New insight into secreted ribonuclease structure: binase is a natural dimer

Abstract

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.

Figure 1. Identification of binase dimers.

(A) The SDS-PAGE in presence of 0.1% SDS, (B) SDS-PAGE of native binase under strong denaturing conditions (1–2% SDS, 2–4 M urea, 3–8 M urea), (C) immunoblot detection and (D) in gel RNase activity assay of lyophilised binase from wild-type B. pumilis 7P (native, N), E. coli JM107 (recombinant, R).

Figure 2. The evidences of binase dimerisation.

(A) The cross-linking analysis of native binase (native, N) and treated by glutaraldehyde (GA) at final concentration 0.5 mg/ml and at increasing protein concentrations: (B) native binase, (C) cross-linked with GA (1–0.05 mg/ml, 2–0.1 mg/ml, 3–0.5 mg/ml, 4–1 mg/ml), (D) native-PAGE of native binase (L – lysozyme (MW  = 14.4 kDa, pI 11.3), R – RNase A (MW  = 13.7 kDa, pI 9.64), N – binase (MW  = 12 and 25 kDa, pI 9.5).

Figure 3. Size exclusion chromatography of binase at pH 8.0 (A, B) and pH 3.0 (C).

A –native binase; B – binase after cross-linking by glutaraldehyde; C - binase denatured by heating for 20 min at 95°C, pH 2.5; I–IV – marker proteins used for molecular weight estimation: BSA (66.2 kDa), trypsin (24 kDa), papain (23.7 kDa), lysozyme (14.4 kDa) correspondingly. Molecular weights of binase peaks were calculated using equation obtained from elution profile of marker proteins.

Figure 4. Western Blot analysis of B. pumilis 7P culture fluid collected after 12 h (exponential phase) and 24 h (stationary phase) of cultivation.

N- lyophilised binase from wild-type B. pumilis 7P.

Figure 5. Putative models of binase dimer in solution.

(A) The model of van der Waals and electrostatic-favored cluster. (B) The model of balanced and electrostatic-favored clusters. Amino acid residues constituting the active centre of enzyme are labelled. The models were generated using atomic coordinates of binase (PDB 1buj) on ClusPro server . Visualisation was performed by Jmol: an open-source Java viewer for chemical structures in 3D (http://www.jmol.org/).