| - | ABSTRACT: BACKGROUND: Ricin is a potent toxin and known bioterrorism, threat with no available antidote. The ricin A-chain (RTA) acts, enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship, between ligand binding and RTA active site conformational change, we used, a fragment-based approach to find a minimal set of bonding interactions, able to induce rearrangements in critical side-chain positions. RESULTS:, We found that the smallest ligand stabilizing an open conformer of the RTA, active site pocket was an amide group, bound weakly by only a few hydrogen, bonds to the protein. Complexes with small amide-containing molecules also, revealed a switch in geometry from a parallel towards a splayed, arrangement of an arginine-tryptophan cation-pi interaction that was, associated with an increase and red-shift in tryptophan fluorescence upon, ligand binding. Using the observed fluorescence signal, we determined the, thermodynamic changes of adenine binding to the RTA active site, as well, as the site-specific binding of urea. Urea binding had a favorable, enthalpy change and unfavorable entropy change, with a deltaH of -13 +/-2, kJ/mol and a deltaS of -0.04 +/-0.01 kJ/K*mol. The side-chain position of, residue Tyr80 in a complex with adenine was reexamined and found not to, involve as large an overlap of rings with the purine as previously, considered, suggesting a smaller role for aromatic stacking at the RTA, active site. CONCLUSIONS: We found that amide ligands can bind weakly but, specifically to the ricin active site, producing significant shifts in, positions of the critical active site residues Arg180 and Tyr80. These, results indicate that fragment-based drug discovery methods are capable of, identifying minimal bonding determinants of active-site side-chain, rearrangements and the mechanistic origins of spectroscopic shifts. Our, results suggest that tryptophan fluorescence provides a sensitive probe, for the geometric relationship of arginine-tryptophan pairs, which often, have significant roles in protein function. Using the unusual, characteristics of the RTA system, we measured the still controversial, thermodynamic changes of site-specific urea binding to a protein, results, that are relevant to understanding the physical mechanisms of protein, denaturation.
| + | |
| - | Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site., Carra JH, McHugh CA, Mulligan S, Machiesky LM, Soares AS, Millard CB, BMC Struct Biol. 2007 Nov 6;7(1):72. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17986339 17986339]
| + | |
