NMR structure of the noncytotoxic α‐sarcin mutant Δ(7‐22): The importance of the native conformation of peripheral loops for activity

Abstract

The deletion mutant ⌬(7-22) of ␣-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 ⌬(7-22) ␣-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 ␣-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 ␣-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.Keywords: ␣-sarcin mutant; ribotoxins; NMR solution structure; ribonucleolytic activity; protein-RNA interaction ␣-Sarcin is the most well-characterized member of the fungal ribotoxin family. This extracellular 150-residue enzyme, secreted by the mold Aspergillus giganteus (Olson and Goerner 1965), has been extensively studied from a structural , dynamic (Pérez-Cañadillas et al. 2002), and electrostatic point of view (Pérez-Cañadillas et al. 1998) by NMR methods. ␣-Sarcin associates with the cell membrane by electrostatic and hydrophobic interactions and is drawn into the cell via endocytosis without the help of known membrane receptors (Olmo et al. 2001). ␣-Sarcin is a very specific ribonuclease that recognizes the ribosome and cleaves a single phosphodiester bond on the 3Ј side of G4325 in the 28S rRNA. This bond is located in a highly conserved purine-rich 14-base sequence, known as the SRL (Endo and Wool 1982;Wool 1984;Glück and Wool 1996), targeted by ribosome-inactivating proteins. A bulged guaReprint requests to: Marta Bruix, Departamento de Espectroscopía y Estructura Molecular, Instituto de Química Física "Rocasolano," Serrano 119, CSIC, 28006 Madrid, Spain; e-mail: mbruix@iqfr.csic.es; fax: 34 (91) 561-9400.Abbreviations: ⌬(7-22), the ␣-sarcin deletion mutant in which residues 7-22 have been removed and replaced by a pair of glycine residues; TRFA, time-resolved fluorescence anisotropy; SRL, sa...

Role of the basic character of α-sarcin’s NH2-terminal β-hairpin in ribosome recognition and phospholipid interaction

“…This involvement might be in terms of a direct interaction with the polymer or most probably due to a modification of the active site accessibility, as has been also suggested before [15,51]. In fact, these solvent accessibility changes were detected before upon deletion of the β-hairpin [22].…”

“…An α-sarcin mutant involving the deletion of this protuberance, α-sarcin Δ (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), retained the same conformation as the wildtype protein, as ascertained from its three-dimensional structure in solution [22].…”

Lactococcus lactis as a vehicle for the heterologous expression of fungal ribotoxin variants with reduced IgE-binding affinity

“…One of them was the wild-type major A. fumigatus allergen Asp f 1 (García-Ortega et al, 2005). Two other ones were the corresponding Δ(7-22) deleted variants of Asp f 1 and α-sarcin, two proteins with highly reduced IgE-reactivity and citotoxicity (García-Ortega et al, 2002García-Mayoral et al, 2004). The fourth one (H137Q) was a mutant version of α-sarcin where only the catalytically essential His-137 has been mutated, being substituted by Gln (Lacadena et al, 1995).…”

“…The second of them (residues 7-22) juts out as a solvent-exposed protuberance and is one of the protein regions with highest conformational flexibility (Pérez-Cañadillas et al, 2000, 2002. Deletion of this β-sheet results in a Δ(7-22) mutant that shows no significant conformational differences except for the deleted region (García-Mayoral et al, 2004) but has lost its ability to specifically recognize the ribosome and is much less cytotoxic (García-Ortega et al, 2002).…”

“…Five of these differences are precisely located at the NH 2 -terminal β-hairpin. Structural and immunogenic studies of Asp f 1, α-sarcin, and two variants where this β-hairpin had been deleted [Asp f 1 Δ(7-22) and α-sarcin -Ortega et al, 2005) showed that the deleted portion is involved in at least one allergenic epitope (García-Mayoral et al, 2004;García-Ortega et al, 2005). In spite of their decreased IgE reactivity, the prevalence of the two deleted proteins among the sera of patients remained essentially unaffected while they still retained most of the IgG epitopes (García-Ortega et al, 2005).…”