Eosinophil cationic protein (ECP) can bind heparin and other glycosaminoglycans through its RNase active site

The eosinophil cationic protein (ECP) is an eosinophil‐secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. During inflammation and eosinophilia disorders, ECP is secreted to the inflammation area, where it would contribute to the immune response. ECP secretion causes also severe damage to the host own tissues. ECP presents a high affinity for heparin and this property might be crucial for its immunomodulating properties, antipathogen action, and its toxicity against eukaryotic cells. ECP, also known as human RNase 3, belongs to the mammalian RNase A superfamily and its RNase activity is required for some of its biological properties. We have now proven that ECP heparin binding affinity depends on its RNase catalytic site, as the enzymatic activity is blocked by heparin. We have applied molecular modeling to analyze ECP binding to heparin representative probes, and identified protein residues at the catalytic and substrate binding sites that could contribute to the interaction. ECP affinity for heparin and other negatively charged glycosaminoglycans (GAGs) can explain not only its binding to the eukaryote cells glycocalix but also the reported high affinity for the specific carbohydrates at bacteria cell wall, promoting its antimicrobial action. Copyright © 2010 John Wiley & Sons, Ltd.     

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. Deepa Sikriwal and Divya Seth contributed equally to this work.     

Characterization of an RNase with two catalytic centers. Human RNase6 catalytic and phosphate-binding site arrangement favors the endonuclease cleavage of polymeric substrates

Background

Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site.

Three-dimensional crystal structure of human eosinophil cationic protein (RNase 3) at 1.75 A resolution

Eosinophil cationic protein (ECP; RNase 3) is a human ribonuclease found only in eosinophil leukocytes that belongs to the RNase A superfamily. This enzyme is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. The protein has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data up to 1. 75 A resolution. The molecule displays the alpha+beta folding topology typical for members of the ribonuclease A superfamily. The catalytic active site residues are conserved with respect to other ribonucleases of the superfamily but some differences appear at substrate recognition subsites, which may account, in part, for the low catalytic activity. Most strikingly, 19 surface-located arginine residues confer a strong basic character to the protein. The high concentration of positive charges and the particular orientation of the side-chains of these residues may also be related to the low activity of ECP as a ribonuclease and provides an explanation for its unique cytotoxic role through cell membrane disruption.     

High-resolution crystal structures of ribonuclease A complexed with adenylic and uridylic nucleotide inhibitors. Implications for structure-based design of ribonucleolytic inhibitors

The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3',5'-ADP, 2',5'-ADP, 5'-ADP, U-2'-p and U-3'-p have been determined at high resolution. The structures reveal that each inhibitor binds differently in the RNase A active site by anchoring a phosphate group in subsite P1. The most potent inhibitor of all five, 5'-ADP (Ki = 1.2 microM), adopts a syn conformation (in contrast to 3',5'-ADP and 2',5'-ADP, which adopt an anti), and it is the beta- rather than the alpha-phosphate group that binds to P1. 3',5'-ADP binds with the 5'-phosphate group in P1 and the adenosine in the B2 pocket. Two different binding modes are observed in the two RNase A molecules of the asymmetric unit for 2',5'-ADP. This inhibitor binds with either the 3' or the 5' phosphate groups in subsite P1, and in each case, the adenosine binds in two different positions within the B2 subsite. The two uridilyl inhibitors bind similarly with the uridine moiety in the B1 subsite but the placement of a different phosphate group in P1 (2' versus 3') has significant implications on their potency against RNase A. Comparative structural analysis of the RNase A, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and human angiogenin (Ang) complexes with these and other phosphonucleotide inhibitors provides a wealth of information for structure-based design of inhibitors specific for each RNase. These inhibitors could be developed to therapeutic agents that could control the biological activities of EDN, ECP, and ANG, which play key roles in human pathologies.     

Crystal structure of eosinophil cationic protein at 2.4 A resolution

Eosinophil cationic protein (ECP) is located in the matrix of the eosinophil's large specific granule and has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA virus, and mammalian cells and tissues. It belongs to the bovine pancreatic ribonuclease A (RNase A) family and exhibits ribonucleolytic activity which is about 100-fold lower than that of a related eosinophil ribonuclease, the eosinophil-derived neurotoxin (EDN). The crystal structure of human ECP, determined at 2.4 A, is similar to that of RNase A and EDN. It reveals that residues Gln-14, His-15, Lys-38, Thr-42, and His-128 at the active site are conserved as in all other RNase A homologues. Nevertheless, evidence for considerable divergence of ECP is also implicit in the structure. Amino acid residues Arg-7, Trp-10, Asn-39, His-64, and His-82 appear to play a key part in the substrate specificity and low catalytic activity of ECP. The structure also shows how the cationic residues are distributed on the surface of the ECP molecule that may have implications for an understanding of the cytotoxicity of this enzyme.     

Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP).

The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single-stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70- to 200-fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double-stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN') and uridine and cytidine 2',3'-cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the 'non-secretory' liver-type enzymes rather than to the 'secretory' pancreatic-type RNases.     

Dynamics of the native and the ligand-bound structures of eosinophil cationic protein: network of hydrogen bonds at the catalytic site

Eosinophil Cationic Protein (ECP) is sequentially and structurally similar to ribonuclease A (RNase A). It belongs to the RNase A family of proteins and the RNA catalysis is essential to its biological function. In the present study, we have generated the dinucleotide-bound structures of ECP by docking the dinucleotides to a number of molecular dynamics (MD) generated ECP structures. The stability of the docked enzyme-ligand complexes was ascertained by extensive MD simulations. The modes of ligand binding are explored by essential dynamics studies. The role of water molecules in the stability of the complex and in the catalysis was investigated. The active site residues form a complex network of connections with the ligand and with a water molecule. The catalytic mechanism of the RNA cleavage is examined on the basis of the active site geometry obtained by the simulations.     

The exo- or endonucleolytic preference of bovine pancreatic ribonuclease A depends on its subsites structure and on the substrate size

The cleavage pattern of oligocytidylic acid substrates by bovine pancreatic ribonuclease A (RNase A) was studied by means of reversed-phase HPLC. Oligocytidylic acids, ranging from dinucleotides to heptanucleotides, were obtained by RNase A digestion of poly(C). They were identified by MALDI-TOF mass spectrometry; it was confirmed that all of them corresponded to the general structure (Cp)(n)C>p, in which C>p indicates a 2',3'-cyclic phosphate. This is a confirmation of the proposed mechanism for RNase A, wherein the so-called hydrolytic (or second) step is in fact a special case of the reverse of transphosphorylation (first step). The patterns of cleavage for the oligonucleotide substrates show that the native enzyme has no special preference for endonucleolytic or exonucleolytic cleavage, whereas a mutant of the enzyme (K7Q/R10Q-RNase A) lacking p(2) (a phosphate binding subsite adjacent, on the 3' side, to the main phosphate binding site p(1)) shows a clear exonucleolytic pattern; a mutant (K66Q-RNase A) lacking p(0) (a phosphate binding subsite adjacent, on the 5' side, to the main phosphate binding site p(1)) shows a more endonucleolytic pattern. This indicates the important role played by the subsites on the preference for the bond cleaved. Molecular modeling shows that, in the case of the p(2) mutant, the amide group of glutamine can form a hydrogen bond with the 2',3'-cyclic terminal phosphate, whereas the distance to a 3',5'-phosphodiester bond is too long to form such a hydrogen bond. This could explain the preference for exonucleolytic cleavage shown by the p(2) mutant.     

Human RNase 7: a new cationic ribonuclease of the RNase A superfamily

Here we report on the expression and function of RNase 7, one of the final RNase A superfamily ribonucleases identified in the human genome sequence. The human RNase 7 gene is expressed in various somatic tissues including the liver, kidney, skeletal muscle and heart. Recombinant RNase 7 is ribonucleolytically active against yeast tRNA, as expected from the presence of eight conserved cysteines and the catalytic histidine–lysine– histidine triad which are signature motifs of this superfamily. The protein is atypically cationic with an isoelectric point (pI) of 10.5. Expression of recombinant RNase 7 in Escherichia coli completely inhibits the growth of the host bacteria, similar to what has been observed for the cationic RNase, eosinophil cationic protein (ECP/RNase 3, pI 11.4). An in vitro assay demonstrates dose-dependent cytotoxicity of RNase 7 against bacteria E.coli, Pseudomonas aeruginosa and Staphylococcus aureus. While RNase 7 and ECP/RNase 3 are both cationic and share this particular aspect of functional similarity, their protein sequence identity is only 40%. Of particular interest, ECP/RNase 3’s cationicity is based on an (over)abundance of arginine residues, whereas RNase 7 includes an excess of lysine. This difference, in conjunction with the independent origins and different expression patterns, suggests that RNase 7 and ECP/RNase 3 may have been recruited to target different pathogens in vivo, if their physiological functions are indeed host defenses.     

Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7

Eosinophil cationic protein (ECP/RNase 3) and the skin derived ribonuclease 7 (RNase 7) are members of the RNase A superfamily. RNase 3 is mainly expressed in eosinophils whereas RNase 7 is primarily secreted by keratinocytes. Both proteins present a broad-spectrum antimicrobial activity and their bactericidal mechanism is dependent on their membrane destabilizing capacities. Using phospholipid vesicles as membrane models, we have characterized the protein membrane association process. Confocal microscopy experiments using giant unilamellar vesicles illustrate the morphological changes of the liposome population. By labelling both lipid bilayers and proteins we have monitored the kinetic of the process. The differential protein ability to release the liposome aqueous content was evaluated together with the micellation and aggregation processes. A distinct morphology of the protein/lipid aggregates was visualized by transmission electron microscopy and the proteins overall secondary structure in a lipid microenvironment was assessed by FTIR. Interestingly, for both RNases the membrane interaction events take place in a different behaviour and timing: RNase 3 triggers first the vesicle aggregation, while RNase 7 induces leakage well before the aggregation step. Their distinct mechanism of action at the membrane level may reflect different in vivo antipathogen functions.     

Molecular modeling of the three-dimensional structure of the bacterial RNase P holoenzyme

Bacterial ribonuclease P (RNase P), an enzyme involved in tRNA maturation, consists of a catalytic RNA subunit and a protein cofactor. Comparative phylogenetic analysis and molecular modeling have been employed to derive secondary and tertiary structure models of the RNA subunits from Escherichia coli (type A) and Bacillus subtilis (type B) RNase P. The tertiary structure of the protein subunit of B.subtilis and Staphylococcus aureus RNase P has recently been determined. However, an understanding of the structure of the RNase P holoenzyme (i.e. the ribonucleoprotein complex) is lacking. We have now used an EDTA-Fe-based footprinting approach to generate information about RNA-protein contact sites in E.coli RNase P. The footprinting data, together with results from other biochemical and biophysical studies, have furnished distance constraints, which in turn have enabled us to build three-dimensional models of both type A and B versions of the bacterial RNase P holoenzyme in the absence and presence of its precursor tRNA substrate. These models are consistent with results from previous studies and provide both structural and mechanistic insights into the functioning of this unique catalytic RNP complex.     

Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3-D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. A molecular mechanics-based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre-tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.     

Eosinophil cationic protein: overview of biological and genetic features

The eosinophil cationic protein (ECP) is a small polypeptide that originates from activated eosinophil granulocytes. A wide range of stimuli has been shown to induce the secretion of ECP. The gene that encodes the human ECP is located on chromosome 14, and the protein shares the overall three-dimensional structure and the RNase active-site residues with other proteins in the RNase A superfamily. Several single-nucleotide polymorphisms in the human ECP gene have been currently described. ECP has many biological functions, including an immunoregulatory function, the regulation of fibroblast activity, and the induction of mucus secretion in the airway. Additionally, the protein is a potent cytotoxic molecule and has the capacity to kill mammalian and nonmammalian cells. The purpose of this article was to review the known biological and genetic characteristics of ECP that contribute to the understanding of this protein's role in the development and progression of a wide variety of diseases.     

Solution structure of the cytotoxic RNase 4 from oocytes of bullfrog Rana catesbeiana

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana. RNase 4 belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Recombinant RC-RNase 4 (rRNase 4), which contains an additional Met residue and glutamine instead of pyroglutamate at the N terminus, was found to possess less catalytic and cytotoxic activities than RNase 4. Equilibrium thermal and guanidine-HCl denaturation CD measurements revealed that RNase 4 is more thermally and chemically stable than rRNase 4. However, CD and NMR data showed that there is no gross conformational change between native and recombinant RNase 4. The NMR solution structure of rRNase 4 was determined to comprise three alpha-helices and two sets of antiparallel beta-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(+/-0.14)A for the backbone atoms, and 1.42(+/-0.19)A for the heavy atoms in residues 3-105. A comparison of the 3D structure of rRNase 4 with the structurally and functionally related cytotoxic ribonuclease, onconase (ONC), showed that the two H-bonds in the N-terminal pyroglutamate of ONC were not present at the corresponding glutamine residue of rRNase 4. We suggest that the loss of these two H-bonds is one of the key factors responsible for the reductions of the conformational stability, catalytic and cytotoxic activities in rRNase 4. Furthermore, the differences of side-chain conformations of subsite residues among RNase A, ONC and rRNase 4 are related to their distinct catalytic activities and base preferences.     

Binding and cleavage of unstructured RNA by nuclear RNase P

Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is processing the 5' leader of pre-tRNAs. Compared to bacterial RNase P, which contains a single small protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more complex, containing nine proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to possess unique RNA binding capabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dependent manner, suggesting that multiple interactions are required for high affinity binding. Mixed-sequence RNAs from protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to poly(U) homopolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiple preferential cleavage sites that do not correspond to identifiable consensus structures or sequences. In addition, pre-tRNA(Tyr), poly(U)(50) RNA, and mixed-sequence RNA cross-link with purified RNase P in the RNA subunit Rpr1 near the active site in "Conserved Region I," although the exact positions vary. Additional contacts between poly(U)(50) and the RNase P proteins Rpr2p and Pop4p were identified. We conclude that unstructured RNAs interact with multiple protein and RNA contacts near the RNase P RNA active site, but that cleavage depends on the nature of interaction with the active site.     

Role of unique basic residues in cytotoxic, antibacterial and antiparasitic activities of human eosinophil cationic protein

Eosinophil granule proteins, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin are members of the RNase A superfamily, which play a crucial role in host defense against various pathogens as they are endowed with several biological activities. Some of the biological activities possessed by ECP have been attributed to its strong basic character. In the current study, we have investigated the role of five unique basic residues, Arg22, Arg34, Arg61, Arg77 and His64 of ECP in its catalytic, cytotoxic, antibacterial and antiparasitic activities. These residues were changed to alanine to generate single and double mutants. None of the selected residues was found to be involved in the RNase activity of ECP. The substitution of all five residues individually was detrimental for the cytotoxic, antibacterial and antiparasitic activities of ECP; however, mutation of Arg22 and Arg34 resulted in the most significant effects. The double mutants also had reduced biological activities. All ECP mutants that had significantly reduced toxicity also had reduced membrane destabilization activity. Our study demonstrates that Arg22, Arg34, Arg61, Arg77 and His64 of ECP are crucial for its membrane destabilization activity, which appears to be the underlying mechanism of its cytotoxic, antibacterial and antiparasitic activities.     

Structural properties of trimers and tetramers of ribonuclease A

Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) x poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) x poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.     

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.     

Structural study of MCPIP1 N-terminal conserved domain reveals a PIN-like RNase

MCP-1-induced protein 1 (MCPIP1) plays an important role in the downregulation of the LPS-induced immune response by acting as an RNase targeting IL-6 and IL-12b mRNAs. A conserved domain located in the N-terminal part of MCPIP1 is thought to be responsible for its RNase activity, but its catalytic mechanism is not well understood due to the lack of an atomic resolution structure. We determined the 3D crystal structure of this MCPIP1 N-terminal conserved RNase domain at a resolution of 2.0 Å. The overall structure of MCPIP1 N-terminal conserved domain shares high structural homology with PilT N-terminal domain. We show that the RNase catalytic center is composed of several acidic residues, verifying their importance by site-specific mutagenesis. A positively charged arm close to the catalytic center may act as an RNA substrate-binding site, since exchange of critical positively charged residues on this arm with alanine partially abolish the RNase activity of MCPIP1 in vivo. Our structure of the MCPIP1 N-terminal conserved domain reveals the details of the catalytic center and provides a greater understanding of the RNA degradation mechanism.