The Action Mode of the Ribosome-Inactivating Protein α-Sarcin

Abstract

Based on the tertiary structure of the ribosome-inactivating protein α-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 β-strand and the last carboxyl β-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids. Instead, a four-strand pleated β-sheet is indispensable for catalyzing both substrates, suggesting that α-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center. The integrity of an amino β-hairpin and that of the loop L3 in α-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo. However, a mutant protein without the β-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids. The functional involvement of the β-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

“…Structural basis of the different ribonucleolytic activity of the ␣-sarcin ⌬(7-22) mutant: Specific and unspecific substrates Different docking studies performed on models of ␣-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 ␣-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 ␣-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 ⌬(7-22) was unable to specifically cleave the phosphodiester bond in the ribosome; consequently, a third segment of ␣-sarcin, the N-terminal ␤-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 ␣-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), ␣-sarcin (Hwu et al 2000), and mitogillin 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 ␤-hairpin in ␣-sarcin produces an enzyme with interesting biological properties (García-Ortega et al 2002): namely, the ⌬(7-22) mutant of ␣-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.…”

An antifungal peptide from passion fruit (Passiflora edulis) seeds with similarities to 2S albumin proteins

“…They are usually found in cereal grains but are also present in onions, spinach and A. thaliana [16] and exert their growth inhibition effects probably by acting on the traffic of phospholipids across membranes [17]. The fourth group encompasses the ribosome-inactivating proteins (RIP) that cause irreversible damages of ribosomes by depuration of rRNA [18]. RIPs are expressed in response to various pathogens, as virus and fungi, mechanical wounding, jasmonic and abscisic acid treatments and abiotic stresses [19][20][21].…”

pH-Dependent Conformational Stability of the Ribotoxin α-Sarcin and Four Active Site Charge Substitution Variants

“…R-Sarcin is stable over a wide range of pH values (3.5-8.5), and its high-resolution NMR structure (27,28), dynamic behavior (29), and individual pK a N values of residues titrating in this pH range have been determined (30,31). Compared to smaller noncytotoxic RNases, R-sarcin's structure is characterized by the presence of longer loops for which important roles related to cytotoxicity have been suggested in (i) modulating the ribonuclease activity (N-terminal β-hairpin) (26,32), (ii) ligand recognition (lysine-rich region of loop 3, N-terminal β-hairpin) (27,28,33,34), and (iii) interaction with biological membranes (loop 2, N-terminal β-hairpin) facilitating the cell entrance by endocytosis (26,28,35). The unique orientation of the different loops of the molecule is stabilized by complex networks of interactions different in nature (27), and it is important to determine their stabilities to better understand the biological activities of R-sarcin.…”