loop (PDB 1EHZ (Mn, 1.9 A˚ resolution) (reproduced by permission of Cambridge University Press from Shi and Moore16, vol. 6, pp 1091–1109) and 1TN2 (Pb/Mg, 3.0 A˚ resolution, after Brown et al.170). The PbII
occupancy is estimated as less than 50%. Dashed lines indicate distances estimated to be less than 4 A˚.
crystallography of nucleic acids, but the sites may not require dehydration of the cation. In addition, ‘‘localized’’ metal sites observed by X-ray crystallography may not always correspond to sites of functional importance, i.e., metal sites whose population actually stabilizes the folded nucleic acid.
8.29.3.3 Specific Site-binding
Sites in which metal ions exchange aqua ligands for ligands from the RNA or DNA have been found in several X-ray crystal structures and predicted from spectroscopic measure- ments. Examples of these sites, the main topic of this chapter, include binding of cisplatin to DNA, metal sites in ribozymes, some metal sites in tRNA (Figure 6), and many other cases. It is important to note that for RNA and DNA oligomers, the thermodynamic affinity of metal ions for RNA or DNA ligands depends not only on the structure of the chelating site and the thermodynamics of bond formation, but also on the balancing factors of metal ion dehydration, displacement of other cations from the site, and transfer of the ion from the counterion condensation layer. These effects have recently been treated theor- etically and in at least one case, compared favorably with experimental measurements.15 An important result from such calculations is that when the unfavorable contributions are taken into account, there is rarely sufficient stabilizing energy to compensate for full dehydration of the cation in the nucleic acid site. Thus, metal ions in specific sites in RNA and DNA rarely have lost more than two to three aqua or other exogeneous ligands in exchange for ligands from the nucleic acid. An exception to this is a highly, dehydrated KI site in a ribosomal RNA domain, described in Section 8.29.4.2. It should be kept in mind that the apparent thermodynamic affinities for such sites in nucleic acids depend on the background of other cations and also contain a substantial electrostatic contribution from the site on the nuclei c aci d.
In the following sections of this chapter, metal sites in nucleic acids and small molecules are categorized by metal type. In each section, known structures of sites in RNA and DNA are described, and results of small-molecule models are summarized.