A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins

Abstract

AbstractFIC proteins regulate molecular processes from bacteria to humans by catalyzing post-translational modifications (PTM), the most frequent being the addition of AMP using ATP as a cofactor, a reaction coined AMPylation. In a large group of AMPylating FIC proteins, which includes single-domain bacterial FIC proteins and animal FICD/HYPE, AMPylation is intrinsically repressed by a structurally conserved glutamate. Curiously, FICD departs from previously characterized bacterial FIC proteins in that it acts bifunctionally to AMPylate and deAMPylate its target, the endoplasmic reticulum BiP/GRP78 chaperone. BiP is a key component of the unfolded protein response (UPR), and is AMPylated under normal conditions where its activity is low, while its activation correlates with its deAMPylation. Currently, a direct signal regulating AMPylation efficiency in bacterial and animal FIC proteins has not been identified. Here, we addressed this question for a FIC protein from the bacterial pathogen Enterococcus faecalis (EfFIC) and for human FICD. We discover that EfFIC catalyzes both AMPylation and deAMPylation within the same active site, suggesting that the conserved glutamate is the signature of AMPylation/deAMPylation bifunctionality. Crystal structures and PTM assays identify a multi-position metal switch implemented by the glutamate, whereby EfFIC uses Mg2+ and Ca2+ to control AMPylation and deAMPylation differentially without conformational change. Remarkably, we find that variations in Ca2+ levels also tune deAMPylation of BiP by human FICD. Together, our results identify metals as diffusible signals that can regulate bifunctional FIC proteins directly, and they suggest that FICD has features of an enzymatic sensor of Ca2+ depletion, a hallmark of the UPR.