Recombinant Ashbya gossypii Peroxisome assembly protein 22 (PEX22)

What is the fundamental role of PEX22 in Ashbya gossypii?

PEX22 in A. gossypii functions as a co-activator for the ubiquitin-conjugating enzyme (E2) PEX4, significantly enhancing its ability to transfer ubiquitin to substrates . Similar to its homologs in other fungi, A. gossypii PEX22 is involved in peroxisome biogenesis and function through the ubiquitination pathway . Specifically, the binding of PEX22 to PEX4 enhances the production of ubiquitinated PEX4 by approximately 2-3 fold, confirming that PEX22 binding influences the transfer of ubiquitin to substrate proteins . This interaction is critical for proper peroxisomal function, which in turn affects various metabolic pathways in this filamentous fungus.

What is known about the PEX4-PEX22 interaction interface?

The interaction between PEX4 and PEX22 has been characterized as direct with a 1:1 stoichiometry, confirmed through isothermal titration microcalorimetry (ITC) . The crystal structure of the PEX415–183:PEX22S complex has been resolved at 2.6 Å resolution . PEX4 adopts a typical UBC (ubiquitin-conjugating) fold containing a core domain of four anti-parallel β-strands (β1–β4), one α-helix (α2), a small 310 helix, an N-terminal helix (α1), and a helix-turn-helix (α3–α4) at the C-terminus . The active site cysteine (Cys115) is situated in a cleft formed by the 310-helix and the loop region between α2 and α3 . Notably, PEX22 binds to PEX4 through a binding site that does not overlap with any other known interaction interface in E2 enzymes, representing a unique mode of E2 regulation .

What expression systems are optimal for recombinant A. gossypii PEX22 production?

For the expression of recombinant A. gossypii PEX22, several important factors should be considered based on experimental evidence. When expressing heterologous proteins in A. gossypii itself, the choice of promoter significantly impacts expression levels . While initial attempts using the S. cerevisiae PGK1 (ScPGK1) promoter yielded inefficient results, substituting it with native A. gossypii promoters from AgTEF and AgGPD improved recombinant protein secretion by up to 8-fold . For heterologous expression of A. gossypii PEX22 in other systems, E. coli has been successfully used for producing the soluble domain (PEX22S) for structural studies . The methodology requires optimization of induction conditions, with IPTG concentration and induction temperature being critical parameters for obtaining correctly folded protein.

What purification strategies yield the highest purity of functional recombinant PEX22?

For high-purity isolation of recombinant A. gossypii PEX22, a multi-step chromatographic approach is recommended. Based on protocols used for structural studies , the following methodology has proven effective:

• Initial capture using affinity chromatography (His-tag or GST-tag approaches)

• Tag removal using a specific protease (TEV protease for His-tags)

• Secondary purification step using ion-exchange chromatography

• Final polishing step with size-exclusion chromatography

Protein purity should be assessed by SDS-PAGE and Western blotting. For functional studies, it's critical to verify that the purified PEX22 retains its ability to enhance PEX4 activity, which can be tested using ubiquitination assays measuring the increase in PEX4 self-ubiquitination in the presence of PEX22 . The presence of PEX22S enhances production of Ub-PEX4 by approximately 2-3 fold, providing a quantitative measure of functional activity .

How can yield and stability of recombinant A. gossypii PEX22 be optimized?

Optimization of yield and stability for recombinant A. gossypii PEX22 requires attention to several key factors. For expression in A. gossypii itself, the carbon source used in culture media significantly affects protein production, with glycerol yielding approximately 1.5-fold higher recombinant protein output compared to glucose . When expressing the protein in E. coli or other heterologous systems, stability can be improved by:

• Expressing specific domains (such as PEX22S) rather than full-length protein

• Including stabilizing additives (glycerol, non-ionic detergents) in buffer solutions

• Optimizing pH and ionic strength based on the protein's theoretical isoelectric point

• Storage at -80°C with flash-freezing in the presence of cryoprotectants

For crystallization purposes, buffer screening is essential to identify conditions that maintain protein solubility while promoting crystal formation .

What crystallization approaches have been successful for A. gossypii PEX22?

Successful crystallization of A. gossypii PEX22 (specifically the soluble domain, PEX22S) has been achieved in complex with PEX415-183 . The crystal structure was determined at 2.6 Å resolution using multiple wavelength anomalous dispersion (MAD) phasing techniques . The crystals were formed in the C2 space group with cell dimensions a=139.3 Å, b=43.1 Å, c=60.4 Å, α=90°, β=100.6°, γ=90° . Researchers should note that crystals for MAD phasing had slightly different cell dimensions: a=138.8 Å, b=43.1 Å, c=60.2 Å, α=90°, β=100.7°, γ=90° .

A comprehensive approach to crystallization would include:

• Initial screening using sparse matrix commercial screens

• Optimization of promising conditions by varying precipitant concentration, pH, and additives

• Seeding techniques to improve crystal quality

• Co-crystallization with PEX4 to stabilize the complex

What biophysical methods are most informative for studying PEX22-PEX4 interactions?

Several biophysical methods have proven valuable for characterizing the PEX22-PEX4 interaction:

• Isothermal Titration Calorimetry (ITC): Directly quantifies the interaction between PEX415–183 and PEX22S, confirming a 1:1 stoichiometry and providing thermodynamic parameters of binding .

• X-ray Crystallography: Provides atomic-level details of the interaction interface, as demonstrated by the 2.6 Å resolution structure of the PEX415–183:PEX22S complex .

• Functional Enzyme Assays: Demonstrates that PEX22S enhances PEX4's self-ubiquitination activity by 2-3 fold, providing functional confirmation of the interaction .

• B-factor Analysis: Examination of residue-average B-factors in the co-crystal structure reveals flexible "hotspots" that may be important for function .

For researchers studying this interaction, combining structural techniques with functional assays provides the most comprehensive understanding of how PEX22 enhances PEX4 activity.

How do mutations in key residues affect PEX22 structure and function?

Mutational analysis of key residues in PEX22 can provide valuable insights into structure-function relationships. While specific mutational data for A. gossypii PEX22 is limited in the provided search results, the approach should focus on:

• Residues at the PEX22-PEX4 interface identified from the crystal structure

• Conserved residues identified through sequence alignment with PEX22 from other species

• Residues in flexible regions (high B-factors) that might play roles in conformational changes

Mutations should be assessed for their effects on:

• Binding affinity to PEX4 (measured by ITC or surface plasmon resonance)

• Enhancement of PEX4 ubiquitination activity

• Protein stability and folding

• In vivo function (complementation assays in pex22Δ strains)

This systematic approach allows correlation of structural features with functional roles.

How can PEX22's enhancement of PEX4 activity be quantitatively measured?

The enhancement of PEX4 activity by PEX22 can be quantitatively assessed through in vitro ubiquitination assays. A well-established approach uses self-ubiquitination of PEX4 as a readout . The protocol involves:

• Setting up reaction mixtures containing purified E1 enzyme, PEX415–183, ubiquitin, and ATP

• Running parallel reactions with and without PEX22S

• Monitoring the formation of ubiquitinated PEX4 species over time by SDS-PAGE and Western blotting

• Quantifying the relative amounts of mono- and poly-ubiquitinated PEX4 species

Using this approach, researchers have observed that PEX22S enhances production of Ub-PEX415–183 by approximately 2-3 fold . Additionally, PEX22S promotes the formation of higher molecular mass species corresponding to PEX415–183 attached to multiple ubiquitin moieties (2, 3, or 4 Ub molecules) . For more specific analysis of the initial ubiquitin transfer rate, the K48R form of ubiquitin can be used to block chain formation, allowing measurement of just the mono-ubiquitination rate .

What is the role of PEX22 in peroxisome biogenesis in A. gossypii?

While the search results don't provide specific details about the role of PEX22 in A. gossypii peroxisome biogenesis, we can infer its function based on homology with S. cerevisiae and other fungi. PEX22 is expected to act as a membrane anchor and activator for PEX4, which is involved in the ubiquitination of PEX5, a key peroxisomal import receptor .

A comprehensive investigation of PEX22's role in A. gossypii peroxisome biogenesis would include:

• Generation of pex22Δ strains and characterization of their peroxisomal phenotypes

• Fluorescence microscopy using peroxisomal marker proteins

• Biochemical fractionation to assess peroxisome integrity and protein import

• Complementation studies with wild-type and mutant forms of PEX22

• Analysis of PEX5 recycling and ubiquitination status in pex22Δ strains

These approaches would help establish whether A. gossypii PEX22 functions similarly to its homologs in other fungi or has unique species-specific roles.

How does the PEX22-PEX4 interaction differ from other E2-coactivator systems?

The PEX22-PEX4 interaction represents a unique E2-coactivator system with several distinguishing features:

• Novel Binding Interface: PEX22 binds to PEX4 through a binding site that does not overlap with any other known interaction interface in E2 enzymes , representing a unique mode of E2 regulation.

• Functional Effect: Unlike some E2-partner interactions that modify substrate specificity, PEX22S primarily enhances the rate of ubiquitin transfer from PEX4 to substrates , increasing activity by 2-3 fold.

• Structural Features: The PEX22S domain exhibits a potentially novel fold with some similarity to Rossmann-like structures, but with distinctive features .

• Membrane Association: In the context of peroxisomes, PEX22 is a membrane protein that recruits and activates the soluble PEX4 , creating a specialized microenvironment for ubiquitination reactions.

Understanding these unique features of the PEX22-PEX4 system provides insights into diverse mechanisms of E2 enzyme regulation beyond the more commonly studied E2-E3 interactions.

How can recombinant A. gossypii PEX22 improve protein production systems?

A. gossypii has emerged as a promising host for heterologous protein production, and understanding PEX22's role may contribute to improving these systems . While direct applications of recombinant PEX22 itself are not extensively documented, several approaches can be considered:

• Engineering improved peroxisome function: Optimizing PEX22 expression or creating modified versions with enhanced PEX4 activation could improve peroxisomal function, which may benefit certain recombinant protein production scenarios, especially for proteins that require peroxisomal post-translational modifications.

• Leveraging regulatory elements: The promoter regions of PEX22 could be harnessed for developing expression systems, especially if they respond to specific metabolic conditions relevant to protein production.

• Enhancing secretion pathways: Understanding how peroxisome biogenesis interacts with secretory pathways in A. gossypii could lead to improved secretion of recombinant proteins, building upon the already demonstrated ability of A. gossypii to secrete native and heterologous enzymes to the extracellular medium .

Current evidence shows that optimizing A. gossypii's native promoters (such as AgTEF and AgGPD instead of heterologous promoters) can improve recombinant protein production by up to 8-fold , indicating the importance of species-specific regulatory elements.

What methodological approaches can improve the study of ubiquitination pathways using the PEX22-PEX4 system?

The PEX22-PEX4 system offers a valuable model for studying ubiquitination pathways. Methodological improvements include:

• Reconstituted in vitro systems: Developing fully reconstituted ubiquitination systems containing purified E1, PEX4, PEX22, and physiological substrates to study the complete reaction pathway.

• Structural biology approaches: Building on the existing crystal structure of the PEX415–183:PEX22S complex to capture additional conformational states, particularly those representing different stages of the ubiquitin transfer reaction.

• Single-molecule techniques: Applying single-molecule FRET or other single-molecule approaches to monitor the dynamics of the PEX22-PEX4 interaction and conformational changes during ubiquitin transfer.

• Chemical biology tools: Utilizing activity-based probes, photo-crosslinking, and other chemical biology approaches to trap transient intermediates in the ubiquitination pathway.

• Mass spectrometry-based proteomics: Developing improved methods for identifying ubiquitination sites and quantifying ubiquitination levels in complex samples.

These methodological advances would not only improve understanding of the PEX22-PEX4 system but could have broader applications in studying ubiquitination pathways in general.

How can structural insights from PEX22 be applied to engineer novel protein interaction modules?

The unique structural features of PEX22 and its interaction with PEX4 provide a foundation for engineering novel protein interaction modules:

• Design of synthetic E2 activators: Using the PEX22-PEX4 interface as a template, researchers could design novel peptides or proteins that selectively activate specific E2 enzymes, potentially creating tools for manipulating ubiquitination pathways.

• Scaffold development: The mixed β-sheet/α-helical fold of PEX22S could serve as a scaffold for presenting functional peptides or protein domains in specific orientations, creating novel binding or catalytic modules.

• Interface transplantation: Key interaction residues from the PEX22-PEX4 interface could be grafted onto other protein scaffolds to create new protein pairs with specific interaction properties.

• Allosteric regulators: Understanding how PEX22 binding enhances PEX4 activity could inform the design of allosteric regulators for other enzymes, particularly those where direct active site modification is challenging.

The crystallographic data showing PEX22S adopting a fold with five parallel β-strands (β3–β2–β1–β4–β5) forming a central β-sheet sandwiched by eight α-helices provides structural details essential for these engineering efforts.

What are the main obstacles in crystallizing full-length A. gossypii PEX22 and how can they be overcome?

Crystallizing full-length membrane proteins like A. gossypii PEX22 presents several challenges:

• Membrane domain insolubility: The membrane-spanning region of PEX22 is hydrophobic and difficult to solubilize without disrupting native structure. This challenge can be addressed by:

  • Using mild detergents optimized through detergent screening

  • Employing lipidic cubic phase crystallization methods

  • Creating fusion proteins with crystallization chaperones

  • Utilizing polymer-bounded nanodiscs to maintain a native-like lipid environment

• Conformational heterogeneity: Full-length PEX22 may exhibit multiple conformations, hindering crystal formation. Approaches to address this include:

  • Introducing stabilizing mutations based on sequence analysis

  • Using conformation-specific antibody fragments as crystallization aids

  • Employing chemical cross-linking to trap specific conformations

• Limited quantity: Obtaining sufficient amounts of pure, homogeneous full-length PEX22 is challenging. Solutions include:

  • Optimizing expression in eukaryotic systems rather than bacteria

  • Using A. gossypii itself as an expression host with its native promoters

  • Implementing high-cell-density fermentation techniques

Previous successful crystallization of the soluble domain (PEX22S) in complex with PEX415-183 at 2.6 Å resolution provides a foundation for these more challenging approaches.

How can contradictory functional data for PEX22 be reconciled across different experimental systems?

When facing contradictory functional data for PEX22 across different experimental systems, researchers should consider:

• Species-specific differences: While the core function of PEX22 as a PEX4 co-activator may be conserved, details of this interaction might differ between species like S. cerevisiae and A. gossypii. Systematic comparative studies using PEX22 from multiple species in identical assay conditions can help identify truly conserved versus species-specific features.

• Experimental context variations: Different in vitro assay conditions (buffer composition, temperature, protein concentrations) can significantly affect observed activities. Standardizing key parameters across laboratories and reporting detailed methods is essential.

• In vitro versus in vivo discrepancies: Activities observed in purified systems may not fully reflect in vivo function where additional factors may be present. Complementary approaches combining in vitro biochemistry with in vivo genetic studies provide more comprehensive understanding.

• Protein preparation differences: Variations in protein expression, purification methods, and storage can affect protein activity and lead to apparently contradictory results. Careful quality control of protein preparations, including assessment of proper folding and absence of aggregation, is critical.

• Data integration approaches: Employing statistical meta-analysis techniques to integrate data from multiple studies can help identify consistent trends despite experimental variability.

What are the best approaches for studying PEX22 in the context of the entire peroxisomal importomer complex?

Studying PEX22 within the context of the entire peroxisomal importomer complex requires sophisticated approaches:

• Proximity labeling techniques: Methods such as BioID or APEX2 can be used with PEX22 as the bait protein to identify proximal interactions within the native peroxisomal membrane environment.

• Cryo-electron tomography: This technique can visualize the peroxisomal importomer in situ, potentially capturing PEX22 in its native context within the membrane and in association with other peroxins.

• Reconstitution systems: Development of in vitro systems with purified components assembled on artificial membranes (liposomes or nanodiscs) to reconstitute functional peroxisomal protein import.

• Integrated structural biology: Combining X-ray crystallography data from individual components (like the PEX415–183:PEX22S complex ) with lower-resolution techniques like small-angle X-ray scattering (SAXS) or cryo-EM to model the entire importomer architecture.

• Genetic interaction mapping: Systematic analysis of genetic interactions between PEX22 and other peroxins can reveal functional relationships within the importomer complex.

• Quantitative proteomics: Techniques like SILAC or TMT labeling combined with mass spectrometry to quantify changes in the peroxisomal proteome when PEX22 function is altered.

These approaches collectively would provide a more comprehensive understanding of how PEX22 functions within the larger context of peroxisomal protein import machinery.