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Several serotypes of Escherichia coli produce protein toxins closely related to Shiga toxin (Stx) from Shigella dysenteriae serotype 1. These Stx-producing E. coli cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in humans, with the latter being more likely if the E. coli produce Stx2 than if they only produce Stxl. To investigate the differences among the Stxs, which are all AB5 toxins, the crystal structure of Stx2 from E. coli O157:H7 was determined at 1.8-Å resolution and compared with the known structure of Stx. Our major finding was that, in contrast to Stx, the active site of the A-subunit of Stx2 is accessible in the holotoxin, and a molecule of formic acid and a water molecule mimic the binding of the adenine base of the substrate. Further, the A-subunit adopts a different orientation with respect to the B-subunits in Stx2 than in Stx, due to interactions between the carboxyl termini of the B-subunits and neighboring regions of the A-sub- unit. Of the three types of receptor-binding sites in the B-pentamer, one has a different conformation in Stx2 than in Stx, and the carboxyl terminus of the A-subunit binds at another. Any of these structural differences might result in different mechanisms of action of the two toxins and the development of hemolytic uremic syndrome upon exposure to Stx2. [ABSTRACT FROM AUTHOR]

Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-α-D-glucopyranoside, MSH or AcCysGlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn[sup 2+] metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a Cterminal domain consisting of two β-sheets and two α-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD[sup +] co-factor of lactate dehydrogenase. The Zn[sup 2+] is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn[sup 2+] providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol Sconjugate amidase based on the structure of MshB. [ABSTRACT FROM AUTHOR]

To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.

Protein phosphatase-1 and protein phosphatase-2B (calcineurin) are eukaryotic serine/threonine phosphatases that share 40% sequence identity in their catalytic subunits. Despite the similarities in sequence, these phosphatases are widely divergent when it comes to inhibition by natural product toxins, such as microcystin-LR and okadaic acid. The most prominent region of non-conserved sequence between these phosphatases corresponds to the beta12-beta13 loop of protein phosphatase-1, and the L7 loop of toxin-resistant calcineurin. In the present study, mutagenesis of residues 273-277 of the beta12-beta13 loop of the protein phosphatase-1 catalytic subunit (PP-1c) to the corresponding residues in calcineurin (312-316), resulted in a chimeric mutant that showed a decrease in sensitivity to microcystin-LR, okadaic acid, and the endogenous PP-1c inhibitor protein inhibitor-2. A crystal structure of the chimeric mutant in complex with okadaic acid was determined to 2.0-A resolution. The beta12-beta13 loop region of the mutant superimposes closely with that of wild-type PP-1c bound to okadaic acid. Systematic mutation of each residue in the beta12-beta13 loop of PP-1c showed that a single amino acid change (C273L) was the most influential in mediating sensitivity of PP-1c to toxins. Taken together, these data indicate that it is an individual amino acid residue substitution and not a change in the overall beta12-beta13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid.