UFC1 Human

What is the functional role of UFC1 in the ufmylation pathway?

UFC1 functions as the E2 conjugating enzyme in the ufmylation pathway, forming a critical link between UBA5 (E1) and UFL1 (E3). The process involves:

• UBA5 activates UFM1 in an ATP-dependent manner

• UFC1 receives the activated UFM1 via a transthiolation reaction, forming a UFC1~UFM1 thioester intermediate

• UFL1, in complex with DDRGK1, facilitates the transfer of UFM1 from UFC1 to target substrates

This pathway plays essential roles in ER homeostasis, protein translation, and DNA damage response mechanisms .

What structural features characterize UFC1 binding interactions?

UFC1 binding is characterized by specific structural elements:

• UFC1 α-helix I (residues 26-48) and β-strand I (residues 54-58) directly interact with the UFL1 N-terminus

• The N-terminal half of UFC1 α-helix II (residues 135-145) shows chemical perturbations during binding, suggesting allosteric regulation

• UFC1 uses the same binding surface to interact with both UBA5 (E1) and UFL1 (E3), creating a competition mechanism that regulates ufmylation efficiency

How do researchers typically express and purify UFC1 for structural studies?

For structural studies of UFC1:

• Generate expression constructs in vectors such as pET15b using Gibson assembly

• Express in bacterial systems (typically E. coli)

• For interaction studies with UFL1, researchers have successfully used fusion constructs like DDRGK1(207-314)-UFL1(1-200)

• Identify and remove disordered regions that hamper protein expression based on structural predictions from tools like AlphaFold2

• Use crystallography, NMR spectroscopy, and biochemical assays for structural characterization of UFC1 and its complexes

What methodologies can resolve the competition dynamics between UBA5 and UFL1 for UFC1 binding?

To investigate UBA5-UFL1 competition for UFC1 binding:

• NMR-based competition experiments: Using 15N-labeled UBA5 C-terminus (residues 347-404) bound to UFC1, researchers can observe changes in the 1H-15N HSQC spectrum upon addition of DDRGK1-UFL1. The shifting of NMR cross-peaks to unbound positions confirms competitive binding .

• Isothermal Titration Calorimetry (ITC): Determine binding affinities (Kd) between UFC1 and its partners. Research shows UFL1-DDRGK1 binds to UFC1 with Kd ≈ 2.4-2.57 μM, which is comparable to UBA5-UFC1 affinity .

• Biochemical competition assays: Using purified components to assess how varying concentrations of UFL1 affect UBA5-UFC1 interactions and vice versa.

• Structural models from AlphaFold2: These can reveal shared binding surfaces without prior experimental information, guiding the design of mutants to specifically disrupt one interaction while preserving the other .

How does the charged state of UFC1 affect its interactions with partner proteins?

The charged state of UFC1 (UFC1~UFM1) significantly impacts its interactions:

• Extended fusion constructs containing the UFM1 binding site of DDRGK1 (DDRGK1ext-UFL1) show approximately 10-fold higher affinity for charged UFC1 (Kd = 0.23 μM) compared to binding to uncharged UFC1 (Kd ≈ 2.57 μM) .

• The UFM1 binding site on DDRGK1 can bind to UFM1 once it is charged on UFC1, enhancing preferential binding to the charged form .

• When UFC1 is no longer charged (after UFM1 transfer), it likely dissociates from the ER environment, becoming available for interaction with UBA5 to initiate another cycle of charging .

• Experimental methods to study these differential interactions include:

  • Pull-down assays comparing binding ratios of charged versus uncharged UFC1

  • ITC measurements to determine precise binding affinities

  • In vitro reconstitution of the ufmylation cascade with labeled components

What approaches can identify critical residues in the UFC1-UFL1 interaction interface?

To identify critical interface residues:

• Computational alanine scanning: Using tools like the Robetta server to predict residues with binding energy contribution (ΔΔGbinding > 1.0 kcal/mol) as potential hotspot residues .

• NMR chemical shift perturbation analysis: 1H,15N-HSQC NMR spectra of 15N-labeled UFC1 with and without UFL1 reveals residues with significant signal attenuation or shifts upon binding .

• Site-directed mutagenesis: Systematically mutating predicted interface residues and measuring effects on binding affinity and enzymatic activity.

• AlphaFold2 structural modeling: This approach successfully identified the binding region of UFL1 to UFC1 without prior experimental data, demonstrating its value in predicting protein-protein interactions involving short motifs or regions that adopt stable structure only upon interaction .

How can researchers effectively study the E2-E3 discharge mechanism in the ufmylation pathway?

To study the discharge mechanism:

• In vitro discharge assays: Monitor the rate of UFC1~UFM1 discharge in the presence of UFL1-DDRGK1 complex or various fusion constructs. Recent studies show that the UFL1-DDRGK1 complex enhances the discharge of charged UFC1 by free lysine .

• Structural comparison with other UBL systems: Compare with known E2-E3 discharge mechanisms in ubiquitin and UBL systems to identify common principles and unique features of ufmylation .

• Time-resolved methodologies: Use stopped-flow techniques or rapid-quench approaches to capture transient intermediates during UFM1 transfer.

• Domain mapping experiments: Test various truncations of UFL1 and DDRGK1 to identify regions necessary for discharge stimulation beyond those present in current fusion proteins .

What structural biology approaches are most effective for studying UFC1 complexes?

Current research demonstrates multiple effective approaches:

• Integrative structural biology: Combining AlphaFold2 predictions with experimental validation has proven particularly powerful, allowing researchers to model UFC1 interactions without prior experimental information .

• X-ray crystallography: Using fusion proteins (e.g., DDRGK1-UFL1) has enabled successful crystallization of otherwise challenging complexes .

• NMR spectroscopy: Particularly valuable for examining binding interfaces and competition dynamics between interaction partners (UBA5 vs. UFL1) .

• Biochemical validation: ITC for precise binding affinities and functional assays to confirm activity of engineered constructs .

The combination of these approaches has been instrumental in revealing key aspects of UFC1's interactions, including the critical role of UFL1's N-terminal helix in binding to UFC1 and the competitive binding between UBA5 and UFL1 .

How can protein engineering approaches optimize UFC1 expression and stability for research?

Effective protein engineering strategies include:

• Structure-guided construct design: Use AlphaFold2 models to identify stable domains and remove disordered regions that hamper expression .

• Fusion protein approaches: DDRGK1(207-314)-UFL1(1-200) fusion constructs have successfully simplified the study of UFL1 activity and enabled crystallographic studies .

• Expression optimization:

  • Clone constructs in vectors optimized for structural biology (e.g., pET15b)

  • Use Gibson assembly for seamless fusion protein creation

  • Verify constructs by DNA sequencing before expression

• Stability screening: Employ thermal shift assays or limited proteolysis coupled with mass spectrometry to identify and improve stable constructs.

How does UFC1 research contribute to understanding ER stress response pathways?

UFC1 and the ufmylation pathway play critical roles in ER homeostasis:

• The UFL1-DDRGK1 complex is associated with the ER membrane, localizing UFC1 activity to this compartment .

• Ufmylation targets include proteins involved in ER stress response, protein translation, and quality control mechanisms .

• Research approaches to explore these connections include:

  • Cellular models of ER stress with UFC1 modulation

  • Identification of ER-specific substrates using proximity labeling approaches

  • Investigation of UFC1's role in unfolded protein response pathways

Understanding these connections provides insights into fundamental cellular processes and potential therapeutic targets for diseases involving ER dysfunction.

What emerging technologies are advancing UFC1 interaction studies?

Several cutting-edge approaches are transforming UFC1 research:

• AI-powered structural prediction: AlphaFold2 and related tools have revolutionized structural biology by accurately predicting protein interactions, including those mediated by short motifs or disordered regions .

• Integrative structural biology: Combining computational predictions with experimental validation accelerates discovery, as demonstrated by the identification of the UFL1 N-terminal helix interaction with UFC1 .

• Engineered fusion constructs: Simplifying complex multi-protein interactions through rational design of fusion proteins has enabled structural studies that were previously challenging .

• High-resolution interaction mapping: NMR approaches provide detailed insights into binding interfaces and competition dynamics between E1, E2, and E3 enzymes .