Editors' ChoicePosttranslational Modifications

Flipping in a kinase fold for AMPylation

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Sci. Signal.  23 Oct 2018:
Vol. 11, Issue 553, eaav7869
DOI: 10.1126/scisignal.aav7869

The pseudokinase SelO mediates AMPylation instead of phosphorylation due to the flipped orientation of ATP in the active site.

The evolutionarily conserved selenoprotein-O (SelO) is predicted to be a pseudokinase because it lacks an aspartate in the catalytic loop. The human form incorporates the selenium-containing amino acid selenocysteine (Sec), although some homologs contain cysteine instead. Based on the crystal structure of the SelO homolog from a plant pathogen bound to a nonhydrolyzable ATP derivative, Sreelatha et al. (see also Sheetz and Lemmon) determined that SelO adopted a protein kinase–like fold. However, compared with its position in conventional protein kinases, the ATP derivative was flipped in the active site of SelO, such that the γ-phosphate (which is typically transferred by kinases to protein substrates) was buried within the kinase domain. Instead of mediating phosphorylation, SelO mediated AMPylation, a posttranslational modification involving the transfer of the α-phosphate of ATP in the form of adenosine monophosphate (AMP) to protein substrates. Structural and mutational analysis suggested that Asp262 substituted for the aspartate found in the catalytic loops of kinases. Under oxidizing conditions, Escherichia coli SelO formed an intracellular disulfide bond that was required for its AMPylation activity, and it AMPylated proteins with roles in oxidative phosphorylation (consistent with the localization of SelO to mitochondria) and redox homeostasis. SelO protein abundance increased in Saccharomyces cerevisiae grown on nonfermentable carbon sources, which induces oxidative stress. Conversely, S. cerevisiae deficient in SelO were more sensitive than wild-type strains to oxidative stress–causing chemicals. AMPylation inhibited the activity of glutaredoxin, an enzyme that reverses S-glutathionylation, a posttranslational modification in which glutathione is transferred to cysteine residues. Loss of SelO in E. coli or yeast led to decreased S-glutathionylation, particularly under oxidizing conditions that would be expected to increase this posttranslational modification. Thus, SelO uses the protein kinase fold to mediate AMPylation rather than phosphorylation.

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