Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification

Abstract

Filamentation induced by cyclic AMP (FIC)-domain enzymes catalyze adenylylation or other posttranslational modifications of target proteins to control their function. Recently, we have shown that Fic enzymes are autoinhibited by an α-helix (α inh ) that partly obstructs the active site. For the single-domain class III Fic proteins, the α inh is located at the C terminus and its deletion relieves autoinhibition. However, it has remained unclear how activation occurs naturally. Here, we show by structural, biophysical, and enzymatic analyses combined with in vivo data that the class III Fic protein NmFic from Neisseria meningitidis gets autoadenylylated in cis, thereby autonomously relieving autoinhibition and thus allowing subsequent adenylylation of its target, the DNA gyrase subunit GyrB. Furthermore, we show that NmFic activation is antagonized by tetramerization. The combination of autoadenylylation and tetramerization results in nonmonotonic concentration dependence of NmFic activity and a pronounced lag phase in the progress of target adenylylation. Bioinformatic analyses indicate that this elaborate dual-control mechanism is conserved throughout class III Fic proteins.adenylylation | AMPylation | posttranslational modification | enzyme regulation | molecular timer

Enzymes Involved in AMPylation and deAMPylation

“…43 NmFic forms a stable tetramer via interactions on two highly conserved surfaces, leading to inactivation of NmFic. 43 Formation of the NmFic dimer and tetramer is tightly controlled by protein concentration. At concentrations of 250 nM and below, NmFic behaves primarily as a monomer and exhibits much higher AMPylation activity.…”

“…43 As a consequence, auto-AMPylation of NmFic leads to a change in positioning of the α -inhibitory loop and release of autoinhibition of catalytic activity. 43 AutoAMPylation occurs primarily in the monomeric form of this protein, indicating that both concentration-dependent self-association and auto-AMPylation play a cooperative role in the regulation of NmFic. 43 Because NmFic is highly conserved in many bacteria, it is predicted that this form of regulation is also conserved in this subset of Fic domains.…”

“…26 Since the original identification of Fic-1 in E. coli , 26,52,53 several Fic-1 homologues have recently been identified and characterized in other bacterial species. 31,36,37,43,54,55 The FicT toxins are typically coexpressed with their antitoxin-containing inhibitory α -helix as part of a toxin–antitoxin system in the cells. 31,36 When expressed without the corresponding antitoxin anti-Fic-1 (AntF), cellular growth is arrested 31,37 (Figure 16).…”

A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism

“…Despite showing that ATP binding and autoadenylylation by CdFic are independent of the inhibitory motif, we cannot rule out the possibility that the ␣ inh of CdFic might still have a function in inhibiting the adenylylation activity against the currently unknown target. However, this would be unusual because the ␣ inh is known to regulate both autoadenylylation and adenylylation in other Fic proteins with known protein targets, such as HYPE (16) and NmFic (62).…”

rAMPing Up Stress Signaling: Protein AMPylation in Metazoans

“…Auto-AMPylation in cis of the Neisseria meningitidis AMPylase NmFic relieves auto-inhibition. This is a prerequisite for target modification, while preventing the formation of an inhibitory tetrameric NmFic complex (Stanger et al, 2016). Self- modification of Tyr 183 and Tyr188, two residues within the α inh helix, leads to partial unfolding of α inh and enhances access to the active site.…”