Free Energy Determinants of Binding the rRNA Substrate and Small Ligands to Ricin A-Chain

Abstract

A continuum model is provided of the free energy terms that contribute to the molecular association of ricin A-chain (RTA) with the rRNA substrate and several small ligands. The model for RTA interactions with the RNA was taken from a previously proposed complex containing a 29-mer oligonucleotide hairpin (. Proteins 27:80-95), and models for the ligands were constructed from x-ray crystallographic structures. The calculated absolute free energies of complex formation for the RTA-RNA assembly and several single-residue substitutions are in good agreement with experimental data, given the approximations of evaluating the strain energy and conformational entropy. The free energy terms were found to resemble those of protein-protein complexes, with the net unfavorable electrostatic contribution offset by the favorable nonspecific hydrophobic effect. Decomposition of the RTA-RNA binding free energy into individual contributions revealed the electrostatic "hot" spots arising from charge-charge complementarity of the interfacial arginines with the RNA phosphate backbone. Base interactions of the GAGA loop structure dominate the hydrophobic complementarity. A linear-scaling model was parametrized for evaluating the binding of small ligands against the rRNA substrate and illustrates the free energy determinant required for designing specific RTA inhibitors.

Arginine Residues on the Opposite Side of the Active Site Stimulate the Catalysis of Ribosome Depurination by Ricin A Chain by Interacting with the P-protein Stalk

“…We show here that G212A binds ribosomes and ribosomal stalk similarly as wild type RTA but depurinates ribosomes and RNA at a lower catalytic rate than wild type RTA. Gly 212 is located between Trp 211 , which is involved in binding to the target adenine (60,61), and Arg 213 , which contributes to binding to the rRNA (62,63) and is on the opposite side from the RTA/RTB contacts (Fig. 2B).…”

Improved formulation of a recombinant ricin A-chain vaccine increases its stability and effective antigenicity

“…The lower adherence found with phosphate buffer may have been due at least in part to the natural affinity of RTA for phosphate groups in its rRNA substrate [28]. Association of phosphate ions with the protein could alter its surface properties, affecting interactions with the adjuvant.…”

Do electrostatic interactions destabilize protein–nucleic acid binding?

“…1,2 However, many calculations have found that electrostatic interaction energies between proteins and nucleic acids are positive, meaning that electrostatic interactions are destabilizing for their binding (see Table I). [3][4][5][6][7][8][9][10][11][12][13] These calculation results are very counterintuitive. They were obtained from solving the PoissonBoltzmann (PB) equation by choosing the molecular surface as the boundary between the solute low dielectric and the solvent dielectric.…”