Inhibition of ricin A-chain (RTA) catalytic activity by a viral genome-linked protein (VPg)

Abstract

Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A-chain (RTA) and a viral genome-linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N-terminal truncated variant, VPg 1-110 , bind to RTA and abolish ricin's catalytic depurination of 28S rRNA in vitro and in a cell-free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases tenfold as temperature elevates (5 °C to 37 °C). RTA-VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = −136.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA-VPg complex, Ea = −62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.

An Electrochemical Approach to Follow and Evaluate the Kinetic Catalysis of Ricin on hsDNA

“…Although the authors could find neither kcat nor Km values for the catalytic activity of ricin on hsDNA as a substrate in the literature, some parameters were attained with synthetic substrates or ribosomes under steady-state catalysis conditions [ 11 , 17 , 18 , 32 ]. In this sense, the kcat/Km value found in this work was reported in the same order of magnitude by Chen et al [ 18 ] (4.5 × 10 5 M −1 s −1 ) for ricin A-chain subunit activity with 14-base stem-loop RNA substrate, and close to that reported by Aitbakieva et al (3.8 × 10 6 M −1 s −1 ) [ 32 ] when using 28S rRNA substrate. In contrast, Tan et al [ 11 ] when using synthetic oligoribonucleotides and as by Sturm and Schramm [ 17 ] when using rabbit reticulocyte ribosomes or yeast ribosomes as substrate, reported higher kcat/Km values of 1.1 × 10 7 M −1 s −1 and 2.6 × 10 8 M −1 s −1 , respectively.…”

“…The depurination reaction concomitant to the catalytic action of ricin was monitored for hsDNA concentrations ranging from 2.5 to 50 µM (Figure 2a), and with the overall reaction dependent on the substrate concentration. The dependence of the initial velocities obtained from fitting data to Equation (1) (vss) on hsDNA concentration follows a regular hyperbolic saturation form (Figure 2b Although the authors could find neither kcat nor Km values for the catalytic activity of ricin on hsDNA as a substrate in the literature, some parameters were attained with synthetic substrates or ribosomes under steady-state catalysis conditions [11,17,18,32]. In this sense, the kcat/Km value found in this work was reported in the same order of magnitude by Chen et al [18] [32] when using 28S rRNA substrate.…”

“…The dependence of the initial velocities obtained from fitting data to Equation (1) (vss) on hsDNA concentration follows a regular hyperbolic saturation form (Figure 2b Although the authors could find neither kcat nor Km values for the catalytic activity of ricin on hsDNA as a substrate in the literature, some parameters were attained with synthetic substrates or ribosomes under steady-state catalysis conditions [11,17,18,32]. In this sense, the kcat/Km value found in this work was reported in the same order of magnitude by Chen et al [18] [32] when using 28S rRNA substrate. In contrast, Tan et al [11] when using synthetic oligoribonucleotides and as by Sturm and Schramm [17] when using rabbit reticulocyte ribosomes or yeast ribosomes as substrate, reported higher kcat/Km values of 1.1 × 10 7 M −1 s −1 and 2.6 × 10 8 M −1 s −1 , respectively.…”

Medical Countermeasures against Ricin Intoxication

“…Aitbakieva et al [ 107 ] demonstrated that VPg1–110, the N-terminal truncated variant of viral genome-linked protein (VPg) from turnip mosaic virus (TuMV), bound to RTA and abolished ricin’s catalytic depurination of 28S rRNA in vitro and in a cell-free rabbit reticulocyte translational system. In the cell-free rabbit reticulocyte translational system, the treatment of RTA with the synthetic VPg1–110 peptide resulted in complete inhibition of RTA activity on ribosomes.…”