Dissecting Structural and Electrostatic Interactions of Charged Groups in α-Sarcin. An NMR Study of Some Mutants Involving the Catalytic Residues

• Published in: Biochemistry (Easton) Vol. 42; no. 45; pp. 13122 - 13133
• Main Authors: García-Mayoral, Ma Flor, Pérez-Cañadillas, José Manuel, Santoro, Jorge, Ibarra-Molero, Beatriz, Sanchez-Ruiz, José Manuel, Lacadena, Javier, Martínez del Pozo, Álvaro, Gavilanes, José G, Rico, Manuel, Bruix, Marta
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.11.2003
• Subjects: Amino Acid Substitution - genetics; Aspergillus - enzymology; Aspergillus - genetics; Binding Sites - genetics; Catalytic Domain - genetics; Endoribonucleases - chemistry; Endoribonucleases - genetics; Fungal Proteins - chemistry; Fungal Proteins - genetics; Glutamic Acid - genetics; Glutamine - genetics; Histidine - genetics; Hydrogen-Ion Concentration; Models, Chemical; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular - methods; Protons; Static Electricity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0349773

The cytotoxic ribonuclease α-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 α-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 α-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 α-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 α-sarcin, which can account for its reduced ribonucleolytic activity and its high specificity.