Cyclophilin A E.Coli

What is Cyclophilin A in E. coli and how does it compare to human cyclophilin?

Cyclophilin A in E. coli, also known as rotamase, is a prokaryotic peptidyl-prolyl cis-trans-isomerase that functions as a homolog of human cyclophilin. This enzyme catalyzes the isomerization of peptide bonds preceding proline residues, a rate-limiting step in protein folding. While functionally similar, E. coli cyclophilin shares only 34% sequence identity with human cyclophilin . Despite this limited sequence similarity, both proteins exhibit nearly identical peptidyl-prolyl isomerase activity with comparable catalytic efficiencies (kcat/Km approximately 1.0 × 10^7 M^-1 × S^-1 at 10°C) . The most notable difference is that wild-type E. coli cyclophilin has significantly lower affinity for the immunosuppressant drug cyclosporin A (Kd = 3.4 μM) compared to human cyclophilin (Kd = 17 nM) .

What are the key structural features of E. coli cyclophilin?

E. coli cyclophilin contains a substrate binding site located in a cleft on the surface of the upper sheet of two orthogonal beta-sheets. Within this cleft, the hydrophobic pocket is formed by the side-chains of five non-polar residues: Phe48, Met49, Phe107, Leu108, and Tyr120, with Phe99 at the bottom . Several structural differences exist between E. coli and human cyclophilins, particularly at the periphery of the upper beta-sheet, where conformations of loops L1, L3, and L4 and the segment connecting alpha1 and beta3 differ significantly due to deletions or insertions . The E. coli protein also has a five-residue insertion in a loop that replaces another loop in the human protein, which contributes to the different architecture of the binding site .

Where is E. coli cyclophilin located within the bacterial cell and what is its potential role?

E. coli cyclophilin, encoded by the gene suggested to be called "rot," is primarily located in the periplasm, as determined by spheroplast fractionation of cells harboring the expression vector for the complete rot gene . This periplasmic localization suggests that E. coli cyclophilin could function in refolding of secreted proteins as they cross the bacterial cell membrane . The periplasmic positioning may be strategically important for assisting in the proper folding of proteins that are destined for secretion or membrane insertion. This differs from human Cyclophilin A, which is predominantly cytosolic but can also be found in mitochondria and can migrate to the nucleus under stress conditions .

What are the optimal expression systems for producing recombinant E. coli cyclophilin?

The cDNA encoding cyclophilin can be efficiently expressed in E. coli under control of the tac promoter. Using this system, active cyclophilin can be produced at levels up to 40% of soluble cell protein, making it an exceptionally high-yield expression system . The expression cassette polymerase chain reaction (PCR) has been successfully used for cloning cyclophilin genes, followed by sequence verification and construction of the expression vector . For researchers seeking to express E. coli cyclophilin, optimizing induction conditions (IPTG concentration, temperature, and duration) is essential for maximizing protein yield while maintaining solubility.

What purification strategies yield the highest purity and activity of E. coli cyclophilin?

Due to the high-level expression achieved with the tac promoter system, E. coli cyclophilin can be purified to homogeneity using a single chromatography column approach . This simplified purification protocol typically involves cell lysis followed by a combination of centrifugation to remove cell debris and a single affinity or ion-exchange chromatography step. For assessing purity, SDS-PAGE analysis is recommended, while activity can be confirmed using standard peptidyl-prolyl isomerase assays with tetrapeptide substrates such as succinyl-Ala-Ala-Pro-Phe-p-nitroanilide . Researchers should carefully optimize buffer conditions during purification to maintain enzyme stability and activity.

How can the peptidyl-prolyl isomerase activity of E. coli cyclophilin be measured and quantified?

The standard method for measuring peptidyl-prolyl isomerase activity of E. coli cyclophilin is the chymotrypsin-coupled assay using tetrapeptide substrates such as succinyl-Ala-Ala-Pro-Phe-p-nitroanilide . In this assay, chymotrypsin selectively cleaves after phenylalanine only when the preceding prolyl peptide bond is in the trans conformation, releasing p-nitroanilide which can be monitored spectrophotometrically. Kinetic parameters (kcat and Km) can be determined by varying substrate concentrations and measuring initial velocities at a fixed temperature (typically 10°C to slow the uncatalyzed isomerization rate) . For more detailed mechanistic studies, NMR spectroscopy can be employed to directly observe cis-trans isomerization in real-time with labeled peptide substrates.

What factors affect the catalytic efficiency of E. coli cyclophilin?

The catalytic efficiency (kcat/Km) of E. coli cyclophilin is approximately 1.0 × 10^7 M^-1 × S^-1 at 10°C, which approaches the upper diffusional limit for enzyme-substrate interactions . Several factors can affect this efficiency:

Why does wild-type E. coli cyclophilin have lower affinity for cyclosporin A compared to human cyclophilin?

Wild-type E. coli cyclophilin exhibits significantly lower affinity for cyclosporin A (CsA) (Kd = 3.4 μM) compared to human cyclophilin (Kd = 17 nM) . This difference is primarily attributed to structural variations at the cyclosporin binding site. Specifically:

• The E. coli cyclophilin has a polar glutamine residue (Gln89) positioned along the pathway to the hydrophobic pocket, which appears to prevent access of the hydrophobic portions of cyclosporin A to the binding cleft .

• The architecture of the binding site in E. coli cyclophilin differs substantially from human cyclophilin, particularly at sites distant from the critical tryptophan residue that corresponds to Trp121 in human cyclophilin .

• A five-residue insertion in a loop of E. coli cyclophilin replaces another loop found in human cyclophilin, contributing to the altered binding site geometry .

These structural differences collectively create steric hindrance that impedes cyclosporin A binding to the wild-type E. coli enzyme.

How do mutations in E. coli cyclophilin affect cyclosporin binding and function?

Strategic mutations in E. coli cyclophilin can significantly enhance its ability to bind cyclosporin A. The most well-characterized mutation is F112W, which increases cyclosporin A binding affinity approximately 20-fold (Kd improves from 3.4 μM to 170 nM) . This mutant E. coli cyclophilin not only binds cyclosporin A with higher affinity but also forms a complex that, like the human cyclophilin-cyclosporin A complex, can inhibit the calcium-dependent phosphatase calcineurin .

How does the substrate binding mechanism of E. coli cyclophilin differ from that of human cyclophilin?

The substrate binding mechanism of E. coli cyclophilin shows both similarities and differences compared to human cyclophilin:

• Similarities: Both enzymes form a binding cleft on the surface of the upper sheet of two orthogonal beta-sheets, creating a hydrophobic pocket for the proline ring of the substrate . In both cases, this pocket accommodates the cis-proline isomer more effectively than the trans isomer.

• Differences: The E. coli enzyme binding site has a slightly different composition and architecture. The walls of the hydrophobic pocket are formed by the side-chains of five non-polar residues (Phe48, Met49, Phe107, Leu108, and Tyr120, with Phe99 at the bottom) . The conformations of loops L1, L3, and L4 and the segment connecting alpha1 and beta3 differ significantly from human cyclophilin due to deletions or insertions .

These structural differences result in the E. coli cyclophilin preferentially recognizing either the cis-proline isomer or a highly distorted form of the trans isomer . This preference is attributed to the largely hydrophobic nature of the binding pocket, which allows the cis isomer to bind more firmly than the trans isomer. The distortion of the trans isomer could lead to better binding, but at an energetic cost .

What is the role of cysteine residues in cyclophilin structure and function?

The role of cysteine residues in cyclophilin structure and function has been extensively studied in human cyclophilin. Contrary to earlier hypotheses suggesting that cysteine residues might be essential for catalysis and cyclosporin A binding, site-directed mutagenesis studies have demonstrated that cysteines play no essential role in these functions .

In human cyclophilin, the four cysteines at positions 52, 62, 115, and 161 were mutated individually to alanine, and the purified mutant proteins retained full affinity for cyclosporin A and equivalent catalytic efficiency as a rotamase . These findings ruled out the previously proposed mechanism involving the formation of tetrahedral hemithioorthoamide during catalysis .

Instead, the current understanding favors mechanisms that may involve other tetrahedral intermediates or, alternatively, a mechanism involving distortion of the bound substrate with a twisted (90°) peptidyl-prolyl amide bond . These insights into human cyclophilin are valuable for understanding E. coli cyclophilin function, as the catalytic mechanisms appear to be conserved despite structural differences.

What experimental approaches can be used to study the temperature-dependent functions of cyclophilins in E. coli?

Research has shown that cyclophilins play important roles in temperature adaptation in E. coli. To study these temperature-dependent functions, several experimental approaches can be employed:

• Growth rate analysis: Comparing the growth rates of wild-type, single cyclophilin mutants, and cyclophilin-null strains at various temperatures, particularly at suboptimal temperatures like 16°C, can reveal temperature-dependent phenotypes .

• Protein aggregation assessment: Cyclophilin-null strains exhibit increased autofluorescence when grown at 16°C, indicating a build-up of aggregated proteins within the bacterial cell . Techniques such as fluorescence microscopy, aggregate isolation, and proteomic analysis of aggregates can help characterize this phenotype.

• Proteomic analysis: Mass spectrometry-based approaches can identify proteins that are disrupted in their folding or abundance when cyclophilins are deleted, particularly under temperature stress conditions .

• In vitro isomerase assays at various temperatures: Measuring the peptidyl-prolyl isomerase activity of purified cyclophilins across a temperature range can provide insights into how temperature affects catalytic efficiency.

• Structural studies: X-ray crystallography or NMR studies of cyclophilin at different temperatures can reveal temperature-dependent conformational changes that might affect function.

These approaches can help elucidate how cyclophilins contribute to protein folding and cellular adaptation under various temperature conditions, which is particularly relevant for understanding bacterial stress responses.

How do prokaryotic and eukaryotic cyclophilins differ in their structural and functional properties?

Despite similar catalytic functions, prokaryotic and eukaryotic cyclophilins exhibit several important differences:

These differences reflect evolutionary adaptations to different cellular environments and functional requirements. While the core catalytic mechanism remains conserved, the peripheral structural elements and binding properties have diverged significantly between prokaryotic and eukaryotic cyclophilins.

What insights can E. coli cyclophilin provide for understanding human cyclophilin function in disease?

E. coli cyclophilin serves as a valuable model system for understanding fundamental aspects of peptidyl-prolyl isomerase function that may be relevant to human disease contexts:

What are the unresolved questions regarding E. coli cyclophilin mechanism and function?

Despite significant advances in understanding E. coli cyclophilin, several important questions remain unresolved:

• Precise catalytic mechanism: While it's established that cysteines are not essential for catalysis , the exact mechanism by which E. coli cyclophilin accelerates peptidyl-prolyl isomerization remains debated. The current hypotheses include mechanisms involving other tetrahedral intermediates or substrate distortion with a twisted peptidyl-prolyl amide bond .

• Physiological substrates: The natural protein substrates of E. coli cyclophilin in the periplasm remain largely unidentified. Identifying these substrates would provide insights into the biological role of this enzyme in bacterial physiology.

• Regulatory mechanisms: How the expression and activity of E. coli cyclophilin are regulated in response to environmental stresses (beyond temperature) is not fully understood.

• Potential antimicrobial target: Given the critical role of cyclophilins in protein folding, especially under stress conditions, the potential of E. coli cyclophilin as an antimicrobial target deserves further investigation.

What novel methodologies are emerging for studying cyclophilin function in bacterial systems?

Several innovative approaches are being developed to advance our understanding of cyclophilin function in bacterial systems:

• Cyclophilin-specific inhibitors: Novel cyclosporin A derivatives are being assessed against recombinant proteins and in in vitro cell infection models to determine if enzymatic activity can be inhibited and if a cyclophilin mutant phenotype can be observed .

• Proteome-wide identification of substrates: Advanced proteomic approaches combining crosslinking, immunoprecipitation, and mass spectrometry can identify the full spectrum of cyclophilin substrates in the bacterial cell.

• Single-molecule approaches: Techniques such as single-molecule FRET can provide real-time visualization of cyclophilin-catalyzed isomerization events, offering unprecedented insights into catalytic mechanisms.

• Computational modeling: Molecular dynamics simulations can model the dynamic interactions between cyclophilin and its substrates, helping to elucidate the catalytic mechanism and substrate specificity determinants.

• In vivo protein folding reporters: Engineered proteins that report on folding state in living bacteria can help assess the role of cyclophilins in protein folding under various stress conditions.