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The cytotoxic ribonuclease alpha-sarcin is the best characterized member of the ribotoxin family. Ribotoxins share a common structural core, catalytic residues, and active site topology with members of the broader family of nontoxic microbial extracellular RNases. They are, however, much more specific in their biological action. To shed light on the highly specific alpha-sarcin activity, we have evaluated the structural and electrostatic interactions of its charged groups, by combining the structural and pK(a) characterization by NMR of several variants with theoretical calculations based on the Tanford-Kirkwood and Poisson-Boltzmann models. The NMR data reveal that the global conformation of wild-type alpha-sarcin is preserved in the H50Q, E96Q, H137Q, and H50/137Q variants, and that His137 is involved in an H-bond that is crucial in maintaining the active site structure and in reinforcing the stability of the enzyme. The loss of this H-bond in the H137Q and H50/137Q variants modifies the local structure of the active site. The pK(a) values of active site groups H50, E96, and H137 in the four variants have been determined by two-dimensional NMR. The catalytic dyad of E96 and H137 is not sensitive to charge replacements, since their pK(a) values vary less than +/-0.3 pH unit with respect to those of the wild type. On the contrary, the pK(a) of His50 undergoes drastic changes when compared to its value in the intact protein. These amount to an increase of 0.5 pH unit or a decrease of 1.1 pH units depending on whether a positive or negative charge is substituted at the active site. The main determinants of the pK(a) values of most of the charged groups in alpha-sarcin have been established by considering the NMR results in conjunction with those derived from theoretical pK(a) calculations. With regard to the active site residues, the H50 pK(a) is chiefly influenced by electrostatic interactions with E96 and H137, whereas the effect of the low dielectric constant and the interaction with R121 appear to be the main determinants of the altered pK(a) value of E96 and H137. Charge-charge interactions and an increased level of burial perturb the pK(a) values of the active site residues of alpha-sarcin, which can account for its reduced ribonucleolytic activity and its high specificity.

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-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 alpha-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 alpha-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 alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.

The cytotoxic ribonuclease alpha-sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a strictly conserved rRNA loop. In order to gain insights on the molecular basis of its highly specific activity, we have previously determined its solution structure and studied its electrostatics properties. Here, we complement those studies by analysing the backbone dynamics of alpha-sarcin through measurement of longitudinal relaxation rates R1, off resonance rotating frame relaxation rates R1 rho, and the 15N[1H] NOE of the backbone amide 15N nuclei at two different magnetic field strengths (11.7 and 17.6 T). The two sets of relaxation parameters have been analysed in terms of the reduced spectral density mapping formalism, as well as by the model-free approach. alpha-Sarcin behaves as an axial symmetric rotor of the prolate type (D(axially)/D(radially)=1.16 +/- 0.02) which tumbles with a correlation time tau(m) of 7.54 +/- 0.02 ns. The rotational diffusion properties have been also independently evaluated by hydrodynamic calculations and are in good agreement with the experimental results. The analysis of the internal dynamics reveals that alpha-sarcin is composed of a rigid hydrophobic core and some exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the mu s to ms time scale are less abundant and in some cases can be assigned to specific motional processes. All dynamic data are discussed in relation to the role of some particular residues of alpha-sarcin in the process of recognition of its ribosomal target.