PDBsum entry 1r4y

Hydrolase

PDB id: 1r4y

Contents

Protein chain

136 a.a. *

* Residue conservation analysis

PDB id:

1r4y

Name:

Hydrolase

Title:

Solution structure of the deletion mutant delta(7-22) of the cytotoxic ribonuclease alpha-sarcin

Structure:

Ribonuclease alpha-sarcin. Chain: a. Synonym: rrna endonuclease. Engineered: yes. Mutation: yes

Source:

Aspergillus giganteus. Organism_taxid: 5060. Gene: sar. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

NMR struc:

25 models

Authors:

M.F.Garcia-Mayoral,L.Garcia-Ortega,M.P.Lillo,J.Santoro,A.Martinez Del Pozo,J.G.Gavilanes,M.Rico,M.Bruix

Key ref:

M.F.García-Mayoral et al. (2004). NMR structure of the noncytotoxic alpha-sarcin mutant Delta(7-22): the importance of the native conformation of peripheral loops for activity. Protein Sci, 13, 1000-1011. PubMed id: 15044731 DOI: 10.1110/ps.03532204

Date:

09-Oct-03 Release date: 06-Apr-04

Protein chain

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

Seq: 177 a.a.

Struc: 136 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

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: 10.1110/ps.03532204

Protein Sci 13:1000-1011 (2004)

PubMed id: 15044731

NMR structure of the noncytotoxic alpha-sarcin mutant Delta(7-22): the importance of the native conformation of peripheral loops for activity.

M.F.García-Mayoral, L.García-Ortega, M.P.Lillo, J.Santoro, A.Martínez del Pozo, J.G.Gavilanes, M.Rico, M.Bruix.

ABSTRACT

The deletion mutant Delta(7-22) of alpha-sarcin, unlike its wild-type protein counterpart, lacks the specific ability to degrade rRNA in intact ribosomes and exhibits an increased unspecific ribonuclease activity and decreased interaction with lipid vesicles. In trying to shed light on these differences, we report here on the three-dimensional structure of the Delta(7-22) alpha-sarcin mutant using NMR methods. We also evaluated its dynamic properties on the basis of theoretical models and measured its correlation time (6.2 nsec) by time-resolved fluorescence anisotropy. The global fold characteristic of ribotoxins is preserved in the mutant. The most significant differences with respect to the alpha-sarcin structure are concentrated in (1) loop 2, (2) loop 3, which adopts a new orientation, and (3) loop 5, which shows multiple conformations and an altered dynamics. The interactions between loop 5 and the N-terminal hairpin are lost in the mutant, producing increased solvent accessibility of the active-site residues. The degree of solvent exposure of the catalytic His 137 is similar to that shown by His 92 in RNase T1. Additionally, the calculated order parameters of residues belonging to loop 5 in the mutant correspond to an internal dynamic behavior more similar to RNase T1 than alpha-sarcin. On the other hand, changes in the relative orientation of loop 3 move the lysine-rich region 111-114, crucial for substrate recognition, away from the active site. All of the structural and dynamic data presented here reveal that the mutant is a hybrid of ribotoxins and noncytotoxic ribonucleases, consistent with its biological properties.

Selected figure(s)

Figure 1.
Figure 1. Representation of the solution structure of the (7-22) -sarcin mutant. (Left) Superposition of the 25 solution structures. (Right) Ribbon diagram of the (7-22) -sarcin mutant structure. Figs. 1-3 Go-were produced with the program MOLMOL (Koradi et al. 1996).

Figure 2.
Figure 2. Structural comparison of the wild-type -sarcin and the (7-22) -sarcin deletion mutant. (Top) Stereoscopic view of the superposition of the two ensembles of their 3D NMR structures; the 20 conformers of the wild type -sarcin are in blue, and those of the (7-22) mutant, in red. (Bottom) Detail of the superposition of the backbone atoms corresponding to some regions of the wild-type -sarcin (blue) and the (7-22) -sarcin deletion mutant (red) structures. (A) Relative orientation of the N-terminal -hairpin and loop 5, (B) segment of loop 2, (C) residues 51-55, and (D) lysine-rich fragment of loop 3.

The above figures are reprinted by permission from the Protein Society: Protein Sci (2004, 13, 1000-1011) copyright 2004.

Figures were selected by the author.