Proton and Phosphorus-31 NMR Study of the Dependence of Diadenosine Tetraphosphate Conformation on Metal Ions*

Adenosine 5’- tetraphospho- 5’ -adenosine (Ap,A) plays a role in cellular metabolism in a wide variety of organisms. Because the divalent cations Mg2+ and Zn2+ are involved in the synthesis and function of Apa, the effect of divalent cations on the dinucleotide’s confor- mation is of interest. ‘H and “P chemical shift exper-iments were carried out as a function of Mg2+ concen- tration and pH. We propose that Mg2+ stabilizes the unusual ring-stacked conformation of Ap,A at pH > 2 by interacting with the @-phosphates. To further probe conformational effects, stable complexes of Ap,A with Co3+ were studied using and “P NMR. eo3+ forms two different bidentate complexes with Ap4A, inde-pendent of whether the other four octahedral coordi- nation sites are occupied by ammonia or trimethylenediamine. NMR results suggest that in one complex the eo3+ is coordinated to two &phosphates and ring stack- ing is stabilized. In the other complex, Cos+ is coordinated to an a-phosphate and its neighboring @-phos-phate and ring stacking is destabilized. These results further support the hypothesis that M e + stabilizes the ring-stacked conformation by interacting symmetri-cally with the two &phosphate groups. The approach of Cornelius et al. (7) Co(NH3).ATP was followed, except that the dinucleotide reaction was run in DzO (99.8% D) instead of water. This was done so that the reaction mixture could be studied immediately by 'H NMR. The synthesis of trimethylenediamine Co(II1) complexes with ATP has been described by Hediger and Milburn (9). We followed their approach with Ap,A instead of ATP, but again working at a 25-50-mg level. 'H NMR spectra were obtained at Wellesley College on a Varian CFT-20 spectrometer modified for protons at 80 MHz. Ninety-degree pulses were applied with no delay time. 31P NMR spectra were obtained at the NMR Resource at the National Magnet Laboratory at Massachusetts Institute of Technology on a Bruker HFX-270 spectrometer operating at 109.3 MHz, Ninety-degree pulses were applied with a delay time of 1 s and broad-band proton decoupling. Temperature was maintained at 24 "C for 'H and 31P spectra.

'H NMR and CD studies have led to the proposal of an unusual ring-stacked conformation for this molecule at neutral pH (2,3). Since the divalent cations Mg2+ and Zn2+ are known to be involved in the synthesis and function of Ap,A, the effect of these cations on the dinucleotide's conformation is of interest. In this report, we focus on Mg?+. Recent CD studies of the family of bisnucleoside oligophosphates of which Ap4A is a member (3) have been interpreted to suggest that Zn2+ disrupts the stacked conformation at neutral pH, but that Mg2+ causes conformational change while maintaining stacking. Our 'H NMR results described below suggest that Mg2+ enhances ring stacking at pH values between 2 and 6 and at least maintains it at pH 7.
* This work was supported by grants (to N. H. K.) from the Dreyfus Foundation and the Research Corporation and by Instrumentation Grant CDP8008634 from the National Science Foundation. The NMR Resource of the Francis Bitter National Magnet Laboratory is supported by Grant RR0095 from the Division of Research Resources of the National Institutes of Health and by National Science Foundation Contract C-670. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To use NMR to probe the site(s) of metal ion interaction with the dinucleotide and the effects of this interaction on overall conformation, we have synthesized stable complexes of A p a with Co3+. ~g2+-nucleotide complexes are not as amenable to such studies since ligand exchange is rapid. O~a h e~a l complexes of Co3+ or C9+ with nucleotides, on the other hand, have dissociation times on the order of lo7 s (4).
The assumption that Co3+ can be used to study the behavior of Mg2+ is based on studies of Cr3+ + ATP and Co3+ .ATP complexes, in which the latter complexes were shown to specifically inhibit a number of enzymes which have Mg2+. ATP or other nucleotides as substrates (5). Furthermore, since the ionic radii of both M e and Co3+ are approximately 0.065 nm (61, the distance from the central metal ion to the ligand are very similar.
We have used 31P NMR to demonstrate coordination of the Co3+ to the dinucleotide and to identify which phosphate groups are bonding to the metal ion. When trivalent cobalt forms complexes with phosphate groups, it produces a decrease in the electron density around the phosphorus atom, thereby causing deshielding effects which shift the coordinated phosphate resonances downfield. Cornelius et al.

EXPERIMENTAL PROCEDURES
ApA was purchased as the trilithium salt from Boehringer Mannheim, and 5'-AMP as the disodium salt from Sigma. Both were used without further purification. Solutions containing metal ions were prepared by mixing weighed quantities of the mono-or dinucleotide with solutions of MgC12 of known concentration. Concentrations of initial metal chloride solutions were determined by titration with EDTA. Concentrations of mono-or dinucleotide solutions were determined by UV absorption spectroscopy (8). In order to minimize intermolecular nucleotide interactions, all solutions used for *H and NMR spectra were prepared so that the concentration of 5'-AMP or Ap4A was 0.008 M. Dilution beyond 0.008 M produced no changes in chemical shifts, suggesting that this concentration was low enough to avoid internucleotide interactions.' An internal reference compound, tetramethylammonium chloride (6 = 3.180 ppm downfield from tetramethylsilane), was used at a concentration of 5 p~ for all 'H spectra. 31P spectra were run with a reference solution of 8.5% D3P04 in D20 in a concentric outer tube. Values are reported for pH although all solutions for NMR spectroscopy were prepared in D'O.
Values were calculated by adding 0.4 to measured pH values. mine cobalt(II1) and the other bis(trimethy1enediamine) cobalt(II1).
Syntheses of the complexes were done using 25 or 50 mg of Ap,A.

Cation Dependence of Diadenosine Tetraphosphate Conformation
The approach of Cornelius et al. (7) Co(NH3).ATP was followed, except that the dinucleotide reaction was run in DzO (99.8% D) instead of water. This was done so that the reaction mixture could be studied immediately by 'H NMR. The synthesis of trimethylenediamine Co(II1) complexes with ATP has been described by Hediger and Milburn (9). We followed their approach with Ap,A instead of ATP, but again working at a 25-50-mg level.
'H NMR spectra were obtained at Wellesley College on a Varian CFT-20 spectrometer modified for protons at 80 MHz. Ninety-degree pulses were applied with no delay time. 31P NMR spectra were obtained at the NMR Resource at the National Magnet Laboratory at Massachusetts Institute of Technology on a Bruker HFX-270 spectrometer operating at 109. 3 MHz, Ninety-degree pulses were applied with a delay time of 1 s and broad-band proton decoupling.
Temperature was maintained at 24 "C for 'H and 31P spectra.

RESULTS AND DISCUSSION
'H NMR studies (2) of the dependence of the chemical shifts of the Ap4A adenine ring protons on pH suggested that this molecule undergoes a conformational transition with increasing pH. Whereas the rings appear to be unstacked below pH 4, they pass through an intermediate stage that has been called "folded" (2) and finally become stacked above pH . There is a possibility that the differences between Ap4A and 5'-AMP H-8 chemical shifts in the presence of Mg2+ could be due to this proton's proximity to different numbers of negatively charged phosphate moieties in the two species rather than to conformational changes. In order to probe this, we monitored the pH dependence of 5'-ADP and 5'-ATP as well as that of 5'-   AMP and found no significant difference in H-8 chemical shift^.^ How the magnesium cation interacts with Ap4A is demonstrated by 31P NMR spectra. Fig. 3 is a graph of chemical shift (with respect to 8.5% D3P04) for the a-and @-phosphate groups of ApA alone in solution and in the presence of several concentrations of MgC12. First, Ap4A must be considered in the absence of Mg2+. As pH increases in the absence of Mg2+, the @-phosphate resonance undergoes significant upfield shifts, whereas the a-phosphate resonance undergoes negligible upfield shifts. The shift of the @-phosphate resonance must be caused by an effect other than deprotonation since deprotonation is known to cause downfield, rather than up-C. Redfield, private communication.

Cation Dependence of Diadenosine Tetraphosphate Conformation 14573
field, shifts. Gorenstein (10) has shown that changes in O-P-0 bond angles lead to upfield shifts on either side of a minimum at 107". Thus, we conclude that the conformational change which accompanies ring stacking must be occurring (at least in part) about the P-0-P bond between the @phosphates. Additional evidence for this conclusion comes from an analysis of Fig. 4. This well-resolved 31P NMR spectrum of Ap4A at pH 7.0 was analyzed as an AA'XX' system, with the a-phosphates A and A' and the @-phosphates X and X'. Whereas it would be desirable to obtain J(XX') values for Ap4A when it is associated with M$+, our 31P NMR spectra of this system was not sufficiently well-resolved to allow calculation of coupling constants. The hypothesis derived from 'H NMR data is, however, well-supported by 31P NMR chemical shift results. In the presence of M$+ at concentration ratios ranging from 0.38 to 3.0, [M$+]/[Ap4A], the change in chemical shift of the @-phosphates is opposite to that in the absence of M$+. For all ratios, the chemical shift is downfield from that of the free dinucleotide at pH 5, 6, and 7 and upfield at lower pH values. Two factors could account for this. It could be postulated that M$+ prevents Ap4A from assuming its ring-stacked conformation at pH values above 5, but this is obviated by 'H NMR results described above, which clearly demonstrate that ring stacking is occurring. The other factor is the association of M$+ with the @-phosphates. Association of metal ions with phosphate groups causes significant downfield shifts (>lo ppm, see below). The comparatively small downfield shifts in the presence of M$+ suggest the formation of a rapidly dissociating complex between the cation and the @-phosphates of Ap4A.
At pH values between 2 and 5 in the presence of M$+, however, the @-phosphates experience relatively constant, small upfield shifts. This is consistent with 'H NMR results which suggest that M$+ enhances the population of a conformation in which there is ring stacking at all pH values above 2. Thus, the @-phosphate chemical shifts in this pH range represent a balance between the upfield shift that accompanies ring stacking and the downfield shift that accompanies binding to M P .
The chemical shift for the a-phosphates is relatively insensitive to pH and M2+. The a-phosphates thus appear neither to bind to M P nor to experience changes in their 0 -P -0 bond angles due to ring stacking.
To confirm our hypothesis that M$+ binds to Ap4A in a 1:l ratio at the @-phosphates, we synthesized coordination complexes of Ap4A (and, for comparison, ATP) with Co3+ as described above. ATP formed complexes with both tetraammine cobalt(II1) and trimethylenediamine cobalt(II1). 31P NMR spectra (not shown) suggest that coordination took place at either the a-and @-phosphates or the @-and yphosphates for the trimethylenediamine cobalt(II1) complex and at the @-and y-phosphates only for the tetraammine cobalt(II1) complex (8).

for C O ( N H~)~. A~~A and
Cotnz.Ap4A indicate that in each case the cobalt ion has formed more than one type of complex with Ap4A. Six distinct resonances can be observed for the Cotnn .Ap4A complexes (Fig. 5B). It is somewhat more difficult to identify six separate signals in the tetraammine complex spectrum (Fig. 5A) due to poorer resolution. However, none of the peaks appear at positions of uncomplexed Ap4A. In light of complexes observed between ATP and Co3+ (7), we have assigned two of the signals to a @. @ complex, whereas the remaining four signals are assigned to an a.@ complex, in which one of the a-phosphate groups and its neighboring @-phosphate groups are not complexed. Table I presents the 31P chemical shifts for Ap4A, Co(NH3),. Ap4A, and Cotn, .Ap4A. In Table 11, differences in chemical shift for phosphates in uncomplexed Ap4A and in the cobalt complexes are shown. For each type of cobalt complex, shifts are reported for two differently coordinated Ap4As, one in which an a-phosphate and its neighboring @-phosphate are associated with the central metal and one in which both @-phosphates are associated. Shifts for the complexed (c) and uncomplexed (u) phosphates are assigned, as well as for phosphates which are not complexed themselves but are adjacent to complexed phosphates (n). The assignments of the six resonances in the 31P NMR spectra of Co(NH,),.Ap4A and Cotnz.Ap4A can be best understood by referring to the following diagram (in which only those phosphate oxygens necessary to show binding sites are We assume that coordination of a phosphate group with Co3+ affects the coordinated group resonance by shifting it down- field. At the same time, adjacent phosphate group resonances experience upfield shifts (7). Thus, in the @.@ complex, both @-phosphate resonances should show downfield shifts, and both a-phosphate resonances should show upfield shifts. If the complex is symmetrical, one resonance should be observed for the a-phosphate and one for the &phosphate. The CY.@ complex should exhibit four resonances, an a-phosphate and a @-phosphate with large downfield shifts, a @-phosphate with an upfield shift, and an a-phosphate with no shift due to complex formation. Peaks a, c, d, and f in both spectra are assigned to the a. @ complex. Peak a is the 8-ppm downfield shifted resonance of the coordinated a-phosphate, peak c is the relatively unchanged resonance of the uncomplexed a-phosphate, peak d is the 7-8-ppm downfield shifted resonance of the complexed P-phosphate, and peak f is the 1-2-ppm upfield shifted peak of its adjacent, uncomplexed @-phosphate. Peaks b and e are assigned to the 8.p complex. Peak b is the 11-ppm downfield shifted resonance of the two coordinated @-phosphates. Peak d is the 3-ppm upfield shifted resonance of the two adjacent, uncoordinated a-phosphates. No residual Ap4A peaks are seen, suggesting that the complex formation reaction went to completion, yielding two products.

A D A (ppm
The identification of two products is confirmed by the 'H NMR spectrum which appears in   has been shown to be in a ring-stacked conformation at pH 6.6, formation of this complex must destabilize the stacking of the adenine rings. The other pair of H-8 and H-2 peaks are slightly upfield of those of free Ap4A at this pH, indicating a slight increase in ring stacking. This complex is likely to be the 8. / 3 complex, whereas the destacked, asymmetric complex is the a.8. The pH behavior of the Cotn2 .Ap4A complexes further confirms the existence of two different species. Free Ap4A assumes an unusual folded conformation in the pH range 3-5 (2). This was shown by downfield shifts of H-8 and H-2 as compared with 5'-AMP. In both cobalt complexes, these downfield shifts are absent. In one case, which we assign to the 8. /3 complex, considerable upfield shifts are seen. This is consistent with our observations for the effect of Mg+ on Ap4A, where ring stacking and its resulting upfield shifts are promoted at pH values above 2. The other complex, a ./3, seems to leave the adenosine groups in a position in which they do not experience any intramolecular interactions and thus behave like 5'-AMP.
Final confirmation of the assignment of the NMR results to the two different isomers of C O ( N H~)~. A~~A and Cotn2. Ap4A awaits successful separation and perhaps x-ray crystallographic analysis. Attempts were made to accomplish separation using high pressure liquid chromatography, but the procedure led to complete degradation of the complexes. Nevertheless, it is clear from the results presented above that two products are formed and that the assignments made are consistent with the hypotheses that Mg2+ forms @. @ complexes. The biological significance of Mg2+ stabilization of the unusual ring-stacked conformation of Ap,A despite changes in pH may be that the cation enables the nucleotide to maintain this conformation in situations in which its local cellular environment experiences pH fluctuations. Free Ap4A does not maintain such conformational stability.