Recombinant Salmo salar Ubiquitin-fold modifier-conjugating enzyme 1 (ufc1)

How does the structure of Salmo salar UFC1 compare to UFC1 from other species?

While complete structural comparison data specific to Salmo salar UFC1 is not extensively documented in the provided research, functional studies indicate conservation of key binding regions. UFC1 maintains a binding pocket that interacts with both UFL1 (E3) and UBA5 (E1) across species .

For comprehensive structural comparison:

• Perform sequence alignment using BLAST or Clustal Omega

• Generate structural models using AlphaFold2, which has demonstrated effectiveness in predicting UFC1 interactions

• Compare binding regions, particularly focusing on residues involved in UFL1 and UBA5 interactions

• Analyze conserved functional domains across species

Research has shown that UFC1 uses the same binding site to interact with both UFL1 and UBA5, a feature likely conserved across species based on functional studies .

What is the optimal buffer system for UFC1 binding experiments?

For binding experiments involving UFC1 and fusion constructs, researchers have successfully used:

This buffer system has been validated for UFC1 binding studies, particularly for analyzing interactions with UFL1 fusion proteins . When conducting ITC (Isothermal Titration Calorimetry) experiments to measure binding affinities between UFC1 and binding partners, this buffer system enables detection of interactions with Kd values in the range of 2-30 μM .

How can I prepare charged UFC1 for interaction studies?

Preparation of charged UFC1 (UFC1~UFM1 conjugate) requires the following methodology:

• Materials Required:

  • UFC1 T106S/C116K mutant (25 μM)

  • UBA5 (10 μM)

  • Strep-tagged UFM1 (40 μM)

  • ATP (5 mM)

  • Buffer: 50 mM sodium bicarbonate buffer (pH 9.8), 100 mM NaCl, 10 mM MgCl₂

• Procedure:

  • Incubate the reaction mixture at 37°C for 4 hours

  • Verify formation of isopeptide-linked UFC1-UFM1 covalent complex by SDS-PAGE

  • Purify the complex using strep-tactin superflow column

  • Elute the complex with Desthiobiotin

  • Further purify using Superdex 75 analytical column to separate UFC1-Strep-tagged UFM1 complex from unreacted Strep-tagged UFM1

  • Concentrate the purified complex and store at -80°C

This protocol has been validated for charged UFC1 preparation with approximately 90% purity, suitable for binding studies with DDRGK1-UFL1 fusion proteins .

What methods are suitable for analyzing UFC1 interactions with binding partners?

Multiple complementary techniques have proven effective for studying UFC1 interactions:

• Isothermal Titration Calorimetry (ITC):

  • Provides direct measurement of binding affinity (Kd)

  • Successfully detected UFC1 binding to DDRGK1-UFL1 with Kd = 2.3 μM

  • Can distinguish effects of mutations (e.g., UFC1 K47E mutation increased Kd to 33.8 μM)

• NMR-based Competition Experiments:

  • Use 15N-labeled proteins (e.g., UBA5 C-terminus) to track chemical shift perturbations

  • Effective for demonstrating competitive binding between UFL1 and UBA5 for the same UFC1 binding site

• Pull-down Assays:

  • Utilize Strep-tagged proteins for affinity purification

  • Successfully demonstrated preferential binding of DDRGK1-UFL1 to charged versus uncharged UFC1

• Functional Ufmylation Assays:

  • Charging reaction: Incubate UFC1 with UBA5, UFM1, ATP, and appropriate buffer

  • Discharge reaction: Add lysine and DDRGK1-UFL1 fusion protein

  • Analyze via non-reducing SDS-PAGE to preserve thioester linkages

These complementary approaches provide a comprehensive analysis of UFC1's interactions within the ufmylation pathway.

How does UFC1 function in the ufmylation cascade?

UFC1 serves as the E2 enzyme in the ufmylation pathway, a post-translational modification system similar to ubiquitination. The complete ufmylation cascade involves:

• Activation (E1): UBA5 activates UFM1 in an ATP-dependent manner

• Conjugation (E2): UFC1 accepts activated UFM1 from UBA5 via a thioester bond

• Ligation (E3): UFL1, in complex with DDRGK1, transfers UFM1 from UFC1 to target substrates

UFC1 plays a critical intermediate role, receiving UFM1 from UBA5 and transferring it to UFL1 for subsequent target modification. Structural studies reveal that UFC1 uses the same binding pocket to interact with both UBA5 and UFL1, suggesting a sequential mechanism where these interactions cannot occur simultaneously .

What experimental evidence demonstrates competition between UFL1 and UBA5 for UFC1 binding?

NMR-based competition experiments provide direct evidence for competitive binding:

• When 15N-labeled UBA5 C-terminus (residues 347-404) is combined with UFC1, significant changes occur in the UBA5 1H-15N HSQC spectrum, visible as chemical shift perturbations

• Addition of DDRGK1-UFL1 to this UBA5-UFC1 complex causes NMR cross-peaks to shift back to their unbound positions

• This confirms that UFL1 displaces UBA5 from UFC1, demonstrating they bind to the same surface

This competitive binding mechanism has important implications for the regulation of ufmylation, as it ensures the sequential action of E1, E2, and E3 enzymes in the pathway.

How do mutations in UFC1 affect its binding and catalytic functions?

Mutagenesis studies have revealed critical residues for UFC1 function:

Similarly, mutations in UFL1 (L11R and F15R) completely abolished binding to UFC1. These findings highlight the importance of specific residues in the interaction interface between UFC1 and its binding partners .

For researchers investigating UFC1 structure-function relationships:

• Target the identified binding pocket for mutagenesis

• Consider the dual binding capability (to both UBA5 and UFL1) when designing mutations

• Assess effects on both binding affinity and catalytic activity

What is the significance of UFC1's preferential binding to the charged form of UFC1?

Extended fusion protein experiments revealed that DDRGK1ext-UFL1 (containing the UFM1 binding site in DDRGK1) shows preferential binding to the charged form of UFC1 (UFC1~UFM1):

This finding suggests that the UFM1 binding site on DDRGK1 can bind to UFM1 both in its free form and when charged on UFC1, facilitating preferential binding to the charged form . This mechanism likely enhances the efficiency of the ufmylation pathway by promoting interaction with the catalytically active charged E2 enzyme.

How can AlphaFold2 modeling be applied to study UFC1 interactions?

AlphaFold2 has proven valuable for modeling UFC1 interactions without prior structural information:

• AlphaFold2 successfully identified the binding region on UFL1 in the full-length protein

• The model revealed that the N-terminal helix of UFL1 is critical for UFC1 binding

• Predictions were experimentally validated through ITC and pull-down experiments

• The accuracy of AlphaFold2 in this context demonstrates its value for studying protein-protein interactions in the ufmylation pathway

For researchers using AlphaFold2:

• Model interactions using full-length proteins initially

• Focus on regions with high predicted confidence scores

• Validate computational predictions with experimental approaches (ITC, NMR, mutagenesis)

• Consider modeling interactions with multiple components of the pathway simultaneously

How does the expression of UFC1 change during Atlantic salmon development and under stress conditions?

While the provided search results don't provide specific data on UFC1 expression patterns in Atlantic salmon, research methods established for other Atlantic salmon proteins can be adapted:

• Developmental studies:

  • Analyze UFC1 expression across different life stages (parr, smolt, adult)

  • Compare expression in various tissues using qPCR and proteomics

  • Consider the impact of smoltification, as this represents a major physiological transition

• Stress response evaluation:

  • Examine UFC1 expression under common stressors:

    • Pathogenic challenges (e.g., sea lice infestation, viral infections)

    • Environmental stressors (temperature changes, hypoxia)

    • Nutritional factors (different dietary fat levels)

These approaches would follow established protocols for gene expression analysis in Atlantic salmon, including real-time PCR for transcriptional analysis and proteomic approaches for protein-level changes.

What considerations should be made when expressing recombinant Salmo salar UFC1 in different expression systems?

When expressing recombinant Salmo salar UFC1:

• Expression system selection:

  • Yeast expression has been successfully used for Salmo salar UFC1

  • Consider E. coli for high yield but potential folding issues

  • Mammalian cells may provide better post-translational modifications

  • Baculovirus systems offer advantages for structural studies

• Codon optimization:

  • Adjust codons based on the expression system to enhance yield

  • Consider the high GC content typical of salmonid genes

• Purification strategy:

  • His-tag has been successfully used for Salmo salar UFC1

  • Consider tag position (N vs C-terminal) based on functional domains

  • Optimize purification buffer conditions based on stability testing

• Functional validation:

  • Verify proper folding through activity assays

  • Compare binding parameters with native protein where possible

  • Assess thermal stability through differential scanning fluorimetry

Note that different expression systems may result in variations in price and lead time .

How can structural knowledge of UFC1 be applied to develop research tools?

Structural insights into UFC1 interactions provide opportunities for developing specialized research tools:

• Designer mutants:

  • Create UFC1 mutants with altered binding specificity

  • Develop variants that selectively bind UFL1 but not UBA5 (or vice versa)

  • Engineer "trap" mutants that stabilize interactions for structural studies

• Biosensors:

  • Develop FRET-based sensors to monitor UFC1 interactions in real-time

  • Create split fluorescent protein systems with UFC1 and binding partners

  • Design assays that report on ufmylation pathway activity

• Inhibitors and activators:

  • Design peptide inhibitors targeting the UFC1 binding interface

  • Develop small molecules that modulate UFC1 activity

  • Create conditional systems for pathway regulation

The identification of the competitive binding mechanism between UFL1 and UBA5 for UFC1 provides a foundation for these applications .

What are the implications of comparative studies between UFC1 from different fish species?

Comparative studies of UFC1 across fish species could address several important questions:

• Evolutionary conservation:

  • Analyze sequence conservation in binding regions versus non-binding regions

  • Identify species-specific adaptations in UFC1 structure

  • Map evolutionary changes to functional differences

• Environmental adaptation:

  • Compare UFC1 from fish adapted to different environmental niches

  • Investigate cold adaptation in UFC1 from polar versus tropical species

  • Examine potential differences between freshwater and marine species

• Methodological approach:

  • Sequence numerous fish UFC1 orthologs

  • Perform phylogenetic analysis and calculate evolutionary rates

  • Use ancestral sequence reconstruction to identify key evolutionary transitions

  • Express and characterize recombinant UFC1 from diverse species

This comparative approach would build on established methods for Atlantic salmon research, incorporating techniques from evolutionary biochemistry .