volume2 number 8 August 1975
Nucleic Acids Research
Multiplicity of cadmium binding sites in nucleotides: X-ray evidence for the
involvement of O21 and O3' as well as phosphate and N7 in inosine
5 '-monophosphate
D. M. L. Goodgame, I. Jeeves, C. D. Reynolds and A. C. Skapski
Chemical Crystallography and Inorganic Chemistry Laboratories, Imperial
College, London, SW7 2AY.
Received 26 June 1975
ABSTRACT
Single-crystal X-ray methods have been used to characterize a hydrated
polymeric cadmium derivative of inosine 5'~m°nophosphate. In the structure
there are two independent cadmium atoms, one of which binds to two ribose
oxygen atoms, an N7 position on a base, and to three water molecules. The
second metal atom binds to a phosphate oxygen, three water molecules, and to
two N7 atoms, which are in cis-positions. For these last, the Cd-N bonds are
appreciably out of the planes of the hypoxanthine bases so that the angle
between these planes is only 31.4°.
INTRODUCTION
During the past few years there has been increasing concern over the
adverse effects on health of the build-up of heavy metals in the environment.
Many of these are sparsely distributed in nature, but undesirable concentrations result from their use in industry and agriculture.
One such toxic metal
is cadmium, which is virtually absent in mammals at birth but which accumul1 2
ates over a life-time ' .
Apart from localized high concentrations of cadmium in the atmosphere
near industrial plant concerned with the refining of the metal or with
electroplating, the most serious source of cadmium intake by the general
population is thought to be food.
Cadmium has been found in a wide range of
fresh and tinned foods , especially sea-foods .
We have recently been examining the interactions of metal ions with
nucleic acid components in order to provide structural information concerning
the binding sites of these metals.
Such definitive information is useful
for evaluating the possible influence of heavy metals on such processes as
cell replication and, thereby v on the question of carcinogenicity or,
conversely, anti-tumour activity (e.g., as with the platinum anti-tumour
drugs ). As it has been suggested
that occupational exposure to cadmium
may increase the risk of cancer in man, and tumours have been induced in rats
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and mice by cadmium treatment , we have included this metal in our studies.
During work with 5'-IMP we have isolated two crystalline derivatives.
One of these, hydrated [Cd5'-IMP(H2O)5 ] , belongs to the structure type
Q
exemplified by a range of related heavy metal derivatives . However, we have
obtained a second compound, of stoichiometry Cd2(5'-IMP)3 .l2H20, in which one
nucleotide is in the form of 5'-IMP2- and the other two are 5'-IMP1". We
report here the results of X-ray studies on this compound.
These demonstrate
the presence of a variety of metal ion binding sites and, notably, the direct
involvement of ribose oxygen atoms.
EXPERIMENTAL
(a) Synthesis
- A solution of the disodium salt of inosine 5'-monophosphate
(Aldrich Chemical Co.) (O.2O5 g) in water (15 ml) was added with stirring to
one of cadmium nitrate tetrahydrate (0.15(4. g) in water (7.5 ml).
A white
gelatinous precipitate which formed immediately was redissolved by addition of
2M HNOj to give a clear solution of pH 3.88. This was heated at 60°C for
15 minutes and then allowed to stand at room temperature for three weeks. The
colourless crystal clusters which formed were filtered off and air-dried.
Analytical results (Pregl method;
Hicroanalytical Laboratory, Imperial
College) were consistent with the formulation:
Cd 2 (C, jH^N^OePKC, oH12N,,08P)2 .
.12H2O : Calc. C, zk.jO; H, k.0\. Found, C, Zk.~5k; H, 3.97#(b) X-Ray
- The crystals are in the form of flat needles. Preliminary
oscillation and Weissenberg photographs showed them to be monoclinic, with
systematic absences hkl : h + k = 2n + 1. These absences are consistent with
space groups C2, Cm or C2/m.
Space group C2 is the only one of the three
which is compatible with the geometry of the nucleotide. Accurate unit-cell
dimensions were determined by measuring high-angle a^ and o/2 reflections on a
diffractometer and performing a least-squares calculation to give
a = 30.377(0, b = 8.760(1), c = 20.885(2) A, P = 106.29(1)°, V = 5 3 3 1 ^ A 3 , .
and Z = If. Intensity data were measured on a Siemens four-circle automatic
diffractometer.
A total of *t260 independent reflections were measured (to
© = 60°), of which 63 were judged to be 'unobserved*.
The structure was
solved by Patterson and Fourier methods, and least-squares refinement using
ani so tropic thermal parameters has reached R = O.Olfl.
DESCRIPTION OF THE STRUCTURE AND DISCUSSION
The principal features of the structure of the compound are shown in
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5-IMPQ)
5IMP(3)
Figure 1.
A view showing the polymeric structure of [Cd2(5'-IMP)3 (H 2 O) 6 ] .
Figure 1.
It consists of units of the type [Cd2(5'-IMP2-)(5'-IMPi-)2 (H 2 O) 6 ]
arranged in a polymeric array.
(There are also a number of non-coordinated
water molecules of crystallisation, ca. 6 distributed over nine positions.
These play a lesser role and have been omitted from Figure 1 for clarity).
There are two independent cadmium atoms, both having a distorted octahedral coordination, but attached to the nucleotides in differing ways.
Cadmium 1 is bonded to a phosphate oxygen atom from 5'-IMP(3), to the N7 atoms
of 5'-IMP(l) and 5'-IMP(2) with these atoms arranged in cis-positions.
octahedron is completed by three coordinated water molecules.
The
Cadmium 2, on
the other hand, is bonded to N7 of 5'-IMP(3), three coordinated water
molecules, and to the two exocyclic ribose oxygen atoms O2 1 and O31 of
5'-IMP(2).
Thus each 5'-IMP unit binds to cadmium in a different way.
feature is that in all cases N7 is employed.
A common
However, whereas for 5'-IMP(l)
this is the only bond formed to cadmium, with 5'-IMP(2) the two ribose
exocyclic oxygen atoms are also utilized, and 5'-IMP(3) uses one phosphate
oxygen atom.
It may be noted that 5'~ IMp (l) and (3) each carry a unit
negative charge, whereas 5'~ IMp (2) is doubly charged.
The differences in the environments of the cadmium atoms are reflected
in the salient bond lengths.
Thus, in the case of cadmium 1 a rather short
Cd-O(phosphate) bond of 2.23 A is accompanied "by two longer Cd-N7 bonds of
2.40 and 2.42 A, while for cadmium 2 a short Cd-N7 bond of 2.27 A is associated with two longer Cd-O(ribose) bonde of 2.42 A (to O2 1 ) and 2.32 A (to 03»).
On the other hand, for both metal atoms all the Cd-O(water) bonds lie in
a narrow range of 2.27-2-32 A.
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The overall structure is stabilized by numerous hydrogen bonds involving
the coordinated water molecules, waters of crystallization, phosphate oxygens,
ribose oxygens, keto oxygen 06, Nl of the base, etc.
There are several structural details which are of interest for assessing
the likely mode of interaction of cadmium ions with nucleic acids.
unusual feature is the binding of cadmium to ribose oxygen atoms.
suggestions have been made
'
The first
Although
, on indirect evidence, that copper ions
can
bind to the ribose moieties in nucleotides, the present result provides the
first direct demonstration that heavy metal-ribose bonding can occur.
There
is therefore the possibility that such interactions may favour the known
catalytic degradation of RNA by heavy metal ions
the cadmium-ribose interaction
.
However, it appears that
is not an unduly strong one, as the
Cd-O(ribose) bonds tend to be rather long and the O2'-Cd-O3' angle is only
68.3 •
Therefore the effects of such interaction are likely to be secondary
to metal-phosphate bonding in influencing RNA degradation.
As regards metal-phosphate bonding, the only structure available for
comparison is that of [Zn(5'-IMP)]n.nH20 'in which each zinc atom binds to
N7 on the base and to three phosphate oxygen atoms, each from a different
nucleotide unit, to the exclusion of water from the coordination sphere.
This maximum utilization of the phosphate groups by zinc is not paralleled by
the cadmium compound reported here, although the sole Cd-O(phosphate) bond is
quite short.
The fact that each N7 atom present is bonded to cadmium is in agreement
with the general trend of structural information becoming available for
9
purine nucleotides . A feature of particular interest concerns the geometry
of the two hypoxanthine bases which are bonded in cis-positions to Cdl.
The
Cd-N7 bonds are very markedly out of the planes of the purine bases, such that
Cdl is 0.92 A out of the base plane of IMP(l) and 0.8l A out of the plane of
IMP(2).
This results in a distortion towards pyramidal geometry at Ny.
The
flexibility at N7 permits the planes of the two purine bases to move a
considerable way towards achieving a parallel base stacking situation.
Thus,
although the N7-Cdl-N7 angle is 79.1°, the angle between the two base planes
is only 31.^°.
We have observed a similar effect in a platinum compound of 5'-IMP
.
In that case, however, the angle between the two bases was somewhat larger
(k3 ) and it was considered unlikely that this would permit the platinum to
bind simultaneously to adjacent purines in a given strand of helical DNA.
Given the strong tendency of the N7 atoms to bind to metals and the even
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snaller departure from ba6e stacking observed in the cadmium compound
described here, one must now seriously consider the possibility that at least
some metals may bind together adjacent purines in nucleic acids.
This, in
turn, could have serious effects on cell replication.
ACKNOWLEDGEMENTS
We thank the Science Research Council for financial support (to I.J. and
C.B.E.) and Dr. P. de Meester for taking some preliminary photographs.
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