UBE2G2 Human

What is UBE2G2 and what is its primary function in cellular metabolism?

UBE2G2 (Ubiquitin-conjugating enzyme E2 G2, also known as UBC7) is an E2 enzyme functioning within the ubiquitin-proteasome system. It plays a crucial role in the endoplasmic reticulum-associated degradation (ERAD) pathway, which identifies and degrades misfolded proteins in the endoplasmic reticulum. Working in concert with cognate E3 ligases, UBE2G2 assembles K48-linked polyubiquitin chains and transfers them to substrate proteins, ultimately leading to their degradation by the proteasome .

The ubiquitination process mediated by UBE2G2 follows a sequential enzymatic cascade:

• Ubiquitin activation by E1 enzyme through ATP-dependent thiolester bond formation

• Transfer of activated ubiquitin to UBE2G2's active site cysteine

• E3 ligase recruitment of both substrate protein and UBE2G2, facilitating polyubiquitin chain assembly and attachment to the substrate

Emerging research suggests that UBE2G2 employs a unique mechanism where it preassembles K48-linked polyubiquitin chains on its active site cysteine before transferring these preassembled chains to substrates in an E3-dependent manner .

What structural characteristics define human UBE2G2?

Human UBE2G2 adopts the canonical E2 enzyme fold with several distinctive features. Both crystallographic (2.56 Å resolution) and NMR spectroscopic studies have revealed its three-dimensional structure, which comprises:

• A single domain consisting of an antiparallel β-sheet with four strands (β1–β4)

• Five α-helices (α1–α5)

• Two 3₁₀-helices (η1 and η2)

• The active site cysteine (Cys89) positioned near one of the 3₁₀-helices (η1)

Analytical ultracentrifugation has confirmed that UBE2G2 exists as a monomer in solution with a molecular weight of approximately 18 kDa .

A particularly significant structural feature revealed by NMR studies is the high mobility of two loop regions (residues 95-107 and 130-135) that flank the active site cysteine. The dynamic nature of these loops suggests they may undergo conformational changes upon interaction with partner proteins during catalysis .

The crystal structure analysis also demonstrated important stabilizing interactions in the protein core, including:

• Hydrophobic interactions among Phe54, Met77, Phe78, Ile154, and Ile158

• A salt bridge between Glu76 and Lys161

Which E3 ligases partner with UBE2G2 and what substrates do they target?

UBE2G2 cooperates with several E3 ubiquitin ligases to facilitate protein degradation via the ERAD pathway. The major known E3 partners include:

Through partnerships with these E3 ligases, UBE2G2 mediates the K48-polyubiquitination and subsequent degradation of diverse substrates including:

• T-Cell Receptor α subunit (TCR-α)

• Thymocyte maturation protein CD3 δ subunit (CD3-δ)

• Human liver cytochrome P450 CYP3A4

• The Pael receptor (implicated in Parkinson's disease)

• Inositol 1,4,5-trisphosphate receptors

These interactions highlight UBE2G2's critical role in cellular protein quality control and various physiological pathways.

How do the dynamic loop regions of UBE2G2 contribute to its catalytic mechanism?

NMR spectroscopy has revealed that two loop regions flanking UBE2G2's active site cysteine (Cys89) display remarkable mobility in solution:

• Loop 1: residues 95-107

• Loop 2: residues 130-135

This high degree of flexibility, confirmed through 15N spin relaxation and residual dipolar coupling analysis, suggests that UBE2G2 likely undergoes significant conformational changes upon binding to protein partners such as E3 ligases, donor ubiquitin, or acceptor substrates .

Within the NMR structural ensemble, specific interactions were observed between His94 and the highly mobile loop residues Asp98 and Asp99. These interactions support a potential catalytic mechanism where His94 functions as a general base activated by the carboxylate side-chains of Asp98 or Asp99 . This arrangement could enhance the nucleophilicity of the active site cysteine or facilitate ubiquitin transfer.

Structural comparisons with other E2-E3 complexes suggest that these loop regions may directly interact with RING domains of E3 ligases. Specifically, the extra loop region of UBE2G2 potentially interacts with both the RING domain and its neighboring regions, contributing to binding specificity and stability of the complex .

Methodologically, researchers can probe the importance of these loops through:

• Site-directed mutagenesis of key loop residues

• Hydrogen-deuterium exchange mass spectrometry to measure loop dynamics

• Cross-linking studies to capture transient interactions

• Molecular dynamics simulations to model conformational changes

What are the key structural and functional differences between human UBE2G2 and yeast Ubc7?

Despite sharing 62% sequence identity, human UBE2G2 and its yeast ortholog Ubc7 exhibit several important differences that may reflect evolutionary adaptations:

The different amino acid composition at positions 76, 154, and 158 results in stronger stabilizing interactions in human UBE2G2 compared to yeast Ubc7:

• Human UBE2G2 forms a salt bridge between Glu76 and Lys161

• UBE2G2 has enhanced hydrophobic interactions involving Phe54, Met77, Phe78, Ile154, and Ile158

These structural distinctions may contribute to differences in stability, flexibility, or interaction specificity between the two orthologs. Researchers can exploit these differences through comparative studies to understand the evolution of ERAD mechanisms across species.

How can researchers optimally express and purify recombinant UBE2G2 for structural and functional studies?

Based on published methodologies, the following optimized protocol can be used to produce high-quality recombinant UBE2G2:

Expression System:

• Construct: Codon-optimized human UBE2G2 gene cloned into a bacterial expression vector

• Vector options:

  • pET-28b with TEV cleavage site replacing thrombin site

  • pET/cMBP-GATEWAY with N-terminal MBP tag and TEV site

• Host: Escherichia coli BL21(DE3)

Purification Protocol:

• For His-tagged constructs:

  • Initial purification via nickel affinity chromatography

  • Tag removal using TEV protease

  • Additional purification as needed

• For MBP-tagged constructs (higher yield):

  • Initial capture on amylose-resin column

  • MBP tag cleavage using His-tagged TEV protease

  • Removal of protease via HisTrap column

  • Further purification via:

    • Ion exchange chromatography (Mono-Q column)

    • Size exclusion chromatography (Superdex 75 column)

Expected Yield: Approximately 8 mg of purified UBE2G2 protein per liter of bacterial culture

For NMR Studies:

• Grow cells in minimal media containing 15N-labeled ammonium chloride and/or 13C-labeled glucose

• Follow purification as above

• Final NMR sample: 1 mM UBE2G2 in buffer containing 20 mM sodium phosphate, 100 mM NaCl, pH 7.0

This methodology provides researchers with highly pure, correctly folded UBE2G2 suitable for crystallization, NMR, enzymatic assays, and protein-protein interaction studies.

What is the role of UBE2G2 in neurodegenerative disorders?

UBE2G2 has been implicated in several neurodegenerative conditions, most notably Parkinson's disease, through its interactions with disease-associated proteins and pathways:

Parkinson's Disease Connection:

• UBE2G2 interacts with Parkin, an E3 ligase encoded by a gene mutated in autosomal recessive juvenile Parkinsonism (AR-JP)

• The UBE2G2-Parkin complex specifically ubiquitinates the Pael receptor

• Pael receptor accumulation is observed in the brains of AR-JP patients

• This suggests that dysfunction in the UBE2G2-Parkin ubiquitination pathway may contribute to disease pathogenesis

Calcium Signaling Regulation:

• UBE2G2 mediates down-regulation of inositol 1,4,5-trisphosphate receptors in neuronal cells

• These receptors are crucial for calcium homeostasis, which is frequently disrupted in neurodegenerative conditions

• Aberrant calcium signaling can trigger neuronal death pathways

Research Methodologies:

• Cell culture models expressing wild-type or mutant Parkin with UBE2G2

• Measurement of Pael receptor ubiquitination and degradation rates

• Analysis of UBE2G2 levels in patient samples

• In vivo studies using UBE2G2 knockout or transgenic models to assess neurodegeneration

Understanding these relationships may provide new therapeutic targets for neurodegenerative diseases by potentially enhancing UBE2G2-mediated clearance of disease-associated proteins.

What techniques are most effective for measuring UBE2G2 enzymatic activity?

Researchers studying UBE2G2 can employ several complementary approaches to assess its enzymatic activity:

1. In vitro Ubiquitination Assays:

• Components required: Purified E1, UBE2G2, appropriate E3 ligase (e.g., gp78 or Parkin), ubiquitin, ATP, buffer system

• Detection methods:

  • Western blotting with anti-ubiquitin antibodies

  • Using fluorescently labeled ubiquitin

  • SDS-PAGE with Coomassie staining to visualize ubiquitin chain formation

2. Thiolester Formation and Discharge Assays:

• Thiolester formation: Monitors the formation of the UBE2G2~Ub thiolester intermediate

  • Detected by non-reducing SDS-PAGE followed by Western blotting

• Discharge assays: Measures the rate at which UBE2G2~Ub transfers ubiquitin to acceptor molecules

  • Provides information about the intrinsic catalytic activity

3. NMR-Based Approaches:

• Advantages: Provides atomic-level details of structural changes during catalysis

• Applications:

  • Study dynamics of loop regions during catalysis

  • Monitor chemical shift perturbations upon ubiquitin binding

  • Track structural changes during interactions with E3 ligases

4. FRET-Based Assays:

• Setup: Using fluorescently labeled ubiquitin and UBE2G2

• Benefits: Allows real-time monitoring of ubiquitin transfer reactions

• Applications: Determining reaction kinetics and affinities

5. Cell-Based Degradation Assays:

• Approach: Measure degradation rates of known UBE2G2 substrates in cells

• Analysis: Western blotting, pulse-chase experiments, or reporter-based systems

• Applications: Assess effects of mutations or inhibitors on UBE2G2 function

These methodological approaches provide a comprehensive toolkit for researchers to investigate the various aspects of UBE2G2 enzymatic function, from basic biochemical properties to cellular roles.

How can researchers investigate the mechanism of K48-linked polyubiquitin chain preassembly by UBE2G2?

The unique ability of UBE2G2 to preassemble K48-linked polyubiquitin chains before transferring them to substrates represents an intriguing mechanistic question. Researchers can investigate this process through:

Biochemical Approaches:

• Chain building assays: Monitor the formation of polyubiquitin chains on the UBE2G2 active site using Western blotting under non-reducing conditions

• Mass spectrometry: Identify the precise structure and length of preassembled chains

• Mutational analysis: Examine the effects of mutations in:

  • Active site residues (Cys89 and surrounding amino acids)

  • Dynamic loop regions (residues 95-107 and 130-135)

  • Putative ubiquitin-binding surfaces

Structural Biology Methods:

• NMR spectroscopy: Monitor chemical shift perturbations as polyubiquitin chains grow on UBE2G2

• Cryo-EM: Capture structural snapshots of UBE2G2 with attached ubiquitin chains

• Hydrogen-deuterium exchange mass spectrometry: Identify regions that change conformation during chain assembly

Advanced Biophysical Techniques:

• Single-molecule FRET: Track the addition of individual ubiquitin molecules to growing chains

• Isothermal titration calorimetry: Measure thermodynamic parameters of ubiquitin addition

Key Controls and Considerations:

• Compare wild-type UBE2G2 with catalytically inactive mutants (e.g., C89A)

• Include parallel experiments with other E2 enzymes that don't perform chain preassembly

• Utilize ubiquitin mutants (e.g., K48R) to confirm linkage specificity

• Examine the effects of E3 ligases on the chain assembly process

What crystallization conditions have proven successful for structural studies of UBE2G2?

The crystal structure of human UBE2G2 was determined at 2.56 Å resolution . Researchers aiming to reproduce or improve upon this structure should consider the following crystallization parameters:

Protein Preparation:

• Construct: Full-length UBE2G2 with minimal cloning artifacts (GGSEF at N-terminus)

• Purity: >95% as assessed by SDS-PAGE

• Concentration: Approximately 10 mg/ml

• Buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl

Crystallization Conditions:

• Method: Hanging-drop vapor diffusion

• Temperature: 20°C

• Drop composition: 1:1 ratio of protein and reservoir solution

• Successful reservoir conditions: Detailed in the following table:

Data Collection Parameters:

• Wavelength: 1.000 Å

• Resolution range: 50-2.56 Å

• Completeness: 97.5% (84.6% in highest resolution shell)

• I/σ(I): 22.2 (4.2 in highest resolution shell)

• Rsym: 5.5% (29.3% in highest resolution shell)

Refinement Statistics:

• Resolution: 49.43-2.56 Å

• Rwork/Rfree: 22.8%/26.2%

• RMSD bond lengths: 0.009 Å

For researchers seeking to obtain co-crystal structures with binding partners, modified approaches may be necessary:

• Consider shorter constructs to reduce loop flexibility

• Explore fusion protein approaches

• Try co-crystallization with stabilizing antibodies or nanobodies

• Attempt cross-linking strategies to capture transient complexes

What structural features distinguish UBE2G2 from other E2 enzymes in the human ubiquitin system?

1. Insertion Loop:

• UBE2G2 contains a characteristic 13-residue sequence insertion downstream from the active site cysteine

• This insertion is shared only with human UBC3 and their yeast orthologs Ubc7p and Cdc34

• May contribute to functional specialization for ERAD pathway roles

2. Dynamic Loop Regions:

• Two highly mobile loops (residues 95-107 and 130-135) flanking the active site

• NMR relaxation data confirms exceptional flexibility in these regions

• This contrasts with many other E2 enzymes that have more rigid active site regions

3. Potential Catalytic Mechanism:

• Interactions between His94 and loop residues Asp98/Asp99 suggest a unique catalytic arrangement

• His94 may function as a general base activated by the carboxylate side-chains of Asp98 or Asp99

• This catalytic setup could be specialized for the polyubiquitin chain preassembly function

4. E3 Binding Interface:

• Structural comparison with UbcH7:E3 complexes reveals both similarities and differences

• The extended loop region unique to UBE2G2 may provide additional E3 contact points

• These differences likely contribute to the selective recognition of specific E3 partners

Researchers can exploit these distinguishing features to develop selective modulators of UBE2G2 function or to engineer E2 enzymes with novel properties for biotechnological applications.

What are the most promising directions for future UBE2G2 research?

Based on current knowledge, several high-priority research directions emerge for advancing our understanding of UBE2G2:

Therapeutic Applications:

• Development of selective UBE2G2 modulators for treating neurodegenerative diseases

• Exploration of UBE2G2 enhancement strategies to improve clearance of disease-associated proteins

• Investigation of UBE2G2's role in other pathological conditions involving protein misfolding

Mechanistic Investigations:

• Comprehensive structural characterization of UBE2G2 in complex with its E3 partners and ubiquitin

• Elucidation of the precise mechanism of polyubiquitin chain preassembly

• Determination of how dynamic loop regions coordinate catalytic activity

• Identification of additional regulatory mechanisms controlling UBE2G2 function

System-Level Studies:

• Global proteomics to identify the complete spectrum of UBE2G2 substrates

• Characterization of the UBE2G2 interactome under various cellular conditions

• Investigation of cross-talk between UBE2G2 and other protein quality control pathways

Technological Innovations:

• Development of UBE2G2-based tools for targeted protein degradation

• Engineering of UBE2G2 variants with enhanced activity or altered specificity

• Creation of biosensors for monitoring UBE2G2 activity in live cells

These research directions will not only advance our fundamental understanding of this important enzyme but may also lead to novel therapeutic strategies for conditions involving protein misfolding and degradation.

How can researchers resolve contradictions in the UBE2G2 literature?

As with many rapidly evolving research areas, studies on UBE2G2 sometimes yield apparently contradictory results. Researchers can address these discrepancies through systematic approaches:

Methodological Standardization:

• Establish consensus protocols for UBE2G2 expression, purification, and activity assays

• Develop reference standards for comparing results across laboratories

• Create community resources such as validated antibodies and cell lines

Comprehensive Meta-Analysis:

• Systematically review existing literature with attention to methodological differences

• Perform statistical analyses to identify factors contributing to discrepant results

• Develop predictive models that accommodate apparently contradictory observations

Collaborative Resolution:

• Form multi-laboratory consortia to replicate key experiments under standardized conditions

• Establish data sharing platforms to facilitate integration of results

• Organize focused workshops to address specific controversies

Technological Refinement:

• Apply cutting-edge techniques to resolve ambiguities:

  • Single-molecule methods to explore heterogeneity in UBE2G2 behavior

  • Cryo-EM to capture transient conformational states

  • Advanced computational modeling to integrate diverse experimental datasets

By embracing these approaches, researchers can transform apparent contradictions into deeper insights about context-dependent mechanisms governing UBE2G2 function.