Pancreatic ribonuclease A (PDB ID: 3LXO) from Bos taurus is an endonuclease that catalyzes the cleavage of single stranded RNA (1). RNA molecules often need to be cleaved into smaller fragments during maturation. Therefore, ribonucleases are vital to the synthesis of both messenger RNAs that carry genetic material for making proteins and noncoding RNAs that have a variety of important functions. The RNA concentration in cells is controlled post-transcriptionally by ribonucleases of varying specificity. Ribonuclease A was studied extensively in the 20th century as an excellent model for developing new experimental methodologies and understanding protein structure, folding and stability, and enzyme catalysis. More recently, a biomedical interest has developed in the therapeutic potential of ribonucleases, especially as anti-viral and anti-tumor drugs. Amphibian ribonucleases, such as onconase (PDB ID: 1ONC), exhibit these anti-viral and anti-tumor cytotoxic catalytic functions both in vitro and in vivo by evading endogenous mammalian ribonuclease inhibitors. Interest in human ribonucleases as drug targets, rather than as drugs themselves, advances their relevance for scientific inquiry. Angiogenin (PDB ID: 4AHL) is a ribonuclease A homolog and its ribonucleolytic activity appears to be a precursor in stimulating neovascularization. Because angiogenesis plays a crucial role in physiological processes like embryonic development, wound healing, and endometrial proliferation, and because angiogenin is involved in a number of pathological conditions such as diabetic retinopathy, psoriasis, arthritis, and tumor growth and metastasis, inhibition of the ribonucleolytic function of angiogenin has enormous therapeutic potential, though to date no clinical drugs have been made successfully to target this important family of enzymes (2).

The three-dimensional structure of ribonuclease A is fully encoded by its amino acid sequence. The single chain of ribonuclease A has a molecular weight of 13690.29 Da and contains 124 amino acid residues, none of which are tryptophan (3). The predominant elements of secondary structure of ribonuclease A are a long four-stranded antiparallel β-sheet and three short α-helices. Ribonuclease A contains three α-helices, one 3/10-helix, seven β-sheets, three β-bridges, seven turns, nine bends, and 17 regions of undefined secondary structure (4). The overall structure of ribonuclease A is kidney-shaped with its active site residues lying in the cleft. Four intrachain disulfide bridges form crosslinks between Cys-26 and Cys-84, Cys-40 and Cys-95, Cys-58 and Cys-110, and Cys-65 and Cys-72 (5). These four disulfide bonds are critical to the stability of the native enzyme. The two disulfide bonds between an α-helix and a β-sheet (Cys-26 to Cys-84 and Cys-58 to Cys-110) contribute more to the thermal stability than do the two disulfide bonds between (Cys-40 to Cys-95) or within (Cys-65 to Cys-72) a surface loop as determined by mutagenesis studies which replaced any pair of cysteine residues with alanine or serine (4).

The isoelectric point of ribonuclease A is 8.64 indicating the presence of a large amount of basic amino acids that influence its polarity (3). Ribonuclease A associates with RNA or mononucleotide inhibitors, such as thymidine-3'-monophosphate in this crystal structure (1). The bioactivity of ribonuclease takes place via the essential catalytic triad composed of residues His-12, His-119, and Lys-41. The catalytic role most often contributed to Lys-41 is stabilization of the excess negative charge that accumulates on the nonbridging phosphoryl oxygens in the transition state during RNA cleavage by Coulombic interactions. Lys-41 was determined to also donate a single hydrogen bond to the transition state during catalysis, which was determined by comparison to the catalytic efficiency of two synthetic ribonuclease mutants with Lys-41 replaced by alkylamino-cysteine. The catalytic activity of an amino-cysteine enzyme, which contains both a positive charge and a potential for N-H hydrogen bonding, was compared to the catalytic activity of a trialkylamino-cysteine, which contains only a positive charge. The low activity of the trialkylamino-cysteine ribonuclease argues against the efficacy of Coulombic forces in transition state stabilization; a hydrogen bond must be donated Lys-41 to support the data (6). Ribonuclease A catalyzes the cleavage of the P-O5’ bond of RNA and a mechanism for the transphosphorylation of RNA and subsequent hydrolysis is shown in Figure 1. In the figure, “B” is His-12, and “A” is His-119. During transphosphorylation the side chain of His-12 acts as a base that abstracts a proton from the 2’-oxygen of a substrate RNA molecule and thereby facilitates the nucleophilic attack of oxygen on the phosphorous atom. This attack displaces a nucleoside. The side chain of His-119 acts as an acid that protonates the 5’’-oxygen and facilitates the displacement. Transphosphorylation results in a 2’,3’-cyclic phosphodiester which is subsequently catalyzed to undergo hydrolysis after each histidine residue is appropriately protonated. After hydrolysis of the substrate, each histidine residue is returned to its initial protonation state to complete the catalytic cycle (2, 4, 6). In native ribonuclease A, Asp-121 interacts with His-119 defining a motif known as the catalytic dyad, in which a histidine residue that mediates general acid/base catalysis forms a hydrogen bond with an aspartate residue. This hydrogen bond has a significant, but non-essential role in catalysis, and thus the major role of the catalytic dyad is to enhance the conformational stability of ribonuclease A. A hydrogen bond between the side chain of Gln-11 and a phosphoryl oxygen of substrate enhances catalysis in a subtle manner by orienting the substrate so as to prevent it from binding in an improper conformation (4).

Ribonuclease A catalyzes the cleavage of the P-O5’ of an RNA strand and the hydrolysis of the P-O2’ of a nucleoside 2’,3’-cyclic phosphodiester on the 3’-side of a pyrimidine residue by binding in the binding pocket as shown in Figure 2 (1, 4). A typical single-stranded RNA molecule binds ribonuclease A at the B1R1P1 subsite (pyrimidine binding pocket) and the B2R2P2 subsite (purine binding pocket), with transphosphorylation and hydrolysis occurring at P1 and R1 (1). The specificity and anchoring of pyrimidines, such as thymine, at the B1 binding site arises primarily from specific hydrogen bonds formed between the base and the backbone amide N-H and the side chain hydroxyl of Thr-45. The His-119 residue displays substantial conformational flexibility occupying two major conformations that have been denoted A and B and are related by an approximately 180º torsion about the Cα-Cβ bond, which may be important to catalysis (7). Investigating ribonuclease A structures in complex with the inhibitor thymidine-3’-monophosphate elucidated major protein-ligand interactions. The thymine base packed against the phenyl ring of Phe-120 further anchoring the ligand in the B1 binding site. The ribose moiety of thymidine-3’-monophosphate associates with the R1 subsite due to hydrogen bonding to water molecules which are in turn hydrogen bonded to the peptide backbone. The 3’-phosphate of thymidine-3’-monophosphate is involved in hydrogen bonds with all three of its terminal oxygen atoms. One oxygen atom participates in a hydrogen bond with the basic nitrogen of the His-119 imidazole ring. Another oxygen forms a hydrogen bond with the main chain nitrogen of Phe-120, and the last oxygen forms a hydrogen bond with the side chain amide nitrogen of Gln-11 (1). Comparison of the overall structures of free weight ribonucleic acid (PDB ID: 7RSA) with ribonuclease A in complex with thymidine-3’-monophosphate (PDB: ID: 3LXO) shows no major differences between the backbone positions of both complexes. This differs from other 3-phosphomonoesters where slight variations in the position of loops are observed, such as in the binding of uridine-3’-monophosphate to ribonuclease A (PDB ID: 1O0N). These slight variations in backbone positions is likely caused by varying conformations of certain amino acids, most notably His-119 A and B (1).

PSI-BLAST is a program used to find proteins with similar primary structure to a protein query. PSI-BLAST assigns a significant E value to the subjects that have sequence homology to the query. The E value is calculated by looking at the total sequence homology and by assigning gaps in which an amino acid or group of amino acids exist in the subject’s sequence but not in the query’s sequence. Total sequence homology decreases the E value, while gaps in the amino acid sequence increase the E value. An E value of less than 0.05 is considered significant for proteins (8). The Dali server is a method for finding proteins with tertiary structure similarity to a query. The Dali server uses a sum-of-pairs method to produce a measure of similarity by comparing intramolecular distances. Structures that have significant similarities are assigned a Dali Z-score above 2, and usually have similar folds (9).

Human ribonuclease A (PDB ID: 2K11) is the same protein found in Homo sapiens instead of Bos taurus. The amino acid sequence of human ribonuclease A is 70% similar to that of bovine ribonuclease A, resulting in a low E value of 4e-76 (8). Human ribonuclease A also has similar folds to bovine ribonuclease A corresponding to a high Z-score of 18.9 (9). Human ribonuclease A, like its bovine counterpart, has a high degree of endonucleolytic activity. A major difference is the strikingly high activity of human ribonuclease A to cleave double stranded RNA molecules and RNA-DNA hybrids. The enhanced activities depend on positively charged residues near, but not in, the active site. Thus, human ribonuclease is richer in positively charged residues. Human ribonuclease is not only a digestive enzyme but also has crucial biological roles, such as triggering the development of immature dendrites. Both human and bovine ribonuclease A have four disulfide bonds between cysteine residues to stabilize the structure. Both ribonucleases contain three α-helices and the seven β-strands that form the twisted β-sheet characteristic of the ribonuclease family (10). The backbones between the two ribonucleases are similar, and their catalytic triads are the same. Additional basic residues contribute to the enzymatic and biological activities of human ribonuclease A, such as Arg-4, Lys-6, Arg-32, Arg-39, and Lys-102. These residues enhance the ability of human ribonuclease to cross cell membranes by binding to negatively charged groups and strongly increase the rate of hydrolysis of double-stranded RNA by stabilizing transient single-stranded regions. The catalytic triad is more rigid in human ribonuclease than in bovine ribonuclease, with the flanking positively charged residues adopting multiple conformations instead of His-119. The similar folds and aspects of secondary structure, the conservation of four stabilizing disulfide bridges, and the conservation of the catalytic triad corresponds to the conservation of endonucleolytic activity between bovine and human ribonuclease A; function is dependent on structure (10).