15494 zyxwvutsrqpo Biochemistry zyxwvut 1994,33, 15494- 15500 Active-Site Mutations of Diphtheria Toxin: Role of Tyrosine-65 in NAD Binding and ADP-Ribosylationf Steven R. Blanke, Kathy Huang, and R. John Collier* Department of Microbiology and Molecular Genetics, Harvard Medical School, and The Shipley Institute of Medicine, Boston, Massachusetts 021 zyxwv I5 Received September 2, 1994; Revised Manuscript Received October 17, 1994@ ABSTRACT: Previous studies have suggested that tyrosine-65 (Tyr-65) of diphtheria toxin (DT) is located at the active site. To investigate the role of Tyr-65 in NAD binding and the ADP-ribosylation of elongation factor-2 (EF-2), we changed this residue to alanine and phenylalanine by site-directed mutagenesis of a synthetic gene encoding the catalytic fragment of DT (DTA). The alanine mutant was greatly diminished in ADP-ribosylation activity (350-fold) and NAD-glycohydrolase activity (88-fold), whereas the phenylalanine mutant was reduced in these activities only slightly. Dissociation constants zyx (&) for NAD binding were 15 pM for wild-type DTA, 26 pM for the phenylalanine mutant, and greater than 800 pM NAD for the alanine mutant. However, both mutant enzymes were found to bind adenosine with nearly equal affinity as wild-type DTA. These results support a model of ADP-ribosylation in which the phenolic ring of Tyr-65 interacts with the nicotinamide ring of NAD, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism. A number of potent bacterial toxins, including diphtheria toxin (DT),' exotoxin A from Pseudomonas aeruginosa (ETA), pertussis toxin, cholera toxin, and heat-labile toxin from Escherichia coli, are proenzymes that are activated upon binding to and entering sensitive eukaryotic cells. In many toxins (including those listed above), the enzyme moiety catalyzes the ADP-ribosylationof a target protein, resulting in deleterious consequences to the intoxicated cell and/or animal host (Collier & Mekalanos, 1980; Moss & Vaughn, 1990). Despite the ability to distinguish their specific macromolecular targets, each of these enzymes may utilize a similar catalytic mechanism. We have studied the catalytic domain of DT as a model for understanding the active-site structure and function of the ADP-ribosyltransferases. DT is organized into three distinct structural domains, which function directly in the three steps of intoxication: receptor binding, membrane translocation, and catalysis (Choe et al., 1992). After secretion from lysogenic strains of Corynebacterium diphtheriae carrying the phage-encoded tox gene, the toxin binds to the DT receptor by the carboxyl- terminal domain (R domain) and is intemalized via receptor- mediated endocytosis (Naglich & Eidels, 1990; Naglich et al., 1992). Acidification of the endosomal compartment is believed to trigger a conformational change in the toxin, allowing the central domain (T domain) to insert into the The work was supported by NIH Grants AI-22021 and AI-22848, PostdoctoralFellowship Award NM8469 (S.R.B.), and NSF REU Grant BIO-9200321 (K. H.). This work was submitted by K.H. zyxwvuts in partial fulfillment of a requirement for the Bachelor of Science Degree with Honors in Biology at Harvard University. * Author to whom correspondence should be addressed. @ Abstract published in Advance ACS Abstracts, December 1, 1994. Abbreviations: ADP, adenosine diphosphate; BSA, bovine serum albumin; DT, diphtheria toxin; EF-2, elongation factor-2; DTA, diphtheria toxin fragment A; DTT, dithiothreitol; EDTA, ethylene- diaminetetraacetic acid; ETA, exotoxin A; NAD, nicotinamide adenine dinucleotide; TCA, trichloroacetic acid; Tris, tris(hydroxymethy1)- aminomethane. 0006-296OJ9410433- 15494$04.50/0 endosomal membrane and the amino-terminal catalytic domain (C domain; equivalent to fragment A, or DTA) to be translocated across the membrane to the cytosol (Moskaug et al., 1991; Blewitt et al., 1985). Two processing events, proteolytic cleavage at Arg-193 and reduction of the disulfide linkage between cysteines-186 and -201, are required for release of DTA into the cytosol (Papini et al., 1993; Tsuneoka et al., 1993). Following these activation steps, DTA catalyzes transfer of the ADP-ribose moiety of NAD to a post-translationally modified histidine residue of elongation factor-2 (EF-2), called diphthamide (Van Ness et al., 1980). This covalent modification inactivates EF-2 and disrupts polypeptide chain elongation, resulting in cell death (Collier & Mekalanos, 1980; Collier, 1982; Collier & Traugh, 1969; Gill et al., 1969; Honjo et al., 1968). Despite limited sequence homology among the ADP- ribosyltransferases,a number of important active-site residues have been discovered (Blanke et al., 1992). Glu-148 was identified as an active-site residue of DTA by photoaffinity labeling experiments with NAD and subsequent site-directed mutagenesis studies (Carroll et al., 1980; Carroll & Collier, 1984; 1985a; Tweten et al., 1985; Wilson et al., 1990). Analogous investigations with ETA revealed Glu-553 to be functionally homologous to Glu-148 of DT and provided an important reference point for identification of sequence homology between the catalytic domains of these toxins (Carroll & Collier, 1988). Among the residues of DT identified in sequence alignments, His-21 and Trp-50 have recently been shown to be important determinants for the binding of NAD (Blanke et al., 1994; Wilson et al., 1994). In the investigations reported here, we have examined the function of Tyr-65 in DTA, which aligns with Tyr-481 in ETA. The crystal structures of these proteins reveal that these tyrosines are located in essentially identical positions within the active-site cleft of the catalytic domains (Choe et al., 1992; Allured et al., 1986). The importance of an active- site tyrosine in DTA had earlier been suggested when the zyxwvutsrqp 0 1994 American Chemical Society