Ternary borate-nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry

Abstract

Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural homology of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl groups, suggested that phosphate ester recognition sites on proteins might exhibit significant affinity for nonenzymatically formed borate esters. (11)B NMR studies and activity measurements on ribonuclease A (RNase A) in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A.3'-deoxycytidine-2'-borate ternary complex that mimics the complex formed between RNase A and a 2'-cytidine monophosphate (2'-CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2'-deoxycytidine mixture, demonstrating the critical importance of the 2'-hydroxyl group for complex formation. Titration of the ternary complex with 2'-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant of RNase A by 2'-CMP. RNase A binding of a cyclic cytidine-2',3'-borate ester, which is a structural homolog of the cytidine-2',3'-cyclic phosphate substrate, could also be demonstrated. The apparent dissociation constant for the cytidine-2',3'-borate.RNase A complex is 0.8 mM, which compares with a Michaelis constant of 11 mM for cytidine-2',3'-cyclic phosphate at pH 7, indicating considerably stronger binding. However, the value is 1,000-fold larger than the reported dissociation constant of the RNase A complex with uridine-vanadate. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters supports enzyme catalysis.

Figure 1

¹¹B NMR spectra for RNase A - borate – 3′-deoxycytidine mixtures. a) 5 mM borate, 2 mM RNase A; b) 5 mM borate, 10 mM 3′-deoxycytidine; c) 5 mM borate, 10 mM 3′-deoxycytidine, 2 mM RNase A. All samples contained 50 mM HEPES, pH 8.65, and the spectra were obtained at T = 5 °C. Total acquisition time for each spectrum was 12.5 h, corresponding to 30,000 transients.

Figure 2

¹¹B NMR spectra. a) 5 mM borate, 2 mM RNase A, 10 mM 2′-deoxycytidine; b) 5 mM borate, 2 mM RNase A, 10 mM 3′-deoxycytidine. All samples contained 50 mM HEPES, pH 8.65, and the spectra were obtained at T = 5 °C.

Figure 3

Effect of temperature on the stability of the ternary complex. ¹¹B spectra of samples containing 5 mM borate, 2 mM RNase A, 60 mM 3′-deoxycytidine at a) T = 5, b) T= 15, c) T=25 °C. Samples also contained 50 mM HEPES, pH 8.65.

Figure 4

Percent of RNase A forming the RNase A•3′-deoxycytidine•borate ternary complex, determined as a function of pH. The sample contained 2 mM RNase A, 5 mM borate, 10 mM 3′-deoxycytidine, in 50 mM HEPES. The fractonal intensity of the ¹¹B signal arising from the ternary complex was calculated, and the results converted to % of RNase A based on the total borate and RNase A concentrations in the sample.

Figure 5

Ligand competition study. a) A series of ¹¹B spectra for a sample initially containing 2.0 mM RNase A, 5 mM borate, 10 mM 3′-deoxycytidine in 50 mM HEPES, pH 7.5. Spectra a through e were obtained with [2′-CMP] concentrations of 0, 1, 2, 5, and 10 mM, respectively. Sample temperature was 5° C. Only the region containing the resonance of the ternary complex is shown. b) Fit of the ligand competition data. The fraction of RNase A in the ternary complex was computed from the fraction of bound borate determined from the ¹¹B resonances.

Figure 6

Active site interactions. a) Interactions of active site RNase A residues with 3′-deoxycytidine-2′-borate are modeled on the basis of the crystal structure of the RNase A complex with cytidine-2′-phosphate (pdb code 1ROB, Lisgarten et al. [26]). b) Overlay of the active site structures for RNase A complexes with 2′-CMP (1ROB) and with 3′-CMP ([27], 1RPF). The 1ROB structure is shown using CPK colors, while for the 3′-CMP complex, the protein residues are green and the 3′-CMP is cyan. Some of the H-bond interactions in the 1ROB structure are indicated as red dotted lines.

Figure 6

Active site interactions. a) Interactions of active site RNase A residues with 3′-deoxycytidine-2′-borate are modeled on the basis of the crystal structure of the RNase A complex with cytidine-2′-phosphate (pdb code 1ROB, Lisgarten et al. [26]). b) Overlay of the active site structures for RNase A complexes with 2′-CMP (1ROB) and with 3′-CMP ([27], 1RPF). The 1ROB structure is shown using CPK colors, while for the 3′-CMP complex, the protein residues are green and the 3′-CMP is cyan. Some of the H-bond interactions in the 1ROB structure are indicated as red dotted lines.

Figure 7

¹¹B spectra of the RNase A complex with cytidine-2′,3′-B(OH)⁻₂. ¹¹B NMR spectra of samples containing 1 mM borate, 2 mM cytidine in 50 mM HEPES, pH 7.0 were obtained in the presence (a) or absence (b) of 2 mM RNase A. Other conditions were: 30,000 transients, corresponding to a total acquisition time of 12.5 h, T = 5 °C. The resonance at δ = −12.3 ppm attributed to cytidine-2′,3′-borate exhibits a very different shift than the resonance arising from the borate monoesters (e.g., Figure 1). The resonance of the 2:1 cytidine-borate complex, which is barely observable under the conditions of the study, is at −7.6 ppm.

Figure 8

Kinetic analysis of RNase A – borate – 3′-deoxycytidine mixtures. Sample contained 1 μM RNase A, 10 mM cCMP, 100 mM HEPES, pH 8.0, as well as 3′-deoxycytidine and borate at the concentrations indicated. Initial velocity was determined from the intensity of the ³¹P resonance of the 3′-CMP product, 3′-dc (3′-deoxycytidine).

Scheme 1

a) Substitution reaction of 3′-deoxycytidine with borate. b) Possible Lys41 facilitation of borate substitution chemistry on RNase A.

Scheme 1

a) Substitution reaction of 3′-deoxycytidine with borate. b) Possible Lys41 facilitation of borate substitution chemistry on RNase A.