Refined structure of monomelic diphtheria toxin at 2.3 Å resolution

Abstract

The structure of toxic monomeric diphtheria toxin (DT) was determined at 2.3 Å resolution by molecular replacement based on the domain structures in dimeric DT and refined to an R factor of 20.7%. The model consists of 2 monomers in the asymmetric unit (1,046 amino acid residues), including 2 bound adenylyl 3′‐5′ uridine 3′ monophosphate molecules and 396 water molecules. The structures of the 3 domains are virtually identical in monomeric and dimeric DT; however, monomeric DT is compact and globular as compared to the “open” monomer within dimeric DT (Bennett MJ, Choe S, Eisenberg D, 1994b, Protein Sci 3:0000–0000). Detailed differences between monomeric and dimeric DT are described, particularly (1) changes in main‐chain conformations of 8 residues acting as a hinge to “open” or “close” the receptor‐binding (R) domain, and (2) a possible receptor‐docking site, a β‐hairpin loop protruding from the R domain containing residues that bind the cell‐surface DT receptor. Based on the monomeric and dimeric DT crystal structures we have determined and the solution studies of others, we present a 5‐step structure‐based mechanism of intoxication: (1) proteolysis of a disulfide‐linked surface loop (residues 186–201) between the catalytic (C) and transmembrane (T) domains; (2) binding of a β‐hairpin loop protruding from the R domain to the DT receptor, leading to receptor‐mediated endocytosis; (3) low pH‐triggered open monomer formation and exposure of apolar surfaces in the T domain, which insert into the endosomal membrane; (4) translocation of the C domain into the cytosol; and (5) catalysis by the C domain of ADP‐ribosylation of elongation factor 2.