ERP27 Human

What is human ERp27 and where is it localized?

Human ERp27 (Endoplasmic Reticulum protein 27.7 kDa) is a two-domain protein located in the endoplasmic reticulum that belongs to the protein disulfide isomerase (PDI) family. Despite being a PDI family member, ERp27 lacks the catalytic domains found in other PDI proteins, making it functionally distinct as a non-catalytic member. The protein is homologous to the non-catalytic b and b' domains of protein disulfide isomerase . ERp27 plays a role in protein folding and quality control processes within the endoplasmic reticulum, though our understanding of these critical processes remains incomplete .

What is the domain organization of human ERp27?

Human ERp27 consists of two domains organized in a pattern similar to sections of the protein disulfide isomerase family. Specifically, ERp27 contains:

• An N-terminal domain (b domain) - Highly soluble and stable in solution

• A C-terminal domain (b' domain) - Contains the peptide binding site

Both domains possess the thioredoxin fold characteristic of PDI family members, confirming ERp27's membership in this protein family . The N-terminal domain has been well-characterized structurally, with its coordinates deposited in the Protein Data Bank (PDB code 2L4C) . The full-length structure has also been determined by X-ray crystallography (PDB code 4F9Z) .

What techniques have been used to determine the structure of ERp27?

The structure of ERp27 has been studied using complementary structural biology techniques:

• Nuclear Magnetic Resonance (NMR) Spectroscopy: The solution structure of the N-terminal (b) domain was determined using high-resolution NMR, with the structural data deposited in the Protein Data Bank (PDB code 2L4C). This technique provided detailed information about the domain's three-dimensional arrangement and dynamic properties .

• X-ray Crystallography: The structure of full-length ERp27 was determined by X-ray diffraction (PDB code 4F9Z), providing insight into the relative orientation of the two domains .

• Computational Simulation: Flexible motion simulations were performed to explore the interdomain dynamics of ERp27, particularly focusing on domain movements around the interdomain linker. These simulations used coarse-grained elastic network modeling through ElNemo and geometric simulation using the FRODA algorithm .

The combination of these approaches has been essential for understanding both the static structure and dynamic behavior of ERp27.

What is known about the interdomain flexibility of ERp27?

ERp27 exhibits significant interdomain flexibility, with the b and b' domains able to undergo substantial relative movements. This flexibility has been characterized through several approaches:

• NMR studies comparing the isolated b domain with full-length ERp27 identified a large contiguous surface on the b domain affected by connection to the b' domain, suggesting extensive interdomain contacts .

• Computational simulations revealed that the domains show considerable freedom to flex (tilt and twist) about the interdomain linker. The three lowest-frequency non-trivial normal modes (modes 7, 8, and 9) demonstrated the largest amplitudes of flexible motion, combining rotation about the interdomain axis and tilting around the interdomain interface .

• Analysis of "tilt/twist" plots of interdomain orientations confirmed that the relative orientation of the domains in ERp27 is distinct from that in yeast PDI, with ERp27 showing a tilt of +34° and twist of -33°, compared with PDI bb' showing a tilt of +51° and twist of +53° .

This flexibility appears functionally important, with the interdomain interface observed in NMR studies being much more extensive than that seen in the X-ray structure, suggesting that crystallization selected one specific interdomain orientation from an ensemble of possible conformations .

What is known about ERp27's substrate binding capabilities?

ERp27 has demonstrated specific substrate binding capabilities, particularly through its b' domain:

• Peptide Binding: ERp27 can bind Δ-somatostatin, which is the standard test peptide used for assessing protein disulfide isomerase-substrate binding. This binding ability has been specifically localized to the second (b') domain of ERp27 .

• Conformational Changes: NMR studies have revealed significant conformational changes in the b'-like domain of ERp27 upon substrate binding. Importantly, these changes are not just localized to the binding site but extend more broadly throughout the domain, suggesting allosteric effects induced by substrate binding .

• Binding Site Conservation: Alignment of human ERp27 and human protein disulfide isomerase has allowed for the putative identification of the peptide binding site of ERp27, indicating conservation of the primary substrate binding site location within the PDI family .

The substrate binding properties of ERp27 suggest it may function as a specialized adaptor or chaperone in the endoplasmic reticulum, potentially recognizing specific substrates or substrate states.

What protein-protein interactions has ERp27 been shown to participate in?

The most well-established protein interaction of ERp27 is with ERp57, another member of the PDI family:

• Interaction with ERp57: ERp27 has been shown to bind ERp57 both in vitro and in vivo. This interaction occurs through a mechanism similar to how ERp57 binds calreticulin .

• Binding Site Localization: The binding site for ERp57 has been mapped to the C-terminal (b') domain of ERp27 .

This interaction with ERp57 is particularly interesting as ERp57 is known to function in glycoprotein folding in conjunction with calnexin and calreticulin. The ability of ERp27 to interact with ERp57 suggests it may play a role in modulating or directing ERp57 function within the endoplasmic reticulum, potentially providing an additional layer of regulation to glycoprotein quality control .

What are the challenges in expressing and purifying ERp27 domains?

Researchers working with ERp27 face several domain-specific challenges in protein expression and purification:

• N-terminal (b) Domain: This domain is relatively straightforward to work with. It expresses in high yield, demonstrates high solubility, and exhibits excellent stability in solution, allowing for collection of high-quality 2D and 3D NMR data .

• C-terminal (b') Domain: In contrast, this domain presents significant challenges:

  • Cannot be expressed in isolation as a soluble protein

  • Attempts to derive it from cleavage of fusion constructs have been unsuccessful

  • Generally requires adjacent domains or C-terminal extensions to remain soluble

• Full-length ERp27: While the complete protein can be readily expressed and produces well-dispersed HSQC spectra, it faces stability issues during extended NMR data collection at temperatures above 30°C, preventing collection of usable 3D data sets needed for full assignment and structure calculations .

These challenges are not unique to ERp27, as ligand-binding b'-type domains of the PDI family are generally difficult to generate and study in isolation. Without adjacent domains or suitable extensions, isolated b' domains tend to oligomerize and aggregate, likely due to exposure of hydrophobic ligand-binding sites .

What NMR approaches are effective for studying ERp27 structure and dynamics?

Several NMR approaches have proven valuable for investigating ERp27:

• Backbone Assignment: For the N-terminal domain, comprehensive backbone assignments were achieved with high completion rates:

  • 100% of backbone amide 15N and 1H resonances

  • 100% of 13Cα and 13Cβ resonances

  • 96.7% of 1Hα resonances

• Relaxation Measurements: 15N-NMR relaxation data collection followed by ModelFree analysis revealed:

  • Limited exchange contributions

  • Slow internal motions

  • An average order parameter (S2) of 0.79 for the domain

  • Specific regions of increased mobility

• Comparative HSQC Analysis: Comparison of HSQC spectra between isolated b domain and full-length ERp27 identified surfaces affected by interdomain connections, providing insights into domain interfaces that complemented crystallographic data .

For the challenging b' domain and full-length protein, experiments at lower temperatures (<30°C) and potentially with stabilizing additives may help overcome solubility limitations during long-term data collection .

How does the interdomain flexibility of ERp27 compare to other PDI family members?

The interdomain flexibility observed in ERp27 has been directly compared to that of yeast PDI bb' domains using computational simulation techniques. Both proteins demonstrate significant flexibility, but with distinct characteristics:

• Normal Mode Analysis: The three lowest-frequency non-trivial normal modes (modes 7, 8, and 9) show the largest amplitudes of flexible motion in both proteins, displaying combinations of rotation about the interdomain axis and tilting around the interdomain interface .

• Domain Orientation Differences: Tilt/twist analysis revealed that ERp27 and yeast PDI adopt significantly different relative domain orientations:

  • ERp27: tilt +34° and twist -33°

  • PDI bb': tilt +51° and twist +53°

This comparison suggests that while the mechanism of interdomain flexibility (rotation and tilting) appears conserved across PDI family members, the preferred conformational states differ, potentially reflecting functional specialization. The extensive interdomain flexibility of ERp27 may allow it to accommodate different binding partners or adopt specific conformations required for interaction with ERp57 or substrates .

What is the functional significance of ERp27's conformational changes upon substrate binding?

NMR studies have revealed that substrate binding induces significant conformational changes in the b'-like domain of ERp27. These changes extend beyond the immediate binding site, suggesting that substrate recognition triggers broader structural reorganization . The functional implications of these conformational changes may include:

• Allosteric Regulation: The extended conformational changes could represent an allosteric mechanism that influences interactions with other proteins such as ERp57 after substrate binding.

• Binding Partner Recruitment: Conformational changes might create or expose surfaces that recruit additional factors needed for substrate processing.

• Substrate Retention/Release Control: The structural reorganization could modulate the affinity for substrates, potentially controlling retention time or facilitating handoff to other chaperones.

Understanding the precise functional consequences of these conformational changes remains an active area of investigation and may provide insights into ERp27's role in endoplasmic reticulum protein quality control .

What strategies can improve structural studies of the b' domain of ERp27?

The b' domain of ERp27 presents significant challenges for structural characterization due to its tendency to aggregate when expressed alone. Based on approaches successful with other PDI family members, several strategies may improve structural studies of this domain:

• Fusion Protein Approaches: Creating fusion constructs with solubility-enhancing partners such as MBP (maltose-binding protein) or GST (glutathione S-transferase) may improve expression and solubility.

• Co-expression with Binding Partners: Co-expressing the b' domain with known interacting partners like ERp57 fragments might stabilize the domain.

• Peptide Occupation: Introducing peptides known to bind the b' domain (such as Δ-somatostatin) during expression or purification may occupy the hydrophobic binding site and prevent aggregation .

• Domain Boundary Optimization: Fine-tuning the domain boundaries to include stabilizing elements from adjacent regions without capturing complete neighboring domains could improve solubility.

• Temperature Reduction: As noted in the literature, full-length ERp27 showed stability issues at temperatures above 30°C for NMR studies, suggesting lower temperature work might be beneficial for b' domain studies as well .

These approaches have proven successful for structural studies of similar domains in related PDI family proteins and may translate effectively to ERp27's b' domain.

What experimental approaches could further elucidate ERp27's role in the ER quality control system?

To better understand ERp27's function within the endoplasmic reticulum quality control system, several experimental approaches could be valuable:

• Identification of Physiological Substrates: Techniques such as crosslinking followed by mass spectrometry could identify proteins that interact with ERp27 in vivo, revealing potential physiological substrates.

• Functional Assays in Cellular Models: CRISPR-based knockout or knockdown of ERp27 in relevant cell types, followed by analysis of ER stress markers, protein secretion profiles, or specific substrate folding, could reveal functional consequences of ERp27 deficiency.

• Structure-Guided Mutagenesis: Based on the determined structures, targeted mutations in the b' domain substrate binding site or the ERp57 interaction interface could help dissect the functional importance of these features.

• Integration with Other ER Quality Control Components: Investigation of how ERp27 functions alongside other ER chaperones and folding factors through techniques such as proximity labeling or co-immunoprecipitation could place ERp27 within the broader context of ER proteostasis networks.

• Tissue-Specific Expression Analysis: Comprehensive examination of ERp27 expression patterns across different tissues and cell types could provide clues about specialized functions in particular cellular contexts.

These approaches would complement the existing structural and biochemical data on ERp27 and potentially reveal its precise role in endoplasmic reticulum protein folding and quality control.