Elucidation of spermidine interaction with nucleotide ATP by multiple NMR techniques

Abstract

Interaction of polyamines with nucleotides plays a key role in many biological processes. Here we use multiple NMR techniques to characterize interaction of spermidine with adenosine 5'-triphosphate (ATP). Two-dimensional (1)H-(15)N spectra obtained from gs-HMBC experiments at varied pH show significant shift of N-1 peak around pH 2.0-7.0 range, suggesting that spermidine binds to N-1 site of ATP base. The binding facilitates N-1 deprotonation, shifting its pK(a) from 4.3 to 3.4. By correlating (15)N and (31)P chemical shift data, it is clear that spermidine is capable of concurrently binding to ATP base and phosphate sites around pH 4.0-7.0. The self-diffusion constants derived from (1)H PFG-diffusion measurements provide evidence that binding of spermidine to ATP is in 1:1 ratio, and pH variations do not induce significant nucleotide self-association in our samples. (31)P spectral analysis suggests that at neutral pH, Mg(2+) ion competes with spermidine and shows stronger binding to ATP phosphates. From (31)P kinetic measurements of myosin-catalyzed ATP hydrolysis, it is found that binding of spermidine affects the stability and reactivity of ATP. These NMR results are important for advancing the studies on nucleotide-polyamine interaction and its impact on nucleotide structures and activities under varied conditions.

Figure 1

ATP (a) and spermidine (b).

Figure 2

A typical ¹H-¹⁵N HMBC spectrum acquired with 20 mM ATP at pH 3.2. It shows well-resolved correlation peaks of H-8 and N-9, H-8 and N-7, H-2 and N-1, H-2 and N-3.

Figure 3

¹⁵N chemical shifts of nucleobase of ATP and ATP-spermidine. The data were from ¹H-¹⁵N HMBC spectra measured at varied sample pH, and ¹⁵N shift values were externally referenced to 90% formamide in DMSO (108.2 ppm). ATP: N-1 (○), N-3 (□), N-7 (△), N-9 (▽); ATP-spermidine: N-1 (●), N-3 (■), N-7 (▲), N-9 (▼). The pK_a values for N-1 protonation / deprotonation are 4.3 and 3.4 respectively.

Figure 4

³¹P chemical shifts of α-, β-, γ-phosphates of ATP and ATP-spermidine at varied sample pH, with external reference of 85% H₃PO₄. ATP: α (□), β (△), γ (○); ATP-spermidine: α (■), β (▲), γ (●). The pK_a values for γ-phosphate are 6.0 and 5.0 respectively.

Figure 5

Diffusion constants of spermidine (△), ATP (○), and ATP-spermidine (●) at varied pH. The data were derived from PFG-STE measurements with 20 mM samples at 25°C.

Figure 6

³¹P spectra of ATP-Mg (a), ATP-spermidine-Mg (b), ATP-spermidine (c) and ATP (d) measured at pH 7.0.

Figure 7

(a) Myosin-catalyzed hydrolysis of ATP (△), ATP-Mg (▲), ATP-spermidine (○) and ATP-spermidine-Mg (●), characterized by decreasing β-signals; (b) Linear relationships of natural logarithms of β-signals vs. time.