PDBsum entry 1de3

Hydrolase

PDB id: 1de3

Contents

Protein chain

150 a.a. *

* Residue conservation analysis

PDB id:

1de3

Name:

Hydrolase

Title:

Solution structure of the cytotoxic ribonuclease alpha-sarcin

Structure:

Ribonuclease alpha-sarcin. Chain: a. Engineered: yes

Source:

Aspergillus giganteus. Organism_taxid: 5060. Strain: mdh 18894. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

20 models

Authors:

J.M.Perez-Canadillas,R.Campos-Olivas,J.Santoro,J.Lacadena,A.Martinez Del Pozo,J.G.Gavilanes,M.Rico,M.Bruix

Key ref:

J.M.Pérez-Cañadillas et al. (2000). The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity. J Mol Biol, 299, 1061-1073. PubMed id: 10843858 DOI: 10.1006/jmbi.2000.3813

Date:

12-Nov-99 Release date: 21-Jun-00

Protein chain

P00655  (RNAS_ASPGI) -  Ribonuclease alpha-sarcin from Aspergillus giganteus

Seq: 177 a.a.

Struc: 150 a.a.

Enzyme reactions

• Enzyme class: E.C.4.6.1.23 - ribotoxin.
• Reaction: a 28S rRNA containing guanosine-adenosine pair + H2O = an [RNA fragment]- 3'-adenosine-3'-phosphate + a 5'-a hydroxy-guanosine-3'-[RNA fragment]

DOI no: 10.1006/jmbi.2000.3813

J Mol Biol 299:1061-1073 (2000)

PubMed id: 10843858

The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity.

J.M.Pérez-Cañadillas, J.Santoro, R.Campos-Olivas, J.Lacadena, A.Martínez del Pozo, J.G.Gavilanes, M.Rico, M.Bruix.

ABSTRACT

alpha-Sarcin selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA, that inactivates the ribosome. The elucidation of the three-dimensional solution structure of this 150 residue enzyme is a crucial step towards understanding alpha-sarcin's conformational stability, ribonucleolytic activity, and its exceptionally high level of specificity. Here, the solution structure has been determined on the basis of 2658 conformationally relevant distances restraints (including stereoespecific assignments) and 119 torsional angular restraints, by nuclear magnetic resonance spectroscopy methods. A total of 60 converged structures have been computed using the program DYANA. The 47 best DYANA structures, following restrained energy minimization by GROMOS, represent the solution structure of alpha-sarcin. The resulting average pairwise root-mean-square-deviation is 0.86 A for backbone atoms and 1.47 A for all heavy atoms. When the more variable regions are excluded from the analysis, the pairwise root-mean-square deviation drops to 0.50 A and 1.00 A, for backbone and heavy atoms, respectively. The alpha-sarcin structure is similar to that reported for restrictocin, although some differences are clearly evident, especially in the loop regions. The average rmsd between the structurally aligned backbones of the 47 final alpha-sarcin structures and the crystal structure of restrictocin is 1.46 A. On the basis of a docking model constructed with alpha-sarcin solution structure and the crystal structure of a 29-nt RNA containing the sarcin/ricin domain, the regions in the protein that could interact specifically with the substrate have been identified. The structural elements that account for the specificity of RNA recognition are located in two separate regions of the protein. One is composed by residues 51 to 55 and loop 5, and the other region, located more than 11 A away in the structure, is the positively charged segment formed by residues 110 to 114.

Selected figure(s)

Figure 5.
Figure 5. Structural comp arison of a-sarcin and restrictocin. (a) Superposition of the lowest energy solution structure of a-sarcin (black, residues 1 to 150) and the crystal structure of restrictocin (RCSB PDB entry 1AQZ, chain A) (gray, residues 1 to 10, 17 to 149). (b) Detail of the superposition of the N-terminal hairpin; the discontinuity in the crystal structure of restrictocin is due to the lack of electron density for residues 11 to 16. (c) Superposition of loop 2 where the largest differences are found.

Figure 6.
Figure 6. Model of the interaction between the 28S rat SRD (Sarcin Ricin Domain) and a-sarcin. The protein is represented by the electrostatic surface and the RNA fragment with a stick model. The phosphodiester chain is colored in red, the recognition guanine G4310 in green and the bases adjacent to the scissile bond, A4324 and G4325, in yellow. Recognition sites and the N-terminal hairpin in the protein structure are labeled. Figure generated with GRASP.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 299, 1061-1073) copyright 2000.

Figures were selected by an automated process.