The action mode of the ribosome-inactivating protein α-sarcin

Abstract

Based on the tertiary structure of the ribosome-inactivating protein alpha-sarcin, domains that are responsible for hydrolyzing ribosomes and naked RNA have been dissected. In this study, we found that the head-to-tail interaction between the first amino beta-strand and the last carboxyl beta-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids. Instead, a four-strand pleated beta-sheet is indispensable for catalyzing both substrates, suggesting that alpha-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center. The integrity of an amino beta-hairpin and that of the loop L3 in alpha-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo. However, a mutant protein without the beta-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids. The functional involvement of the beta-hairpin and the loop L3 in the sarcin stem/loop RNA of ribosomes is demonstrated by a docking model, suggesting that the two structures are in essence naturally designed to distinguish ribosome-inactivating proteins from RNase T1 to inactivate ribosomes.

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

“…Different docking studies performed on models of α‐sarcin ‐RNA complexes (Szewczak et al 1993; Szewczak and Moore 1995; Pérez‐Cañadillas et al 2000; Hwu et al 2000), restrictocin‐RNA complexes (Yang and Moffat 1996; Nayak et al 2001; Yang et al 2001), and kinetic studies on α‐sarcin (Takeda et al 1997) have suggested the participation of two distant regions in its specific interaction with the ribosome. First, the lysine‐rich region of α‐sarcin's loop 3 participates in the substrate recognition by interacting with the negatively charged phosphodiester bond around the bulged G4319.…”

“…Second, it has also been suggested that residues from Trp 51 to Gly 55 in loop 2 and residues from loop 5 interact with the ribosome at the conserved GAGA tetraloop (4323–4326) which is about 15 Å away from the bulged guanine (4319). Ribonucleolytic assays against a reticulocyte lysate showed that Δ(7‐22) was unable to specifically cleave the phosphodiester bond in the ribosome; consequently, a third segment of α‐sarcin, the N‐terminal β‐hairpin, was suggested to directly interact with some region of the ribosome (Hwu et al 2000; García‐Ortega et al 2002). Ribonucleolytic assays against other substrates lacking the characteristic structural elements of native ribosomes, such as naked rRNA, SRL, poly(A), and small dinucleotides show that the mutant is able to more efficiently hydrolyze these ligands (García‐Ortega et al 2002).…”

“…Inserting the targeting motifs of ribotoxins into other enzyme structures would allow the generation of specific targeted enzymes. In this sense, some groups have formed chimeric constructions, such as the insertion into the RNase T1 sequence of an α‐sarcin segment known to be essential in the ribosome recognition process (Chen and Lin 2002); in addition, several deletion mutants of restrictocin (Nayak et al 2000), α‐sarcin (Hwu et al 2000), and mitogillin (Kao and Davies 1999) were prepared to identify regions in fungal ribotoxins contributing to ribosome targeting and modulation of the catalytic activity. It was recently shown that the deletion of part of the long β‐hairpin in α‐sarcin produces an enzyme with interesting biological properties (García‐Ortega et al 2002): namely, the Δ(7‐22) mutant of α‐sarcin lacks the specific ability of the wild type to degrade rRNA in intact ribosomes and exhibits increased nonspecific ribonuclease activity and decreased interaction with lipid vesicles.…”

Antifungal Proteins

“…RIPs are RNA N-glycosidases that depurinate rRNA, resulting in the arrest of protein synthesis due to ribosome damage (7,39,65,94,144,167). Plant RIPs inhibit mammalian, bacterial, fungal, and plant protein syntheses in vitro and in vivo (67).…”

Biomedical Vignette

“…Hwu et al [10] found that ␣-sarcin shares a similar catalytic center as ribonuclease T1 because a four-strand pleated ␤-sheet is indispensable for catalyzing the hydrolysis of ribosomes or ribonucleic acids. The integrity of an amino ␤-hairpin and that of loop L3 in ␣-sarcin are also crucial for recognizing and hydrolyzing ribosomes.…”