Cation binding to nucleic acids can be viewed at two extremes. First, there exists a ‘‘cloud of cations’’, usually monovalents, that condense with and are displaced along the length of the polynucleic acid.40,41 They are fully solvated and are exchangeable, serving mainly to electro- statically stabilize the polyanion. At the other extreme are tightly bound, partially dehydrated cations that bind at specific locations—usually to nucleic acids (especially RNA motifs) with well- defined tertiary structures.33,40,42 More recently, evidence has emerged for an intermediate state (especially for DNA) where fully hydrated ions exhibit preferential binding to certain sequences (Figure 4). These often lie in the minor groove of AT-rich sequences and the major groove of GC-rich sequences.41,43 This mode is especially observed for divalent ions.
Crystallographic characterization of the B-DNA sequence, CGCGAATTCGCG, has been reported at1.5 A˚ resolution, with one Mg2þ per duplex.44 Improved structures of this B-form DNA in the presence of Mg2þand Ca2þhave been recently solved with atomic resolutions of 1.1 A˚
and 1.3 A˚, respectively45(Figure 5). Duplexes in the crystal lattice are surrounded by 13 Mg2þand 11 Ca2þ, respectively. Each cation generates a different DNA crystal lattice and stabilizes different end-to-end overlaps and lateral contacts between duplexes. Mg2þion allows the two outermost base pairs at either end to interact laterally via minor-groove H bonds, turning the 12-mer into an effective 10-mer. A Mg2þion coordinates in the major groove contributing to the kinking of the duplex at one end, while the Ca2þ ions reside in the minor groove, coordinating to the bases through their hydration shells. Mg2þ ions were also found at the periphery of the minor groove, bridging phosphate groups from opposite strands and contacting the groove at one border of the A tract. Several other Mg2þ ions bridge oxygen atoms of bases via coordinated water molecules, forming an extensive network of H bonds. Only two direct Mg–phosphate oxygen bonds have been identified with an MgO distance of2.1 A˚. A comparison of Mg2þand Ca2þcoordination modes reveals some important distinctions, with the cations residing in the major and minor grooves, respectively, while calcium ions are more likely to form inner-sphere complexes. This is consistent with the lower hydration enthalpy of Ca2þ relative to Mg2þ.3,9 In both cases, the divalent ions mediate contacts between multiple strands. Thus, it is possible that Mg2þ ions mediate specific contacts between DNA and small molecules (groove binding drugs) or proteins.
Sequence-specific binding and groove-specific bending ofB-DNA by Mg2þand Ca2þhas been observed crystallographically.45–47 The 1 A˚ resolution structures of the decamers CCAACGTTGG and CCAGCGCTGG reveal binding of Mg2þ and Ca2þ to the major and minor grooves of DNA, respectively, as well as nonspecific binding to backbone phosphate oxygen atoms. Minor-groove binding involves H-bonds interactions between cross-strand DNA
(a) M+, B-form
Narrow minor groove Narrow minor groove Wide minor groove (b) M2+, B-form
Minor groove
Major groove
(c) M2+, B-form
(d) M2+, B-form (e) M2+, A-form (f) M2+, A-form
Figure 4 The correlation between DNA groove width and cation coordination sites is illustrated. The metal species shown are (a) Rbþ, (b) Mg(H2O)62þ, (c) Mg(H2O)62þ, (d) Ca(H2O)82þ, (e) Mg(H2O)62þ, and (f) Co(NH3)63þ. (reproduced with permission from Hudet al.41; # 2001, Society of Biological Inorganic
Chemistry).
base atoms of adjacent base pairs and the metal-bound water ligands. In the major groove, the hydrated metal ion H bonds with O and N atoms from adjacent bases.
Divalent Mg interacts with DNA in a more specific manner, since waters of solvation can both donate and accept H bonds to base atoms. Mg2þ is drawn electrostatically to both major and minor grooves of DNA, as well as to the sugar–phosphate backbone. At GGCC/GGCC sequences, binding of magnesium to the major groove GG residues, and to the minor groove at the GC step, results in a bending of the helix toward the major groove. Ca2þion has been found to form polar covalent bonds bridging adjacent N7 and O6 atoms of GG bases. In addition to having higher affinity to DNA, Ca2þ binding to DNA results in greater DNA bending and thermal stabilization than Mg2þ.45–47
Of considerable importance are the structural motifs termed telomeres that have been identified at the ends of chromosomal DNA. Such motifs provide chromosomal stability and have been implicated in cell replications and a putative role in the mechanism of aging. Such structures (illustrated inFigure 6) are formed when four guanine bases form stable four-stranded helices.
Such tetrads are stabilized principally by potassium ion, although sodium ion stabilization has also been characterized.48,49 A cubic coordination motif is generated by the eight O6 atoms from the guanine bases.
8.5.4.2 RNA
Highly structured RNA domains are often stabilized by either Kþ,50or especially Mg2þ.42Divalent magnesium may bind in either an outer-sphere or by an inner-sphere mode (Figure 7).51The former requires hexahydrated magnesium [Mg(H2O)6]2þto interact with RNA and base centers by hydro- gen bonding to the waters of solvation. These waters are consequently stabilized to exchange with bulk water. Often, binding is to the O6 and N7 atoms of guanosine.51,52In a few cases the hydrated metal ion is stabilized by a negative electrostatic potential surface, with no direct bonding contacts.
For inner-sphere ligation, common ligands to Mg2þinclude the phosphate oxygens, N7 on purine bases, base keto groups such as O6 of guanosine and O4 of uracil, and the ribose 20-OH.51Typically from 1 to 3 waters are displaced and octahedral coordination in maintained.
Mg1 Mg1
1
Mg2 Mg2
Mg3 Mg3
Mg4 24
1
Mg4
Mg2b Mg2b
Mg5 Mg5
Mg4C Mg4C
Mg1b Mg1b
Mg5C Mg5C
Mg2a
Mg3a Mg3a
Mg2a
Mg4a Mg4a
Mg5b Mg5b
13 13
12 12
Figure 5 Stereoview of the B-DNA, CGCGAATTCGCG, in the presence of Mg2þ. Filled bonds indicate direct ion–DNA contacts, broken lines indicate hydrogen bonds (reproduced with permission from Minasovet al.45).
Several binding motifs on RNA have been characterized. These include the major groove of tandem G–U pairs (Figure 7) which provides a surface with strong negative potential and a series of potential ligands to the metal ion, and H-bound acceptors for metal-bound H2O. Such sites readily bind hydrated magnesium ion.53 The deep major groove of A-form RNA, and internal loops such as the loop E motif (Figure 8) are regions of high negative charge density and are attractive binding sites for hydrated Mg2þ. The loop E motif shows a number of magnesium ions that crosslink neighboring backbones, forcing them into close proximity, and include an unusual binuclear cluster.54
The adenine–adenine platform RNA motif has been shown to define a monovalent ion-binding site that is important for ribozyme folding (Figure 8).40,42 Such binding motifs complement the general association of counter ions that are required to minimize electropositive repulsion between phosphates, and are likely to be more common than current evidence suggests. Selectivity mechanisms for ion binding include Coulomb attraction and van der Waals interactions arising from size variations and changes in RNA conformation. However, differences in solvation energies for bound and free solution ions play a major role. The calculated selectivity (Kþ>Naþ>Rbþ>Csþ>Liþ)40 reflects experimental hydration energies.55 Experimentally, the preferred ion is Kþ. The binding site for Kþis illustrated inFigure 8 and can be contrasted with the G tetrad identified in telomeric DNA (Figure 6).
8.5.4.3 Drug–Mg2þ–DNA Ternary Complexes
Many DNA-binding drugs use a divalent cofactor to mediate binding of the two partners. The metal cofactor typically bridges the phosphate of the DNA backbone with a metal chelate motif on the ligand. The metal cofactor may also perturb the conformation of the ligand and/or the DNA to optimize the drug–DNA interaction. For example, mithramycin (Figure 9) is a member of the aureolic acid group of antitumor antibiotics. They contain an aureolic acid core that chelates Mg2þ. Divalent magnesium appears to promote the selective binding to a cognate DNA sequence (50-XXGCXX-30) by a coupled mechanism requiring local conformation changes in the DNA backbone and a structural rearrangement of the mithramycin dimer.56Molecular modeling suggested that sequence selectivity arises from preferential coordination of Mg2þ to d(GpC) domains in the minor groove of Z-type DNA. Magnesium stabilizes a local Z-type conformation, which opens up binding pockets on the DNA for the sugar chains. Additionally, Mg2þwas also found to realign and stabilize the MTC dimer in a manner that optimizes the position of both the aureolic acid core and the sugar chains for DNA binding.
Similar examples of synergy between drug and DNA binding through a metal cofactor have been identified. In each case the drug contains a chelate motif (Figure 9) for a metal cofactor (typically Mg2þ), such as quinobenzoxazine and derivatives, quinolones (norfloxacin), and UK-1.57–60
N N
N N N
N N
N
N N
N N N
N N N
N N N
O
O
O O O
O O
O
O H
H
H H H H
H H
H H H
H
Syn
Syn
Anti (a) Anti
(b) (c)
G
G
G
G G
G G
G G G G
G G
G G
G T
T
T T
T T
T T
5´
5´
5´
5´
K+
3.4Å
2.80Å 3.15Å
Figure 6 Stabilization of telomeric DNA by Kþ. (a) A guanine tetrad within an antiparallel quadruplex.
Strand orientations (50to 30) are represented by (X) or (), respectively. Structural requirements at the ribose rings are indicated. (b) A proposed structure for the overlapping ends of telomeric DNA. (c) An octacoord- inate potassium chelation cage created by the O6 oxygen atoms of the stacked guanine bases. Interatomic distances are derived from fiber diffraction studies and model building (reproduced with permission from
Sundquist and Klug48;#1989, Society of Biological Inorganic Chemistry).
H2N
H2N
H2N N
N N
NH2
NH2
N N
N N
N
N N N
N
H H H
H H H
HH H H H H H
H H
H H H H
H H
H H
H H H
H H H H H
H
H
H O
O O
O O OO O
O
O
O
O O
O O O O
O
O O
O O
O O
P
P
P Mg
N Mg
N N N
N
O O
O O
O O O
O O P
O
(a)
(b)
(c)
3´
5´
5´-NN GU NN-3´
3´-NN GU NN-5´
major groove edge
O
N
N N H N
H2N
6 7
sugar
A104
A73 U74
G72
G102
G75
G78
U77
O58 A78
C97
C98
Rp
Rp
G100
A101
5´ GCGAGAGUAGGG GUGAUGGUAGCC
A101
G109 A98
N Mg
Figure 7 (a) A variety of magnesium binding sites on RNA structures, showing outer-sphere binding through solvent waters, and direct inner-sphere binding to phosphates. (b) A tandem GU motif that is favorable for major groove binding of metal cofactors through the O6 and N7 of guanosine. Outer-sphere binding by Os(NH3)63þ, a mimic of Mg(H2O)62þ, is shown. (c) The loop E motif possesses a narrow minor groove that appears able to stabilize binuclear magnesium centers (reproduced with permission from Pyle42;
#2002, Society of Biological Inorganic Chemistry).