Leucine 145 of the ribotoxin α‐sarcin plays a key role for determining the specificity of the ribosome‐inactivating activity of the protein

Abstract

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as ␣-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of ␣-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type ␣-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of ␣-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of ␣-sarcin on ribosomes.

The1H,13C,15N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy

“…In a-sarcin, mutating this Leu to Phe has been shown to affect the pKa of the nearby His and to affect the enzyme kinetics. 34 The interaction of the phenolic O of Tyr33 and the charged N f of the catalytic Lys38 observed in most of the NMR solution structures could stabilize the charge on the latter and modulate the enzyme's activity. This interaction is neither observed in crystal structures of ECP with a bound substrate mimics (1H1H) 24 nor without (1QMT nor 1DYT) nor in the solution structures of the ECP homologs human RNase 1 (2k11) 35 nor bovine pancreatic RNase A (2AAS).…”

The Therapeutic Potential of Fungal Ribotoxins

“…These mutational studies have revealed the involvement of several residues, which are conserved among the different microbial extracellular RNases, in catalysis. Thus, it is well known that His137 and Glu96 are the only residues that are essential for the cleavage reaction performed by α-sarcin [35,45,[70][71][72][73][74][75], whereas His-50, Tyr-48, Arg-121, and Leu-145 mostly contribute to the stabilization of the transition state [45][46][47][48], as stated above. Most of these residues are located in the central β-sheet and their side-chains point towards the concave face of the protein structure where the substrate is supposed to dock [29].…”

“…Mutational studies have also revealed that three other active site residues, Tyr48, Arg121, and Leu145, although not essential, appear to be determinants of the ribotoxin activity of α-sarcin [46][47][48]. Studies on the crystal structures of complexes of restrictocin with inhibitors led to the proposal that these ribotoxins may use base flipping to enable cleavage at the correct site of the SRL substrates [49].…”

Conserved asparagine residue 54 of α-sarcin plays a role in protein stability and enzyme activity

“…This inactive character of the N54Y variant could be explained by considering the aromatic nature of the amino acid involved in this mutation and its proximity to the active site of the protein (Figure 1), since, as stated above, this mutant also displayed spectral characteristics corresponding to the native natural protein. Remarkably, the Asn 54 NH chemical shift also represented one of the largest differences observed when comparing wild-type a-sarcin with a mutant where the conserved Leu 145 in these ribotoxins was mutated to Phe, the corresponding residue found in the other less specific but more active microbial ribonucleases such as RNases T1 and U2 (Masip et al, 2003). In line with this idea, Asn 54 is disturbed upon mutation of the asarcin catalytic residues (His 50, Glu 96, Arg 121, and His 137) (Lacadena et al, 1999;Masip et al, 2001).…”

“…17 kDa) and basic proteins, displaying very similar amino acid sequences (approx. Site-directed mutagenesis studies have been carried out in order to identify most of the residues responsible for its ribonucleolytic activity (Lacadena et al, 1999;Martínez-Ruiz et al, 2001;García-Ortega et al, 2001, 2002Masip et al, 2001Masip et al, , 2003, as well as some of those involved in its interaction with cell membranes , 2002Masip et al, 2001). These ribotoxins are endocytosed by their target cells upon interaction with the membranes, leading to cell death via apoptosis that is directly related to their catalytic activity on ribosomes .…”