Functional role of glutamine 28 and arginine 39 in double stranded RNA cleavage by human pancreatic ribonuclease

Abstract

Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR.

Figure 1. Sequence alignment of HPR with other ribonucleases.

The sequences of ribonucleases were taken from protein data bank their PDB Ids being: HPR, human pancreatic ribonuclease (1DZA); BS-RNase, bovine seminal ribonuclease (1BSR); RNase A: bovine pancreatic ribonuclease (3JW1). The Swiss-Prot ID of DPR, Douc Pancreatic Ribonuclease is Q8SPN4.1. In the parenthesis is given the percent amino acid sequence similarity between different ribonucleases with respect to HPR. The secondary structures are shown at the top of sequences as, α-helices in brown filled box and β-strands in green filled arrow while the loop residues as black line. The identical residues are shown in orange and the conserved cysteine residues are highlighted in light blue. The active site residues, His12 and His119 are shaded in green while the residues under investigation, Gln28, Gly38 and Arg39 in the current study are highlighted in red.

Figure 2. Michelis-Menten curves and catalytic efficiencies of HPR and its variants on poly(A).ploy(U).

The ribonuclease activity of HPR and its variants was analysed on the double stranded RNA substrate, poly(A).poly(U) as described. A. Michelis-Menten curves; B. Catalytic efficiencies (k _cat/K _m).

Figure 3. Michelis-Menten curves and catalytic efficiencies of HPR and its variants on poly(C).

The ribonuclease activity of HPR and its variants was analysed on the single stranded RNA substrate, poly(C) as described. A. Michelis-Menten curves; B. Catalytic efficiencies (k _cat/K _m).

Figure 4. CD spectra of HPR and its variants.

CD spectra were recorded in the far-UV region (200–250 nm) at pH 7.4 and 25°C. The spectra are presented as mean residue ellipticity, expressed in degrees.cm².dmol⁻¹. Panel A: CD spectra of HPR, Q28A, Q28L, G38D and R39A. Panel B: CD spectra of HPR, Q28A/G38D, Q28A/R39A, Q28L/R39A, R39A/G38D and Q28A/G38D/R39A. Panel C: [θ]₂₂₂ of HPR and its variants.

Figure 5. Thermal denaturation profiles of HPR and its variants.

Heat induced unfolding curves of HPR and its variants are shown as plots of f _D values vs temperature. f _D is the fraction of the protein in denatured state as defined in the text. Panel A: denaturation profiles of HPR, Q28A, Q28L, G38D and R39A. Panel B: denaturation profile of HPR, Q28A/G38D, Q28A/R39A, Q28L/R39A, R39A/G38D and Q28A/G38D/R39A.

Figure 6. Effect of HPR and its variants on thermal transition profile of double stranded DNA poly (dA−dT).poly(dA−dT).

The thermal transition profiles of DNA alone or with protein were studied spectrophotometrically at 260 nm in 10 mM MOPS buffer containing 50 mM NaCl (pH 7.5). Melted fraction of DNA (F_t) was plotted against temperature. Panel A: thermal transition profile of DNA in the presence of HPR, Q28A, Q28L, G38D, R39A and RNase A. Panel B: thermal transition profile of DNA in the presence of HPR, Q28A/G38D, Q28A/R39A, Q28L/R39A, R39A/G38D, Q28A/G38D/R39A and RNase A.

Figure 7. Cartoon model of HPR (golden) superimposed over RNase A (light green).

The structure was drawn by taking atomic coordinates from Protein Data Bank in PyMol. PDB IDs of HPR and RNase A are 1DZA and 3JW1, respectively. All residues are shown in ball and stick model. The residues under investigation, Gln28, Gly38 and Arg39 are shown in yellow. Asp38 of RNase A is shown in red, active site residues His12 and His119 in light pink and the ligand, uridine-5'-monophosphate in grey.

Figure 8. In silico analysis of HPR variants.

The structures were drawn in PyMOL software using the coordinates of 1DZA (19). All important residues are shown in ball and stick model. The three panels show effect of respective mutations on various interactions in HPR variants. A. Gln28; B. Gly38; and C. Arg39. The hydrogen bond and van der Waal interactions are shown in blue and red dotted lines, respectively.