Structural study of Rv2757c (Endoribonuclease) from Mycobacterium tuberculosis H37Rv

Abstract

Mycobacterium tuberculosis is one of the oldest pathogenic bacterial species ever human being experienced. In 2013, there were 9.0 million new TB cases and 1.5 million TB deaths. Among these casualties, 210,000 deaths were estimated to be from multi drug-resistant TB. There are some antibiotics for TB

however these antibiotics have shown the emergence of drug-resistant M.tb. SBDD (Structure-Based Drug Discovery) enables a novel drug to be developed more effectively based on the structural information of the target protein. Toxin-Antitoxin system was selected to develop a new antibiotics for TB. TA systems were classified into five classes based on the genetic architecture and the nature of regulation of TA systems. Type II TA systems consist of an antitoxin protein that sticks to and neutralizes the toxins activity by direct binding interaction. The Rv2757c toxin composed of 138 amino acids has been estimated to a VapC toxin which has a ribonuclease activity. VapBC family occupies 20 of 38 functional TA systems of M.tb. VapC toxins has highly conserved acidic amino acids which are thought to be putative catalytic residues in PIN domain. To determine the structure of the Rv2757c toxin, the gene of the Rv2757c toxin was cloned in pCold vector. Consequentially, the protein was over-expressed in E.coli cells. The proteins were purified with IMAC affinity chromatography and SEC. The purified protein was used in formation of crystals using crystal-screening kits. The well-made crystals appeared in 2-3 days in optimized reservoir solution consisting of 20% (w/v) PEG-8000, 0.1 M CHES/sodium hydroxide pH 9.5. X-ray diffraction data was collected using synchrotron radiation on ADSC Q315r detector at beamline PAL-5C (SBII) (Pohang, South Korea) at λ= 0.97944. Crystal of the Rv2757c toxin belonged to the monoclinic space group C2 (C121), with unit cell parameters of a=61.465 Å, b=44.813 Å, c=57.486 Å, and α=γ=90, β=99.87°. The crystal structure of the Rv2757c toxin shows that it consists of seven α-helices, five β-strands and one 310-helix, and the sequence of β-α-α-β-α-α-β-α- 310 -α-β-α-βhelices and strands. Four mutants of the Rv2757c were purified to determine if the conserved residues are necessary in ribonuclease activity of the Rv2757c toxin. The wild type and four mutants of the Rv2757c toxin were used in agarose gel electrophoresis with synthesized mRNA and fluorescence spectroscopy with synthesized mRNA and fluorescent spectroscopy with fluorescent-labeled RNA substrate. Consequently, the Rv2757c toxin protein showed ribonuclease activity in vitro, and two residues (D97, D115) of the Rv2757c toxin were predicted to be in part of ribonuclease activity.