A quantitative characterization of interaction between prion protein with nucleic acids

Abstract

Binding of recombinant prion protein with small highly structured RNAs, prokaryotic and eukaryotic prion protein mRNA pseudoknots, tRNA and polyA has been studied by the change in fluorescence anisotropy of the intrinsic tryptophan groups of the protein. The affinities of these RNAs to the prion protein and the number of sites where the protein binds to the nucleic acids do not vary appreciably although the RNAs have very different compositions and structures. The binding parameters do not depend upon pH of the solution and show a poor co-operativity. The reactants form larger nucleoprotein complexes at pH 5 compared to that at neutral pH. The electrostatic force between the protein and nucleic acids dominates the binding interaction at neutral pH. In contrast, nucleic acid interaction with the incipient nonpolar groups exposed from the structured region of the prion protein dominates the reaction at pH 5. Prion protein of a particular species forms larger complexes with prion protein mRNA pseudoknots of the same species. The structure of the pseudoknots and not their base sequences probably dominates their interaction with prion protein. Possibilities of the conversion of the prion protein to its infectious form in the cytoplasm by nucleic acids have been discussed.

Keywords: Binding constant (Kd); Fluorescence anisotropy (r); Poly A; Prion protein; Pseudoknots; Small highly structured RNAs (shsRNAs).

Fig. 1

Structures of different small highly structured RNAs (shsRNAs) used in this study. The shsRNAs RQ11+12, RQT 157 and MNV contain 197, 157 and 86 nucleotides respectively. The sequence and other details are described in Table 1. The free-energy of formation and the secondary structures are made through RNAstructure website. The RNA pseudoknot structures are also projected through Heuristic Modeling vsfold5.

Fig. 2

a. The structural variations of pseudoknots in prion protein mRNA in different species. The secondary and pseudoknot structures and the free energy were calculated by RNAstructure and vsfold5 as described in. b. A schematic drawing of the classical pseudoknot secondary structure as described by Wills (45) in human. Normally, the pseudoknot contains two stems and three loops. The sequence of the different pseudoknots used in this study were presented in Table 2.

Fig. 3

Increase in the relative tryptophan fluorescence anisotropy of α-PrP in 0.1 M Tris-HCl, pH 7.2 with the increase in the concentrations of different small highly structured RQ 11+12, RQT 157 and MNV having 197, 157 and 86 nucleotides respectively. Excitation, 280 m, emission, 350 nm. The titration experiment was performed with three replications and plotted the average values with error-bars as standard-deviations.

Fig. 4

Interaction between human and mouse recombinant prion protein (α-PrP and moPrP respectively) with different pseudoknots. a. binding of α-PrP with human prion protein mRNA pseudoknot Hm 45 in 100 mM Tris-HCl pH 7.2 and in 100 mM acetate buffer, pH 5 measured from the increase in the fluorescence anisotropy of the tryptophan groups in the protein. Difference of anisotropy values of α-PrP in buffer and in the presence of nucleotides were plotted against nucleotide concentration. b. Binding of α-PrP with three different prion protein mRNA pseudoknots Hm45 (human), Cm47 (cattle) and Ym44 (yeast) at pH 5. c. comparison of binding interaction of mouse prion protein mRNA pseudoknots with α-PrP and moPrP at pH 5. d. comparison of binding of human pseudoknot Hm45 with α-PrP and moPrP. e. binding of moPrP with mouse, human and yeast mRNA pseudoknots at pH 5. Protein concentrations were 220 nM for all the experiments. Temperature of measurements 20 °C. Data points were fitted with a rectangular hyperbola (R² is over 0.9 for all the curves) and the apparent K_D values are presented in Table 3. These titration experiments were performed with a set of three technical replications and plotted the average values with error-bars as standard-deviations.

Fig. 5

Binding interaction of α-PrP with PolyA (a) and tRNA (b) either in 100 mM acetate buffer pH 5 or in 100 mM Tris-HCl buffer pH7.2. Experiments were carried out as mentioned earlier. c. The effect of salt on the interaction between α-PrP and tRNA at pH 7.2 and 5. The effect of NaCl on the complex formed between α-PrP (0.22 µM) and tRNA (12 µM) when saturation values of fluorescence anisotropy were attained in buffers. Small aliquots of 4 M NaCl were added to the solution to attain the desired NaCl concentrations. Temperature 20 °C. All these titration experiments were performed with a set of three technical replications.

Fig. 6

Circular dichroism spectra of α-PrP (a) and moPrP 121–231 fragment (b) in Tris-HCl pH 7.2 (red) and acetate buffer pH 5 (green). in the panel a, in set, represents the structure (PDB ID: 1OEI) of pH-dependent folding of octapeptide repeats region of human prion protein. The result shows that the secondary structures of the full-length prion protein and the globular fragment of the protein do not change a significant extent from neutral to pH 5. c, the exposure of nonpolar groups from the interior of globular structure of prion protein in acidic pH detected by a hydrophobic binding fluorescent probe bis-ANS. The fluorescence intensity of bis-ANS is pH independent. Spectra were taken after incubation of bis-ANS (8 µM) with moPrP121-231(2 µM) for 30 min at the pHs indicated in the figure. From these spectra, ratios of fluorescence intensities at 495 nm (indicative of the dye binding to the newly exposed hydrophobic groups from the interior of the protein) to the fluorescence intensity at 520 nm (for protein in buffer) has been plotted. d. The prion protein sequence alignment of various mammalian species, indicating a highly conserved sequence in helix-1 region (left panel). There are also two salt bridges present in the helix − 1 (right panel). A schematic diagram is presented in the right hand panel, indicating the formation of bridge between (electrostatic interaction and hydrogen-bond) arginine (R) and aspartic acid (D).

Fig. 7

The amino acid sequence alignment at β₂-α₂ loop region (panel-a) and three-dimensional (3D) structure of full-length human prion protein. It was assumed that the β₂-α₂ loop region is extremely flexible and this loop region varies substantially between species. The structure also influenced by the residue types in the 2 amino acid sequence positions 170 (S or N) and 174 (N or T). The corresponding full-length α-PrP 3D structure with dis-ordered-loop (panel-b), and S170N mutated rigid-loop variant (panel-c) (PDB ID: 1QLZ and 1E1S respectively).