Functional Role of Glutamine 28 and Arginine 39 in Double Stranded RNA Cleavage by Human Pancreatic Ribonuclease

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

Role of unique basic residues of human pancreatic ribonuclease in its catalysis and structural stability

Human pancreatic ribonuclease (HPR) and bovine RNase A belong to the RNase A superfamily and possess similar key structural and catalytic residues. Compared to RNase A, HPR has six extra non-catalytic basic residues and high double-stranded RNA (dsRNA) cleavage activity. We mutated four of these basic residues, K6, R32, K62, and K74 to alanine and characterized the variants for function and stability. Only the variant K74A had an altered secondary structure. Whereas R32A and K62A had full catalytic activity, the mutants K6A and K74A had reduced activity on both ssRNA and dsRNA. The mutations of K62 and K74 resulted in reduction in protein stability and DNA double helix unwinding activity of HPR; while substitutions of K6 and R32 did not affect either the stability or helix unwinding activity. The reduced catalytic and DNA melting activities of K74A mutant appear to be an outcome of its altered secondary structure. The basic residues studied here, appear to contribute to the overall stability, folding, and general catalytic activity of HPR

Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis

Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P-1. Although RNase A also contains a P-1 subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P-1 subsite residues Arg36, Asn39, and Gln40 of EDN in its catalytic activity. The individual mutation of Arg36, Asn 39, and Gln40 resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P-1 subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates

Transition temperatures of HPR and variants.

<p>The transition temperatures were derived from the thermal denaturation curves of HPR and its variants. Each experiment was done three times and the standard errors are given.</p

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

<p>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 (<i>k</i>_cat/<i>K</i>_m).</p

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

<p>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. <i>Panel A</i>: thermal transition profile of DNA in the presence of HPR, Q28A, Q28L, G38D, R39A and RNase A. <i>Panel B</i>: thermal transition profile of DNA in the presence of HPR, Q28A/G38D, Q28A/R39A, Q28L/R39A, R39A/G38D, Q28A/G38D/R39A and RNase A.</p

Transition temperatures (<i>T</i>_m) for the melting of DNA in the presence of HPR and variants.

<p>The transition temperatures were derived from the thermal denaturation curves of DNA in the absence and presence of HPR and its variants. Each experiment was done three times and the standard errors are given.</p

Sequence alignment of HPR with other ribonucleases.

<p>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.</p

Steady state kinetics of HPR and variants on poly(A).poly(U).

<p>The kinetic parameters were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017159#s4" target="_blank">Materials and Methods</a>. Each experiment was done three times and the standard errors are given.</p

<i>In silico</i> analysis of HPR variants.

<p>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.</p