“Salmo salar ribonuclease: structural and functional evidences of an auto inhibition mechanism”

• Main Authors: I. Russo Krauss, A. Merlino, E. Pizzo, A. Bosso, FAELLA, RITA, C. Carluccio, M. Trapani, R. Troisi, L. Mazzarella, F. Sica
• Other Authors: Associazione Italiana Cristallografia, Russo Krauss, I., Merlino, A., Pizzo, E., Bosso, A., Faella, Rita, Carluccio, C., Trapani, M., Troisi, R., Mazzarella, L., Sica, F.
• Format: Conference Object
• Language: English
• Published: 2019
• Subjects: Salmo salar
• Online Access: http://hdl.handle.net/11588/769859

The pancreatic-type ribonucleases (ptRNases) constitute one of the most investigated family of homologous proteins [1]. PtRNases have been found in various organs and tissues of mammals, amphibians, reptiles, birds [2] and, more recently, fish. In particular, seven new RNases were identified in two teleosts: five from Zebrafish (ZF RNases) and two from Salmo Salar (Ss-RNase-1 and Ss-RNase-2) [3-5]. We have focused our attention on the latter enzyme, which is the most active fish RNase, although its activity is much lower than that of the other members of the ptRNase family. The crystallographic structure of Ss-RNase-2 (SS2) shows the typical V-shape structure of pancreatic-like ribonucleases, with three helices and six β-strands connected by loops and turns. The electron density in the active site region is discontinuous. In particular, the position of His 113, one of the residues of the catalytic triad that is usually very well defined, cannot be unambiguously identified. Furthermore, the segment Val117-Ile121, which is well-structured and anchored to the protein body, partially obstructs the active site. These structural features suggest that SS2 is in a sort of inhibited state. It is reasonable to hypothesize that, in consequence of an interaction with a specific ligand, the C-terminal segment moves to free the active site. To define the activation mechanism of SS2, we have designed and characterized two deletion mutants: SS2-des117-121, in which the chain segment that in the wild-type protein obstructs the active site has been removed, and SS2-des119-126, in which the elimination of the last eight residues of the chain might allow the sliding of the obstructing segment out of the active site. Interestingly, the crystallographic structure of SS2-des117-121 shows a well-defined active site, almost free of obstructions. Furthermore, the enzymatic activity assays show an improvement of the specific activity of SS2-des117-121 with respect to the wild-type protein. On the bases of these findings, we have proposed ...