Recombinant Bovine E3 UFM1-protein ligase 1 (UFL1), partial

What is UFL1 and what role does it play in the UFMylation pathway?

UFL1 (UFM1-specific ligase 1) functions as the only identified E3 ligase in the UFM1 conjugation system, a novel type of ubiquitin-like modification pathway. Similar to the ubiquitination cascade, UFMylation involves a three-step enzymatic reaction with UBA5 (E1), UFC1 (E2), and UFL1 (E3) . UFL1 is responsible for the final step that transfers the activated UFM1 from UFC1 to target substrates, thereby mediating numerous hormone signaling pathways and endocrine regulation under various physiological and pathological stresses, including ER stress, genotoxic stress, oncogenic stress, and inflammation . As the sole E3 enzyme in this pathway, genetic knockout of UFL1 results in complete loss of UFMylation in cells, underscoring its critical importance in this modification system .

Why is recombinant UFL1 challenging to express and purify?

Expression and purification of recombinant UFL1 present significant challenges due to its inherent instability when expressed alone. Research has shown that UFL1 expressed independently in bacterial systems like Escherichia coli forms soluble aggregates that elute in the void fraction during size exclusion chromatography . Multiple approaches using alternative buffers, additives, and solubility-enhancing tags have proven unsuccessful .

The key breakthrough came with the recognition that UFL1 requires an interacting partner, UFBP1 (also known as DDRGK1), for stability and activity. When co-expressed with UFBP1, UFL1 forms a stable heterodimeric complex that no longer aggregates . This requirement for a binding partner explains why earlier attempts to characterize UFL1 biochemically were challenging, as robust E3 activity is only observed when UFL1 is in complex with UFBP1 .

What are the structural domains of UFL1?

UFL1 possesses a distinctive structural arrangement that was not immediately evident from sequence analysis alone. AlphaFold structural predictions revealed that UFL1 contains:

• An N-terminal helix (amino acids 1-25)

• Five consecutive proteasome component (PCI)-like Wing-Helix (WH) domains

• A sixth partial WH domain

Interestingly, the partial WH domain is completed by a complementary region from UFBP1, which contributes a C-terminal WH and partial WH domain at the interface with UFL1. This structural complementarity explains why UFL1 and UFBP1 are functionally interdependent . The N-terminal helix of UFL1 has been identified as crucial for binding to UFC1 (the E2 enzyme), highlighting its importance in the UFMylation cascade .

What is the optimal approach for expressing and purifying active recombinant bovine UFL1?

Based on current research, the most effective approach for obtaining active recombinant UFL1 involves co-expression with its cofactor UFBP1/DDRGK1. The recommended protocol includes:

• Co-expression system design: Express full-length UFL1 together with UFBP1 lacking its transmembrane sequence in a bacterial expression system .

• Purification strategy: Implement a three-step purification process:

  • Initial affinity chromatography (using a 6xHis tag)

  • Size exclusion chromatography to confirm proper complex formation

  • Additional chromatography step for higher purity

• Validation of complex formation: Verify proper complex formation using techniques such as mass photometry to confirm a 1:1 stoichiometry of UFL1:UFBP1 .

• Alternative fusion approach: For structural studies, researchers have successfully used a fusion construct approach by directly linking portions of DDRGK1 (UFBP1) to UFL1. For example, DDRGK1:87-314-UFL1:1-200 creates a stable construct that maintains functional properties .

These approaches address the inherent instability of UFL1 when expressed alone and enable the production of functional protein for biochemical and structural studies.

How can researchers assess the activity of recombinant UFL1 in vitro?

To evaluate the enzymatic activity of recombinant UFL1, researchers should implement the following methodological approaches:

• Complete UFMylation reconstitution assay:

  • Combine purified UBA5 (E1), UFC1 (E2), UFL1-UFBP1 complex (E3), ATP, and UFM1

  • Include appropriate substrate proteins (e.g., MRE11, Histone H4, or RPL26)

  • Monitor UFM1 conjugation to substrates via Western blotting

• Single turnover discharge assay:

  • Pre-charge UFC1 with UFM1 using UBA5 and ATP

  • Inactivate UBA5 with EDTA to prevent further charging

  • Add UFL1-UFBP1 complex along with free lysine

  • Monitor the discharge rate of UFM1 from UFC1 as a measure of E3 activity

• Binding affinity measurements:

  • Use isothermal titration calorimetry (ITC) to measure binding affinities between:

    • UFL1-UFBP1 complex and UFC1

    • UFL1-UFBP1 complex and charged UFC1 (UFC1~UFM1)

  • Compare these values to understand mechanistic details of the reaction

The table below summarizes binding affinity measurements for different UFL1 constructs:

These data demonstrate that extended constructs containing the UFM1-binding site in DDRGK1 exhibit preferential binding to charged UFC1, providing insights into the mechanistic aspects of the UFMylation cascade .

How does UFL1 compete with UBA5 for binding to UFC1, and what are the implications for the UFMylation cascade?

UFL1 and UBA5 (the E1 enzyme) compete for binding to the same surface on UFC1 (the E2 enzyme), revealing a crucial regulatory mechanism in the UFMylation cascade. This competition has been demonstrated through multiple experimental approaches:

• NMR competition experiments: NMR studies using 15N-labeled UBA5 C-terminus (UBA5 347-404) bound to UFC1 showed that addition of DDRGK1-UFL1 caused NMR cross-peaks in UBA5 to shift to their unbound position, confirming that UFL1 displaces UBA5 from UFC1 .

• Binding interface analysis: Both UBA5 and UFL1 utilize their helical regions to bind to the same pocket on UFC1, with UFL1's N-terminal helix competing with UBA5's C-terminal helix .

• Affinity measurements: The binding affinity of UBA5's C-terminal helix to UFC1 has been reported to be approximately 1-2 μM, which is similar to the affinity of DDRGK1-UFL1 for uncharged UFC1 (Kd ≈ 2.57 μM) .

The resolution to this competition appears to be through preferential binding of UFL1-UFBP1 to charged UFC1 (UFC1~UFM1). Extended constructs of UFL1-UFBP1 that include DDRGK1's UFM1-binding region show a 10-fold increase in affinity for charged UFC1 (Kd ≈ 0.23 μM) compared to uncharged UFC1 . This preferential binding provides a mechanism for UFL1 to outcompete UBA5 after UFC1 has been charged with UFM1, ensuring the directional flow of the UFMylation cascade.

Additional regulatory factors likely include:

• Conformational changes in UFC1 upon UFM1 charging

• Subcellular localization differences (UBA5 and UFC1 are cytosolic, while UFL1 is ER-membrane associated through UFBP1)

This competition mechanism represents a crucial control point in the UFMylation pathway that ensures proper substrate modification.

What is the molecular basis for UFL1-UFBP1 interdependence, and how does this impact experimental approaches?

The interdependence between UFL1 and UFBP1 (DDRGK1) is rooted in their unique structural relationship. AlphaFold structural predictions and subsequent experimental validation have revealed that:

• Complementary structural domains: UFL1 contains five complete Wing-Helix (WH) domains and a sixth partial WH domain. This partial domain is completed by a complementary region from UFBP1, which possesses a C-terminal WH and partial WH domain at the interface with UFL1 .

• Minimal stability requirements: Structure-guided mutagenesis and biochemical reconstitution have demonstrated that these complementary partial WH domains represent the minimal requirement for the expression and purification of stable UFL1-UFBP1 complexes .

• Functional implications: Beyond stability, UFBP1 was initially described as a substrate but is now recognized as playing an essential structural role in substrate UFMylation. Removal of the N-terminal domain of UFBP1 impacts UFMylation of various substrates including MRE11, Histone H4, and RPL26 .

This interdependence has profound implications for experimental approaches:

• Expression strategies: Researchers must co-express UFL1 with UFBP1 to obtain stable, functional protein for biochemical studies .

• Fusion protein design: For structural studies, fusion proteins connecting portions of UFBP1 to UFL1 have proven effective. The crystal structure of this interaction was solved using such a fusion construct .

• Functional analysis: The UFL1-UFBP1 complex functions as a scaffold-type E3 ligase, lacking catalytic cysteines and instead acting analogously to RING-type E3 ligases by bringing together the charged E2 and substrate to activate transfer .

Understanding this interdependence is crucial for researchers designing experiments with recombinant UFL1, as attempts to work with UFL1 alone will likely result in unstable, non-functional protein.

How do disease-causing mutations in the UFMylation pathway affect UFL1 function?

• UFC1 mutations: Disease-causing mutations in UFC1 (the E2 enzyme that works with UFL1) reduce but do not completely abolish the activity of UFC1. This partial retention of activity may explain the survivability of affected individuals .

• Functional consequences: Since UFL1 is the sole E3 enzyme in the UFMylation pathway, any impairment in its function or in the function of its partner enzymes (UBA5, UFC1) would impact all downstream UFMylation events. Genetic knockout of UFL1 results in complete loss of UFMylation in cells .

• Physiological impacts: UFL1-mediated UFMylation plays critical roles in:

  • ER stress responses

  • Genotoxic stress management

  • DNA damage response

  • Protein translation

  • ER homeostasis

Therefore, mutations affecting UFL1 function would likely disrupt these essential cellular processes, contributing to disease pathogenesis.

For researchers studying recombinant bovine UFL1, understanding the impact of mutations would require:

• Structure-guided mutagenesis to create disease-relevant variants

• Functional assays comparing wild-type and mutant UFL1 activity

• Analysis of substrate specificity changes in mutant forms

Such studies could provide valuable insights into the molecular basis of UFMylation-related disorders and potentially identify novel therapeutic targets.

What are the critical factors for successful structural analysis of UFL1-UFBP1 complexes?

Structural characterization of UFL1-UFBP1 complexes presents unique challenges that researchers should address through careful experimental design:

• Construct optimization:

  • For X-ray crystallography, fusion constructs linking portions of DDRGK1 (UFBP1) to UFL1 have proven successful. For example, researchers have solved crystal structures using constructs like DDRGK1:87-314-UFL1:1-200 .

  • For NMR studies, carefully designed fragments focusing on specific interaction domains can overcome size limitations.

• Expression and purification considerations:

  • Ensure co-expression of UFL1 with UFBP1 to maintain stability

  • Implement rigorous quality control via size exclusion chromatography to confirm proper complex formation

  • Consider using techniques like mass photometry to validate 1:1 stoichiometry

• Complementary structural approaches:

  • Leverage computational methods like AlphaFold2 to generate initial models

  • Validate predictions with experimental techniques (X-ray crystallography, NMR)

  • Use biochemical assays to confirm structural insights

• Domain mapping:

  • Focus on the critical N-terminal helix of UFL1 (a.a. 1-25) for understanding UFC1 interactions

  • Analyze the complementary partial WH domains at the UFL1-UFBP1 interface

  • Consider the role of UFBP1's UFM1-binding region in preferential binding to charged UFC1

Researchers who successfully addressed these considerations have made significant breakthroughs in understanding the structural basis of UFL1 function, including solving the first crystal structure of the critical UFL1-UFBP1 interaction .

How can researchers troubleshoot inconsistent activity of recombinant UFL1 in reconstitution assays?

When encountering variable or inconsistent activity with recombinant bovine UFL1 in reconstitution assays, researchers should systematically evaluate the following potential issues:

• Complex formation and stability:

  • Verify proper UFL1-UFBP1 complex formation using size exclusion chromatography

  • Confirm appropriate stoichiometry (ideally 1:1) using techniques like mass photometry

  • Check for protein degradation using SDS-PAGE analysis before each assay

• Component activity verification:

  • Test the activity of each component (UBA5, UFC1, UFM1) individually

  • Confirm that UBA5 can activate UFM1 in an ATP-dependent manner

  • Verify that UFC1 can accept UFM1 from UBA5

  • Use positive controls for each step of the cascade

• Buffer and reaction conditions optimization:

  • Evaluate the effect of different buffer compositions, pH values, and ionic strengths

  • Optimize ATP and magnesium concentrations

  • Consider temperature sensitivity of the components

  • Test different incubation times for each step of the reaction

• Substrate-specific considerations:

  • Different substrates (e.g., MRE11, Histone H4, RPL26) may require specific conditions

  • The N-terminal domain of UFBP1 impacts UFMylation of certain substrates, so ensure appropriate UFBP1 constructs are used

  • Consider whether additional cofactors might be required for specific substrate recognition

• Extended construct testing:

  • If standard UFL1-UFBP1 complexes show inconsistent activity, try extended constructs

  • DDRGK1ext-UFL1 constructs that include the UFM1-binding region of DDRGK1 show preferential binding to charged UFC1 and may enhance activity

By systematically addressing these factors, researchers can identify and resolve sources of variability in UFL1 activity assays, leading to more reproducible and reliable experimental results.

What are the emerging insights into the mechanism of substrate recognition by UFL1-UFBP1 complexes?

Understanding how UFL1-UFBP1 complexes recognize and select specific substrates remains one of the most challenging aspects of UFMylation research. Current evidence suggests a complex mechanism:

• Scaffold-type E3 ligase mechanism: The UFL1-UFBP1 complex functions as a scaffold-type E3 ligase, lacking catalytic cysteines. This is analogous to RING-type E3 ligases, which function by binding both the charged E2 and substrate to facilitate transfer .

• Role of UFBP1 in substrate recognition: Initially described as a substrate itself, UFBP1 plays a crucial role in substrate UFMylation. Removal of the N-terminal domain (NTD) of UFBP1 impacts UFMylation of various substrates including MRE11, Histone H4, and RPL26, suggesting its involvement in substrate recognition or positioning .

• Preferential binding to charged E2: Extended UFL1-UFBP1 constructs that include the UFM1-binding region of DDRGK1 show significantly higher affinity for charged UFC1 (UFC1~UFM1) compared to uncharged UFC1. This suggests a mechanism where the complex preferentially engages with the activated E2 to facilitate UFM1 transfer to substrates .

• Additional binding partners: Research has identified other interactors of UFL1, such as CDK5RAP3 through yeast two-hybrid screening, suggesting potential roles for additional proteins in substrate recognition or regulation . Another partner, LZAP, also possesses a UFM1 binding site, potentially contributing to the substrate recognition mechanism .

• Subcellular localization: UFL1 is associated with the ER membrane through its interaction with DDRGK1, while UFC1 and UBA5 are freely present in the cytosol. This localization may restrict UFL1 activity to specific cellular compartments and therefore limit the pool of potential substrates .

Future research directions should focus on:

• Structural studies of UFL1-UFBP1 in complex with substrates

• Systematic identification of substrate recognition motifs

• Investigation of additional cofactors that may contribute to substrate specificity

How might research on bovine UFL1 inform therapeutic approaches for diseases linked to UFMylation dysfunction?

Research on bovine UFL1 and the UFMylation pathway has significant implications for potential therapeutic approaches targeting UFMylation-related diseases:

• Disease relevance: The UFMylation pathway plays crucial roles in multiple cellular processes including:

  • DNA damage response

  • Protein translation

  • ER homeostasis

  • Response to cellular stresses (ER stress, genotoxic stress, oncogenic stress)

  Disruption of these processes can contribute to various pathological conditions, including cancer, neurodegenerative disorders, and inflammatory diseases.

• Mechanistic insights: Understanding the detailed mechanisms of UFL1 function, particularly:

  • The competition between UFL1 and UBA5 for UFC1 binding

  • The regulatory role of the N-terminal helix of UFL1

  • The structural basis of UFL1-UFBP1 interdependence

  • Preferential binding to charged E2

  These insights provide potential intervention points for therapeutic development .

• Structure-guided drug design opportunities: The elucidation of UFL1's structure, particularly:

  • The N-terminal helix crucial for UFC1 binding

  • The complementary partial WH domains at the UFL1-UFBP1 interface

  • The UFM1-binding site in DDRGK1

  These structural features offer potential targets for small molecule modulators that could either enhance or inhibit UFMylation in a context-dependent manner .

• Biomarker potential: Recombinant bovine UFL1 research provides tools for developing assays to monitor UFMylation activity, which could serve as biomarkers for disease states characterized by UFMylation dysfunction.

• Species conservation considerations: While bovine UFL1 serves as a valuable research model, therapeutic development would require careful translation to human systems, taking into account any species-specific differences in structure or function.

Future therapeutic approaches might include:

• Small molecule inhibitors targeting the UFL1-UFC1 interaction

• Compounds that modulate the UFL1-UFBP1 complex formation

• Strategies to enhance UFMylation in conditions where it is deficient

• Targeted approaches to inhibit UFMylation of specific substrates implicated in disease