Recombinant Adenosine monophosphate-protein transferase FICD homolog (CBG05963)

What is Adenosine monophosphate-protein transferase FICD homolog (CBG05963)?

Adenosine monophosphate-protein transferase FICD homolog (CBG05963) is a full-length protein (507 amino acids) derived from the nematode Caenorhabditis briggsae. It belongs to the Fic domain protein family and is also known as fic-1, Protein adenylyltransferase fic-1, or De-AMPylase fic-1 . Animal genomes typically encode a single ortholog of the widely disseminated Fic domain proteins that carry out AMPylation in bacteria . The protein functions as both an AMPylase and a deAMPylase, carrying out two functionally antagonistic reactions in the endoplasmic reticulum (ER): BiP AMPylation and deAMPylation .

How does the oligomeric state of FICD affect its enzymatic activities?

The antagonistic activities of FICD (AMPylation and deAMPylation) are directly influenced by its oligomeric state. Research has demonstrated that FICD in its dimeric form functions predominantly as a deAMPylase, while the monomeric form primarily acts as an AMPylase . Under standard experimental conditions, both wild-type and mutant FICD appear principally dimeric as assessed by size exclusion chromatography . This oligomeric regulation provides a sophisticated mechanism for controlling FICD's opposing enzymatic functions within the cell.

Researchers investigating the relationship between FICD structure and function should consider introducing mutations that affect dimerization, such as L258D, which can prevent the formation of complexes with different stoichiometries (e.g., BiP:FICD 2:2 and 1:2 mixtures) . This approach allows for more controlled experimental conditions when studying the specific activities of monomeric versus dimeric forms.

What is the molecular basis for FICD's substrate specificity?

The substrate specificity of FICD, particularly toward BiP (a chaperone protein), is mediated by its TPR motifs. Crystallographic studies at 2.6 Å have revealed that the TPR motifs of FICD bind specifically to the conserved hydrophobic linker of BiP, thus conferring specificity for BiP in its ATP-bound state . Mutation studies have identified several key residues in FICD that are critical for BiP AMPylation, with mutations in the TPR region (including N99A, D103A, K124A, and K127A) reducing AMPylation activity by >98% .

Moreover, FICD contains an inhibitory α-helix with a conserved sequence motif (S/T)xxxE(G/N) near the ATP-binding site that keeps the enzyme intrinsically inactive for AMP transfer . Substitution of the conserved glutamate in this inhibitory motif by glycine (E234G in FICD) has been shown to stimulate AMPylation activity in vitro . This structural information provides crucial insights for researchers designing experiments to study FICD's substrate interactions and enzymatic mechanisms.

How can researchers distinguish between FICD's AMPylation and deAMPylation activities in experimental settings?

Distinguishing between FICD's AMPylation and deAMPylation activities requires careful experimental design. Researchers can:

• Manipulate FICD's oligomeric state: Since dimers favor deAMPylation and monomers favor AMPylation, introducing mutations that affect dimerization can help isolate each activity .

• Use point mutations in the inhibitory α-helix: The E234G mutation in FICD stimulates AMPylation activity in vitro, allowing researchers to enhance this specific function .

• Modify autoAMPylation sites: T168A and T183A substitutions affect FICD's autoAMPylation and slightly reduce both AMPylation and deAMPylation activities, providing another control mechanism .

• Control BiP's conformational state: The T229A mutation in BiP inhibits its ATPase activity, keeping the chaperone in the ATP state, which is the preferred substrate for FICD-mediated AMPylation .

By systematically applying these approaches, researchers can selectively study either AMPylation or deAMPylation in controlled experimental settings.

What expression systems are most effective for producing recombinant FICD homologs?

For recombinant expression of FICD homologs, Escherichia coli has proven to be an effective host system. According to the available data, recombinant FICD homolog (CBG05963) has been successfully expressed in E. coli with an N-terminal His-tag . This approach yields functional protein suitable for biochemical and structural studies.

When expressing FICD in E. coli, researchers should consider several optimization strategies:

• Use of specialized E. coli strains designed for recombinant protein expression

• Optimization of induction conditions (temperature, IPTG concentration, induction time)

• Addition of appropriate tags (His-tag is commonly used) for purification purposes

• Co-expression with chaperones if protein solubility is an issue

The expression of full-length protein (1-507 amino acids) allows for comprehensive functional studies, though researchers might consider expressing specific domains separately for targeted structural or interaction studies .

What are the optimal storage conditions for maintaining FICD protein stability and activity?

Proper storage of recombinant FICD proteins is critical for maintaining their stability and enzymatic activities. Based on the available information, the following storage recommendations should be considered:

• Store at -20°C/-80°C upon receipt

• Aliquot the protein to avoid repeated freeze-thaw cycles, as this can significantly reduce activity

• For working aliquots, store at 4°C for up to one week

The protein can be provided in either liquid form or as a lyophilized powder. Lyophilized FICD is typically prepared from a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 . For reconstitution, it is recommended to:

• Briefly centrifuge the vial prior to opening

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) when preparing aliquots for long-term storage

It's worth noting that the shelf life of liquid form is generally 6 months at -20°C/-80°C, while the lyophilized form can remain stable for up to 12 months under the same conditions .

What experimental design considerations are essential when studying FICD-mediated AMPylation?

When designing experiments to study FICD-mediated AMPylation, researchers should consider the following key factors:

A robust experimental design should incorporate appropriate controls, including FICD mutants with altered activities, substrate proteins in defined conformational states, and reaction conditions that mimic the ER environment.

How are FICD proteins implicated in human disease?

Recent research has identified a significant connection between FICD dysfunction and serious human diseases. Studies have reported on five individuals from three different consanguineous families with infancy-onset diabetes mellitus and severe neurodevelopmental delay associated with de-regulated AMPylation . This finding highlights the critical role that properly regulated AMPylation plays in human physiology and development.

The ER-resident FICD protein's function in regulating BiP through AMPylation directly impacts ER homeostasis, which is crucial for proper cellular function. Disruption of this regulatory mechanism can lead to ER stress and associated pathologies . As BiP is a major chaperone involved in protein folding and quality control in the ER, dysregulation of its activity through aberrant AMPylation/deAMPylation can have far-reaching consequences for cellular proteostasis.

Researchers investigating disease connections should consider studying FICD variants identified in patients and analyzing their effects on AMPylation activity, substrate specificity, and ER homeostasis to better understand the molecular mechanisms underlying these conditions.

What approaches can researchers use to identify novel substrates of FICD?

While BiP is the best-characterized substrate of FICD, evidence suggests that FICD may have additional targets. Potential additional AMPylation substrates include the eukaryotic elongation factor 1A2 (EEF1A2) and uridine 5′ monophosphate synthase (UMPS) . To identify and validate novel FICD substrates, researchers can employ several approaches:

• Structural analysis: FICD's TPR motifs are essential for BiP AMPylation and are also required for AMPylation of EEF1A2, but not for UMPS . Researchers can use this information to predict other potential substrates with similar binding motifs.

• Sequence similarity network analysis: This approach can reveal common ancestry of multidomain proteins and help identify homologs in complex multidomain families with varied domain architectures . This could be particularly useful for identifying proteins that might interact with FICD.

• Covalent tethering techniques: The use of cosubstrate analogs (TReNDs) has been successful in stabilizing the transient interaction between FICD and BiP for structural studies . Similar approaches could be employed to capture and identify novel FICD-substrate interactions.

• Proteomic approaches: Mass spectrometry-based methods to detect AMPylated proteins in cells with modified FICD expression or activity could reveal the broader substrate landscape.

When pursuing these approaches, researchers should consider the impact of FICD's oligomeric state and the conformational state of potential substrates, as these factors significantly influence FICD's substrate specificity.

What are the most promising avenues for future research on FICD homologs?

Based on the current state of knowledge, several promising research directions for FICD homologs emerge:

• Therapeutic potential: Given the connection between FICD dysfunction and human diseases like infancy-onset diabetes, exploring the therapeutic potential of modulating FICD activity represents an important avenue for translational research.

• Structural dynamics: Further investigation into how FICD transitions between monomeric and dimeric states and how this affects the balance between AMPylation and deAMPylation activities could reveal novel regulatory mechanisms.

• Evolutionary conservation: Comparative studies of FICD homologs across species could provide insights into the evolutionary conservation of AMPylation/deAMPylation mechanisms and their biological significance.

• Interactome mapping: Comprehensive identification of FICD-interacting proteins beyond known substrates could reveal broader roles in cellular signaling networks and homeostasis maintenance.

• Development of specific inhibitors: Design and development of small molecules that can selectively modulate FICD's AMPylase or deAMPylase activities could provide valuable tools for both basic research and potential therapeutic applications.