Three-dimensional structure of human RNase 1ΔN7 at 1.9 Å resolution

Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the amino-terminal sequence resulted in RNase 1ΔN7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 A resolution. The molecule displays the α + β folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1ΔN7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1ΔN7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.

[1]  G. Gilliland.  Crystallographic Studies of Ribonuclease Complexes , 1997 .

[2]  M. Seno,et al.  The three-dimensional structure of human RNase 4, unliganded and complexed with d(Up), reveals the basis for its uridine selectivity. , 1999, Journal of molecular biology.

[3]  D. Newton,et al.  X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin at 1.83 A resolution. , 1996, Journal of molecular biology.

[4]  D. Newton,et al.  Natural and engineered cytotoxic ribonucleases: therapeutic potential. , 1999, Experimental cell research.

[5]  M. Seno,et al.  Recombinant human pancreatic ribonuclease produced in E. coli: importance of the amino-terminal sequence. , 1995, Biochemical and biophysical research communications.

[6]  K. Dyer,et al.  Molecular cloning and characterization of a novel human ribonuclease (RNase k6): increasing diversity in the enlarging ribonuclease gene family. , 1996, Nucleic acids research.

[7]  E. Boix,et al.  Crystal structure of eosinophil cationic protein at 2.4 A resolution. , 1999, Biochemistry.

[8]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[9]  J F Riordan,et al.  Amino acid sequence of human tumor derived angiogenin. , 1985, Biochemistry.

[10]  M. Seno,et al.  Tissue-specific expression of pancreatic-type RNases and RNase inhibitor in humans. , 1997, DNA and cell biology.

[11]  M. Ribó,et al.  A versatile negative-staining ribonuclease zymogram. , 1994, Analytical biochemistry.

[12]  J. Fontecilla-Camps,et al.  Crystal structure of ribonuclease A.d(ApTpApApG) complex. Direct evidence for extended substrate recognition. , 1994, The Journal of biological chemistry.

[13]  M. V. Nogués,et al.  Structure and function of ribonuclease A binding subsites. , 1991, Essays in biochemistry.

[14]  J. Pous,et al.  Three-dimensional crystal structure of human eosinophil cationic protein (RNase 3) at 1.75 A resolution. , 2000, Journal of molecular biology.

[15]  M. Seno,et al.  Inhibition of cell growth by a fused protein of human ribonuclease 1 and human basic fibroblast growth factor. , 1999, Protein engineering.

[16]  A. Kobata,et al.  Fractionation by lectin affinity chromatography indicates that the glycosylation of most ribonucleases in human viscera and body fluids is organ specific. , 1986, Archives of biochemistry and biophysics.

[17]  G. Schieven,et al.  Differences in glycosylation pattern of human secretory ribonucleases. , 1988, The Biochemical journal.

[18]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[19]  G. D'alessio,et al.  Ribonucleases : structures and functions , 1997 .

[20]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[21]  M. Frazier,et al.  Ribonucleases expressed by human pancreatic adenocarcinoma cell lines. , 2000, European journal of biochemistry.

[22]  J. Batra,et al.  Human pancreatic ribonuclease--deletion of the carboxyl-terminal EDST extension enhances ribonuclease activity and thermostability. , 1997, European journal of biochemistry.

[23]  A. Wlodawer,et al.  Structure of phosphate-free ribonuclease A refined at 1.26 A. , 1988, Biochemistry.

[24]  J. Deisenhofer,et al.  A structural basis of the interactions between leucine-rich repeats and protein ligands , 1995, Nature.

[25]  W. Ardelt,et al.  Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity. , 1996, Journal of molecular biology.

[26]  K. Acharya,et al.  Molecular recognition of human angiogenin by placental ribonuclease inhibitor—an X‐ray crystallographic study at 2.0 Å resolution , 1997, The EMBO journal.

[27]  K. Dyer,et al.  Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity. , 1998, Nucleic acids research.

[28]  M. Lombardi,et al.  The differential pattern of tissue-specific expression of ruminant pancreatic type ribonucleases may help to understand the evolutionary history of their genes. , 1999, Gene.