The long-range goals of this grant are to understand the actions of ADP-ribosylating exotoxins in detail; and, where feasible, to apply our knowledge of such toxins to develop better methods of disease control. Work described is focused primarily on diphtheria toxin (DT) and exotoxin A of P. aeruginosa (PT), two related toxins that block protein synthesis in mammalian cells by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). With respect to DT, we propose: (i) to provide the biochemical component of a collaborative project to determine the 3-dimensional structure of the toxin (and perhaps fragments and mutant forms); (ii) to probe the structure of the catalytic center (particularly the NAD binding site) with the goal of elucidating the mechanism of catalysis of ADP-ribosylation of EF-2; (iii) to investigate the insertion of the B moiety into membranes, with a view to understanding the mechanism by which B promotes transmembrane transfer of the A chain; (iv) to characterize the receptor binding site on the toxin and examine its relationship with the polyphosphate binding site (P-site); (v) to study the structure, specificity, and biological significance of the hig-affinity binding site of DT for endogenous dinucleotides. With respect to PT, we propose: (i) to provide the biochemical support for determination of the 3-dimensional structure through an existing collaboration; (ii) to clone and sequence the structural gene for the toxin; and (iii) to pursue detailed structure-activity studies similar to those outlined for DT. A variety of biochemical and biophysical methods applicable to these problems will be supplemented by the use of monoclonal antibodies, molecular cloning and mutagenesis. The results will be relevant to: (i) detailed understanding of bacterial pathogenesis at the molecular/sub-molecular level; (ii) cell surface receptor-ligand interactions; (iii) mechanisms of insertion and traversal of membranes by proteins; and (iv) potential mechanisms of directing toxic proteins or moieties thereof to specific cells.
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