Fic and non-Fic AMPylases: protein AMPylation in metazoans

Abstract

Protein AMPylation refers to the covalent attachment of an AMP moiety to the amino acid side chains of target proteins using ATP as nucleotide donor. This process is catalysed by dedicated AMP transferases, called AMPylases. Since this initial discovery, several research groups have identified AMPylation as a critical post-translational modification relevant to normal and pathological cell signalling in both bacteria and metazoans. Bacterial AMPylases are abundant enzymes that either regulate the function of endogenous bacterial proteins or are translocated into host cells to hijack host cell signalling processes. By contrast, only two classes of metazoan AMPylases have been identified so far: enzymes containing a conserved filamentation induced by cAMP (Fic) domain (Fic AMPylases), which primarily modify the ER-resident chaperone BiP, and SelO, a mitochondrial AMPylase involved in redox signalling. In this review, we compare and contrast bacterial and metazoan Fic and non-Fic AMPylases, and summarize recent technological and conceptual developments in the emerging field of AMPylation.

Keywords: ampylases; chaperone and neurodegeneration; filamentation induced by cAMP; non-Fic; post-translational modification.

Figure 1.

Mechanisms of target AMPylation. (a) Reaction scheme of Rab1 AMPylation by non-Fic AMPylase DrrA. Asp150, Asp110 and Asp249 are involved in coordination of the divalent cation; Asp112 is involved in deprotonation of the incoming target side chain (Tyr77 of Rab1). (b) Reaction scheme of CDC42/Rac1 AMPylation by Fic domain containing bacterial AMPylase VopS. The conserved His acts as a proton sink and deprotonates Thr35 of Cdc42/Rac1. The figure emphasizes the significance of Fic motif in coordinating the phosphates of the ATP molecule and catalysing AMP transfer. ATP molecule is depicted in green, and red arrows depict the reaction steps during AMPylation. This figure has been modified from Hedberg C. and Itzen A [8] and Gavriljuk et.al. [9].

Figure 2.

Targeting of Ras GTPase family members by bacterial AMPylases.

Figure 3.

Domain organization schematic of metazoan AMPylases.

Figure 4.

Flipped orientation of the ATP molecule in SelO. In the active site of a canonical kinase the adenine ring is buried deep in the catalytic cleft while the phosphates are exposed. Kinases transfer the distal phosphate (farthest from the adenine ring) onto its substrates (phosphorylated) with concomitant ADP release. In SelO, the orientation of the ATP molecule is flipped, with the phosphates buried deep in the binding cleft. SelO transfers the proximal phosphate (closest to the adenine ring) onto its target (AMPylated) with pyrophosphate being the other product of the AMPylation reaction. The dashed lines represent cleavage of the bond in a phosphorylation or AMPylation reaction.

Figure 5.

HYPE crystallizes as an asymmetric dimer. (a) A cartoon representation of a HYPE monomer. Key structural features are highlighted. Asterisk represents missing electron density for 6 residues within the linker. The same structural features are present in the other HYPE monomer but has not been shown here for clarity. (b) HYPE residues involved in the formation of interfaces 1 and 2 are labelled. The HYPE monomers are depicted in green and red. Black solid lines (Dimer Interface 1 inset) denote hydrogen bonds between residues. Residues making up Dimer Interface 2 interact weakly, mostly through Vander-Waal's and electrostatic interactions.

Figure 6.

HYPE switching between AMPylation and deAMPylation states in response to ER conditions. HYPE AMPylates BiP when the unfolded protein load in the ER is low and deAMPylates BiP under conditions of stress that often increase unfolded protein load. AMPylated BiP cannot function as a chaperone and is pooled into a reservoir of non-functional BiP that can be activated under conditions of stress by deAMPylation. It is currently hypothesized that Glu234 mediates the switch between AMPylation and deAMPylation competent HYPE conformations. The bold arrows indicate the movement of α-inh that harbours Glu234. During AMPylation, Glu234 disengages from the catalytic site and allows the alignment of key residues in the catalytic FIC motif and Thr518 of BiP. When cells require functional BiP to tackle increasing loads of unfolded polypeptides, Glu234 engages with Arg374 and coordinates the attack of a water molecule (acting as a nucleophile) on the bond between Thr518 and AMP. The smaller arrows indicate a hypothetical electron transfer between various moieties involved in the proposed catalytic mechanism. This figure is adapted from Preissler et al. [77].

Figure 7.

Consequences of HYPE functions in the cytoplasm and ER. The arrows pointed outwards from the green and red zoom-in circles depict events regulated by HYPE mediated AMPylation in cytoplasm and ER, respectively.

Figure 8.

Pictorial representation of approaches used to capture and identify FicD targets. Panels (a) and (b) represent approaches that modify targets using synthetic nucleotides followed by a click-chemistry-based capture of modified targets while in (c) the enzyme is modified using TReND and forms a binary complex that then AMPylates target proteins.

Figure 9.

FIC-1 targets and their role in various physiological processes.