Structural Basis for Cyclosporin Isoform-Specific Inhibition of Cyclophilins from Toxoplasma gondii.

Cyclosporin (CsA) has antiparasite activity against the human pathogen Toxoplasma gondii. A possible mechanism of action involves CsA binding to T. gondii cyclophilins, although much remains to be understood. Herein, we characterize the functional and structural properties of a conserved (TgCyp23) and a more divergent (TgCyp18.4) cyclophilin isoform from T. gondii. While TgCyp23 is a highly active cis-trans-prolyl isomerase (PPIase) and binds CsA with nanomolar affinity, TgCyp18.4 shows low PPIase activity and is significantly less sensitive to CsA inhibition. The crystal structure of the TgCyp23:CsA complex was solved at the atomic resolution showing the molecular details of CsA recognition by the protein. Computational and structural studies revealed relevant differences at the CsA-binding site between TgCyp18.4 and TgCyp23, suggesting that the two cyclophilins might have distinct functions in the parasite. These studies highlight the extensive diversification of TgCyps and pave the way for antiparasite interventions based on selective targeting of cyclophilins.

Serine 106 preserves the tertiary structure, function, and stability of a cyclophilin from Staphylococcus aureus.

SaCyp, a staphylococcal cyclophilin involved in both protein folding and pathogenesis, has a Ser residue at position 106 and a Trp residue at position 136. While Ser 106 of SaCyp aligned with a cyclosporin A (CsA) binding Ala residue, its Trp 136 aligned with a Trp or a Phe residue of most other cyclophilins. To demonstrate the exact roles of Ser 106 and Trp 136 in SaCyp, we have elaborately studied rCyp[S106A] and rCyp[W136A], two-point mutants of a recombinant SaCyp (rCyp) harboring an Ala substitution at positions 106 and 136, respectively. Of the mutants, rCyp[W136A] showed the rCyp-like CsA binding affinity and peptidyl-prolyl cis-trans isomerase (PPIase) activity. Conversely, the PPIase activity, CsA binding affinity, stability, tertiary structure, surface hydrophobicity, and Trp accessibility of rCyp[S106A] notably differed from those of rCyp. The computational experiments also reveal that the structure, dimension, and fluctuation of SaCyp are not identical to those of SaCyp[S106A]. Furthermore, Ser at position 106 of SaCyp, compared to Ala at the same position, formed a higher number of non-covalent bonds with CsA. Collectively, Ser 106 is an indispensable residue for SaCyp that keeps its tertiary structure, function, and stability intact.Communicated by Ramaswamy H. Sarma.

Chaperoning activity of the cyclophilin family prevents tau aggregation.

Tauopathies, such as Alzheimer's disease, are characterized by the misfolding and progressive accumulation of the microtubule associated protein tau. Chaperones, tasked with maintaining protein homeostasis, can become imbalanced with age and contribute to the progression of neurodegenerative disease. Cyclophilins are a promising pool of underinvestigated chaperones with peptidyl-prolyl isomerase activity that may play protective roles in regulating tau aggregation. Using a Thioflavin T fluorescence-based assay to monitor in vitro tau aggregation, all eight cyclophilins, which include PPIA to PPIH prevent tau aggregation, with PPIB, PPIC, PPID, and PPIH showing the greatest inhibition. The low thermal stability of PPID and the strong heparin binding of PPIB undermines the simplistic interpretation of reduced tau aggregation. In a cellular model of tau accumulation, all cyclophilins, except PPID and PPIH, reduce insoluble tau. PPIB, PPIC, PPIE, and PPIF also reduce soluble tau levels with PPIC exclusively protecting cells from tau seeding. Overall, this study demonstrates cyclophilins prevent tau fibril formation and many reduce cellular insoluble tau accumulation with PPIC having the greatest potential as a molecular tool to mitigate tau seeding and accumulation.

Discovery and molecular basis of subtype-selective cyclophilin inhibitors.

Although cyclophilins are attractive targets for probing biology and therapeutic intervention, no subtype-selective cyclophilin inhibitors have been described. We discovered novel cyclophilin inhibitors from the in vitro selection of a DNA-templated library of 256,000 drug-like macrocycles for cyclophilin D (CypD) affinity. Iterated macrocycle engineering guided by ten X-ray co-crystal structures yielded potent and selective inhibitors (half maximal inhibitory concentration (IC50) = 10 nM) that bind the active site of CypD and also make novel interactions with non-conserved residues in the S2 pocket, an adjacent exo-site. The resulting macrocycles inhibit CypD activity with 21- to >10,000-fold selectivity over other cyclophilins and inhibit mitochondrial permeability transition pore opening in isolated mitochondria. We further exploited S2 pocket interactions to develop the first cyclophilin E (CypE)-selective inhibitor, which forms a reversible covalent bond with a CypE S2 pocket lysine, and exhibits 30- to >4,000-fold selectivity over other cyclophilins. These findings reveal a strategy to generate isoform-selective small-molecule cyclophilin modulators, advancing their suitability as targets for biological investigation and therapeutic development.

Non-Immunosuppressive Cyclophilin Inhibitors.

Cyclophilins, enzymes with peptidyl-prolyl cis/trans isomerase activity, are relevant to a large variety of biological processes. The most abundant member of this enzyme family, cyclophilin A, is the cellular receptor of the immunosuppressive drug cyclosporine A (CsA). As a consequence of the pathophysiological role of cyclophilins, particularly in viral infections, there is a broad interest in cyclophilin inhibition devoid of immunosuppressive activity. This Review first gives an introduction into the physiological and pathophysiological roles of cyclophilins. The presentation of non-immunosuppressive cyclophilin inhibitors will commence with drugs based on chemical modifications of CsA. The naturally occurring macrocyclic sanglifehrins have become other lead structures for cyclophilin-inhibiting drugs. Finally, de novo designed compounds, whose structures are not derived from or inspired by natural products, will be presented. Relevant synthetic concepts will be discussed, but the focus will also be on biochemical studies, structure-activity relationships, and clinical studies.

Cyclophilins and Their Functions in Abiotic Stress and Plant-Microbe Interactions.

Plants have developed a variety of mechanisms and regulatory pathways to change their gene expression profiles in response to abiotic stress conditions and plant-microbe interactions. The plant-microbe interaction can be pathogenic or beneficial. Stress conditions, both abiotic and pathogenic, negatively affect the growth, development, yield and quality of plants, which is very important for crops. In contrast, the plant-microbe interaction could be growth-promoting. One of the proteins involved in plant response to stress conditions and plant-microbe interactions is cyclophilin. Cyclophilins (CyPs), together with FK506-binding proteins (FKBPs) and parvulins, belong to a big family of proteins with peptidyl-prolyl cis-trans isomerase activity (Enzyme Commission (EC) number 5.2.1.8). Genes coding for proteins with the CyP domain are widely expressed in all organisms examined, including bacteria, fungi, animals, and plants. Their different forms can be found in the cytoplasm, endoplasmic reticulum, nucleus, chloroplast, mitochondrion and in the phloem space. They are involved in numerous processes, such as protein folding, cellular signaling, mRNA processing, protein degradation and apoptosis. In the past few years, many new functions, and molecular mechanisms for cyclophilins have been discovered. In this review, we aim to summarize recent advances in cyclophilin research to improve our understanding of their biological functions in plant defense and symbiotic plant-microbe interactions.

p53 Is Potentially Regulated by Cyclophilin D in the Triple-Proline Loop of the DNA Binding Domain.

The multifunctional protein p53 is the central molecular sensor of cellular stresses. The canonical function of p53 is to transcriptionally activate target genes in response to, for example, DNA damage that may trigger apoptosis. Recently, p53 was also found to play a role in the regulation of necrosis, another type of cell death featured by the mitochondrial permeability transition (mPT). In this process, p53 directly interacts with the mPT regulator cyclophilin D, the detailed mechanism of which however remains poorly understood. Here, we report a comprehensive computational investigation of the p53-cyclophilin D interaction using molecular dynamics simulations and associated analyses. We have identified the specific cyclophilin D binding site on p53 that is located at proline 151 in the DNA binding domain. As a peptidyl-prolyl isomerase, cyclophilin D binds p53 and catalyzes the cis-trans isomerization of the peptide bond preceding proline 151. We have also characterized the effect of such an isomerization and found that the p53 domain in the cis state is overall more rigid than the trans state except for the local region around proline 151. Dynamical changes upon isomerization occur in both local and distal regions, indicating an allosteric effect elicited by the isomerization. We present potential allosteric communication pathways between proline 151 and distal sites, including the DNA binding surface. Our work provides, for the first time, a model for how cyclophilin D binds p53 and regulates its activity by switching the configuration of a specific site.

Structure of the activated human minor spliceosome.

The minor spliceosome mediates splicing of the rare but essential U12-type precursor messenger RNA. Here, we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-angstrom resolution. The 5' splice site and branch point sequence of the U12-type intron are recognized by the U6atac and U12 small nuclear RNAs (snRNAs), respectively. Five newly identified proteins stabilize the conformation of the catalytic center: The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome, the RBM48-ARMC7 complex binds the γ-monomethyl phosphate cap at the 5' end of U6atac snRNA, the U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 small nuclear ribonucleoprotein (snRNP), and CRIPT stabilizes U12 snRNP. Our study provides a framework for the mechanistic understanding of the function of the human minor spliceosome.

Structural and functional analyses of the PPIase domain of Arabidopsis thaliana CYP71 reveal its catalytic activity toward histone H3.

Arabidopsis thaliana CYP71 (AtCYP71) is a chromatin-remodeling protein that promotes shoot apical meristem (SAM) differentiation. The N terminus of AtCYP71 contains a noncanonical WD domain, and the C terminus contains an enzymatic peptidyl-prolyl isomerase (PPIase) cyclophilin (CYP) domain. To date, there has been no characterization of CYP71, and its mode of action remains unknown. Here, we report the crystal structure of the CYP domain of AtCYP71 at 1.9 Å resolution. The structure shows key differences when compared to the canonical CYP fold of human CypA. To the best our knowledge, this is the first A. thaliana CYP structure with a conserved active site loop. Using nuclear magnetic resonance spectroscopy, we demonstrate that the CYP domain is active toward histone H3. Our findings suggest that the PPIase activity of the CYP domain is important for the function of AtCYP71 in chromatin remodeling during organogenesis.

N-Terminal Segment of TvCyP2 Cyclophilin from Trichomonas vaginalis Is Involved in Self-Association, Membrane Interaction, and Subcellular Localization.

In Trichomonas vaginalis (T. vaginalis), cyclophilins play a vital role in dislodging Myb proteins from the membrane compartment and leading them to nuclear translocation. We previously reported that TvCyP1 cyclophilin from T. vaginalis forms a dimer and plays an essential role in moving the Myb1 transcription factor toward the nucleus. In comparison, TvCyP2 containing an extended segment at the N-terminus (N-terminal segment) formed a monomer and showed a different role in regulating protein trafficking. Four X-ray structures of TvCyP2 were determined under various conditions, all showing the N-terminal segment interacting with the active site of a neighboring TvCyP2, an unusual interaction. NMR study revealed that this particular interaction exists in solution as well and also the N-terminal segment seems to interact with the membrane. In vivo study of TvCyP2 and TvCyP2-∆N (TvCyP2 without the N-terminal segment) indicated that both proteins have different subcellular localization. Together, the structural and functional characteristics at the N-terminal segment offer valuable information for insights into the mechanism of how TvCyP2 regulates protein trafficking, which may be applied in drug development to prevent pathogenesis and disease progression in T. vaginalis infection.

Structural and functional insights into CWC27/CWC22 heterodimer linking the exon junction complex to spliceosomes.

Human CWC27 is an uncharacterized splicing factor and mutations in its gene are linked to retinal degeneration and other developmental defects. We identify the splicing factor CWC22 as the major CWC27 partner. Both CWC27 and CWC22 are present in published Bact spliceosome structures, but no interacting domains are visible. Here, the structure of a CWC27/CWC22 heterodimer bound to the exon junction complex (EJC) core component eIF4A3 is solved at 3Å-resolution. According to spliceosomal structures, the EJC is recruited in the C complex, once CWC27 has left. Our 3D structure of the eIF4A3/CWC22/CWC27 complex is compatible with the Bact spliceosome structure but not with that of the C complex, where a CWC27 loop would clash with the EJC core subunit Y14. A CWC27/CWC22 building block might thus form an intermediate landing platform for eIF4A3 onto the Bact complex prior to its conversion into C complex. Knock-down of either CWC27 or CWC22 in immortalized retinal pigment epithelial cells affects numerous common genes, indicating that these proteins cooperate, targeting the same pathways. As the most up-regulated genes encode factors involved in inflammation, our findings suggest a possible link to the retinal degeneration associated with CWC27 deficiencies.

Identification of Salmonella Typhimurium Peptidyl-prolyl cis-trans Isomerase B (PPIase B) and Assessment of their Role in the Protein Folding.

BACKGROUND: Peptidyl-prolyl cis-trans isomerase (PPIases) enzyme plays a vital role in protein folding. It catalyses the cis-trans isomerisation of peptide bonds, an essential step for newly synthesized protein to acquire its correct functional conformation in both prokaryotes and eukaryotes. OBJECTIVE: The present study showed the biochemical and molecular characterisation of cyclophilins (PpiB), a type of peptidyl-prolyl isomerases proteins from the pathogenic bacteria Salmonella Typhimurium. METHODS: Salmonella Typhimurium is one of the leading serovars responsible for human and animal salmonellosis globally, with the majority of human cases originating through the food chain. Here successful expression and purification of PpiB protein have been demonstrated and LC-MS based analyses showed high protein score and similarity with other PPi protein. Further the enzymatic activity of the purified recombinant PpiB was determined using Succinyl-Ala-Phe-Pro- Phe-p nitroanilide as substrate and enzyme-catalysed reaction. RESULT: Km and Vmax were calculated and found to be Vm = 1.023 ± .06400 min/µg, Km = 0.6219 ± 0.1701 µM, respectively. We have reported for the first time the presence of Salmonella PPIase-B (PpiB) protein isoforms in salmonella genome having PPi activity. CONCLUSION: Taken together, our data clearly showed that Salmonella Cyclophilin B (PpiB) protein is active and involved in diverse biological processes and highly similar to the different domain of Cyclophilin proteins.

In-Cell Trityl-Trityl Distance Measurements on Proteins.

Double-electron electron resonance (DEER) can be used to track the structural dynamics of proteins in their native environment, the cell. This method provides the distance distribution between two spin labels attached at specific, well-defined positions in a protein. For the method to be viable under in-cell conditions, the spin label and its attachment to the protein should exhibit high chemical stability in the cell. Here we present low-temperature, trityl-trityl DEER distance measurements on two model proteins, PpiB (prolyl cis-trans isomerase from E. coli) and GB1 (immunoglobulin G-binding protein), doubly labeled with the trityl spin label, CT02MA. Both proteins gave in-cell distance distributions similar to those observed in vitro, with maxima at 4.5-5 nm, and the data were further compared with in-cell Gd(III)-Gd(III) DEER obtained for PpiB labeled with BrPSPy-DO3A-Gd(III) at the same positions. These results highlight the challenges of designing trityl tags suitable for in-cell distance determination at ambient temperatures on live cells.

Removal of an atypical region from a staphylococcal cyclophilin affects its structure, function, stability, and shape.

SaCyp, a cyclophilin having 197 amino acid residues, acts both as a protein-folding catalyst and a virulence factor in Staphylococcus aureus. Interestingly, a region, homologous to the SaCyp region carrying 121-148 amino acid residues, is present in many putative cyclophilins but absent in well-studied cyclophilins. To determine the exact roles of this unusual region in SaCyp and related proteins, we have investigated a deletion mutant (rCypΔ) of a recombinant SaCyp (rCyp) using various probes. The data reveal that rCypΔ has significantly less catalytic activity and possesses altered structure and hydrophobic surface compared to rCyp. Conversely, the deletion substantially increased inhibitor binding affinity and altered the shape of rCyp. However, both proteins were unfolded by a non-two-state mechanism in the presence of urea. Additionally, the stability of rCyp was significantly reduced due to the deletion of the residues 121-148. Our MD simulation study also indicated the considerable alteration in structure, shape, and fluctuations of SaCyp due to the removal of the region carrying 121-148 residues. Hence, the atypical region located in SaCyp might be vital for maintaining its unique structure, function, stability, and shape.

Bifunctional Small-Molecule Ligands of K-Ras Induce Its Association with Immunophilin Proteins.

Here we report the design, synthesis, and characterization of bifunctional chemical ligands that induce the association of Ras with ubiquitously expressed immunophilin proteins such as FKBP12 and cyclophilin A. We show this approach is applicable to two distinct Ras ligand scaffolds, and that both the identity of the immunophilin ligand and the linker chemistry affect compound efficacy in biochemical and cellular contexts. These ligands bind to Ras in an immunophilin-dependent fashion and mediate the formation of tripartite complexes of Ras, immunophilin, and the ligand. The recruitment of cyclophilin A to GTP-bound Ras blocks its interaction with B-Raf in biochemical assays. Our study demonstrates the feasibility of ligand-induced association of Ras with intracellular proteins and suggests it as a promising therapeutic strategy for Ras-driven cancers.

19F NMR relaxation studies of fluorosubstituted tryptophans.

We present 19F longitudinal and transverse relaxation studies for four differently fluorosubstituted L-tryptophans, which carry single F atoms in the indole ring, both in the context of the free amino acid and when located in the cyclophilin A protein. For the free 4F-, 5F-, 6F-, 7F-L-Trp, satisfactory agreement between experimentally measured and calculated relaxation rates was obtained, suggesting that the parameters used for calculating the rates for the indole frame are sufficiently accurate. We also measured and calculated relaxation rates for four differently 19F-tryptophan labeled cyclophilin A proteins, transferring the parameters from the free amino acid to the protein-bound moiety. Our results suggest that 19F relaxation data of the large and rigid indole ring in Trp are only moderately affected by protein motions and provide critical reference points for evaluating fluorine NMR relaxation in the future, especially in fluorotryptophan labeled proteins.

Biophysical and biochemical characterization of a thermostable archaeal cyclophilin from Methanobrevibacter ruminantium.

The archaeal protein folding machinery is quite similar to that found in eukaryotes, especially in terms of shared components like chaperones. Cyclophilins are chaperones found in both eukaryotes and archaea, which catalyze the reversible cis-trans isomerization around peptidyl-prolyl imide bond (PPIase activity). Eukaryotes possess multiple cyclophilin genes, many of which have acquired divergent functions. Archaea, having a single copy of this gene, may help better in comprehending the role of cyclophilins in maintaining cellular proteostasis. However, no cyclophilin homologs from archaea have been characterized as yet, limiting comparison with their eukaryotic counterparts. In the present work, we characterize in detail a cyclophilin from the archaea, Methanobrevibacter ruminantium (MrCyp). We explore the functional and structural characteristics of MrCyp using various biophysical techniques. MrCyp exhibits both the PPIase and aggregation prevention activity. Analysis of folding/unfolding data and measurement of ∆GNUH2O and Tm suggest that the protein is thermodynamically stable. MrCyp helps in increasing cell viability of E. coli cells. These features imply that MrCyp could be a promising candidate for co-expression mediated enhancement in the yield and quality of over-expressed proteins in heterologous expression systems such as E. coli. This is the first study of its kind, reporting the detailed functional characterization of an archaeal cyclophilin.

Enzyme activity and structural features of three single-domain phloem cyclophilins from Brassica napus.

Cyclophilins (CYPs) are a group of ubiquitous prolyl cis/trans isomerases (PPIases). It was shown that plants possess the most diverse CYP families and that these are abundant in the phloem long-distance translocation stream. Since phloem exudate showed PPIase activity, three single-domain CYPs that occur in phloem samples from Brassica napus were characterised on functional and structural levels. It could be shown that they exhibit isomerase activity and that this activity is controlled by a redox regulation mechanism, which has been postulated for divergent CYPs. The structure determination by small-angle X-ray scattering experiments revealed a conserved globular shape. In addition, the high-resolution crystal structure of BnCYP19-1 was resolved and refined to 2.0 Å resolution, and the active sites of related CYPs as well as substrate binding were modelled. The obtained data and results support the hypothesis that single domain phloem CYPs are active phloem PPIases that may function as chaperones.

Genome-Wide Identification and Characterization of the Cyclophilin Gene Family in the Nematophagous Fungus Purpureocillium lilacinum.

Purpureocillium lilacinum has been widely used as a commercial biocontrol agent for the control of plant parasitic nematodes. Whole genome analysis promotes the identification of functional genes and the exploration of their molecular mechanisms. The Cyclophilin (CYP) gene family belongs to the immunophillin superfamily, and has a conserved cyclophilin-like domain (CLD). CYPs are widely identified in prokaryotes and eukaryotes, and can be divided into single- and multi-domain proteins. In the present study, 10 CYP genes possessing the CLD, named PlCYP1-P10, were identified from the genome of P. lilacinum strain 36-1. Those 10 PlCYPs were predicted to have different cellular localizations in P. lilacinum. Phylogenetic and gene structure analysis revealed the evolutionary differentiation of CYPs between Ascomycotina and Saccharomycotina fungi, but conservation within the Ascomycotina fungi. Motif and gene structure distributions further support the result of phylogenetic analysis. Each PlCYP gene had a specific expression pattern in different development stages of P. lilacinum and its parasitism stage on eggs of Meloidogyne incognita. In addition, the 10 PlCYP genes exhibited different expression abundances in response to abiotic stresses, among which PlCYP4 was highly expressed at a high temperature (35 °C), while PlCYP6 was up-regulated under 5 mM of H2O2 stress. Furthermore, the heterologous expression of PlCYP4 and PlCYP6 in Escherichia coli enhanced the cellular tolerance against a high temperature and H2O2. In summary, our study indicates the potential functions of PlCYPs in virulence and the stress response, and also provides a frame for further analysis of the CYP gene family in Ascomycotina fungi.

Cyclophilin B control of lysine post-translational modifications of skin type I collagen.

Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin.