Structure and functional studies of the ribonuclease binase Glu43Ala/Phe81Ala mutant

Abstract

Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

“…We have shown that binase exists on SDS-PAGE in both its monomeric and dimeric forms ( Fig. 1A ), which fits with previous data showing that binase forms dimers in a crystal and probably in solutions with high protein concentrations [8] , [15] . The question is addressed to the ratio of binase monomers and dimers in solution in the absence of denaturing agents.…”

“…2B , Fig. 2C ) as suggested previously [8] , [15] . Moreover, binase is quite stable: it does not dissociate completely even in the presence of 2% SDS or 8 M urea ( Fig.…”

“…Bacilli produce at least 2 types of RNases, the “high-molecular weight” RNases [32] and small guanyl-specific RNases, among which binase, the thermostable enzyme from B. pumilus (originally named B. intermedius ), and barnase from B. amyloliquefaciens are the best characterised [4] , [11] , [25] , [27] . Despite classical reports describing the monomeric form of binase [11] , [27] , many studies have demonstrated the dimerisation of binase in crystals [8] , [13] , [14] , [33] . Actually, the ability to form dimers is not surprising for RNases and was shown to endow them with additional biological properties such as allostery, anti-tumour and immunosuppressive activity, improved stability and control over the accessibility and specificity of active sites [17] , [34] , [35] .…”

“…The three-dimensional structure of binase was refined by Pavlovsky et al [13] and Polyakow et al [14] . In the crystal structures of binase, the characteristic dimer is present with the active site of one subunit being blocked owing to interactions within the dimer [8] . Since the dimers were found in crystals grown under four different conditions, it can be suggested that the enzyme exists as a dimer in solution as well [15] .…”

High-Throughput Small-Molecule Crystallography at the ‘Belok’ Beamline of the Kurchatov Synchrotron Radiation Source: Transition Metal Complexes with Azomethine Ligands as a Case Study

“…Examples of protein structures solved and refined against experimental diffraction data collected at the 'Belok' beamline include: uridine phosphorylase, laccases, ribonuclease, etc. [43][44][45][46][47]. In the powder diffraction mode, the emphasis is placed on real-time studies on new phase emergence and phase transitions for functionally sound materials [48,49].…”