The Cellular Peptidyl-Prolyl cis/trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

Kaposi''s sarcoma-associated herpesvirus (KSHV) causes Kaposi''s sarcoma and primary effusion lymphoma. KSHV-infected cells are predominantly latent, with a subset undergoing lytic reactivation. Rta is the essential lytic switch protein that reactivates virus by forming transactivation-competent complexes with the Notch effector protein RBP-Jk and promoter DNA. Strikingly, Rta homolog analysis reveals that prolines constitute 17% of conserved residues. Rta is also highly phosphorylated in vivo. We previously demonstrated that proline content determines Rta homotetramerization and function. We hypothesize that proline-directed modifications regulate Rta function by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine- or phosphothreonine-proline (pS/T-P) motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses. Our data show that KSHV Rta protein contains potential pS/T-P motifs and binds directly to Pin1. Rta transactivation is enhanced by Pin1 at two delayed early viral promoters in uninfected cells. Pin1''s effect, however, suggests a rheostat-like influence on Rta function. We show that in infected cells, endogenous Pin1 is active during reactivation and enhances Rta-dependent early protein expression induced by multiple signals, as well as DNA replication. Surprisingly, ablation of Pin1 activity by the chemical juglone or dominant-negative Pin1 enhanced late gene expression and production of infectious virus, while ectopic Pin1 showed inhibitory effects. Our data thus suggest that Pin1 is a unique, dose-dependent molecular timer that enhances Rta protein function, but inhibits late gene synthesis and virion production, during KSHV lytic reactivation.     

A Legionella pneumophila Peptidyl-Prolyl cis-trans Isomerase Present in Culture Supernatants Is Necessary for Optimal Growth at Low Temperatures

Several Legionella pneumophila proteins were highly expressed in low-temperature supernatants. One of these proteins was the peptidyl-prolyl isomerase PpiB. Mutants lacking ppiB exhibited reduced growth at 17°C. Since PpiB lacked a signal sequence and was present in 17°C supernatants of type II and type IV secretion mutants, this protein may be secreted by a novel mechanism.     

Structural and functional studies of FkpA from Escherichia coli, a cis/trans peptidyl-prolyl isomerase with chaperone activity

The protein FkpA from the periplasm of Escherichia coli exhibits both cis/trans peptidyl-prolyl isomerase (PPIase) and chaperone activities. The crystal structure of the protein has been determined in three different forms: as the full-length native molecule, as a truncated form lacking the last 21 residues, and as the same truncated form in complex with the immunosuppressant ligand, FK506. FkpA is a dimeric molecule in which the 245-residue subunit is divided into two domains. The N-terminal domain includes three helices that are interlaced with those of the other subunit to provide all inter-subunit contacts maintaining the dimeric species. The C-terminal domain, which belongs to the FK506-binding protein (FKBP) family, binds the FK506 ligand. The overall form of the dimer is V-shaped, and the different crystal structures reveal a flexibility in the relative orientation of the two C-terminal domains located at the extremities of the V. The deletion mutant FkpNL, comprising the N-terminal domain only, exists in solution as a mixture of monomeric and dimeric species, and exhibits chaperone activity. By contrast, a deletion mutant comprising the C-terminal domain only is monomeric, and although it shows PPIase activity, it is devoid of chaperone function. These results suggest that the chaperone and catalytic activities reside in the N and C-terminal domains, respectively. Accordingly, the observed mobility of the C-terminal domains of the dimeric molecule could effectively adapt these two independent folding functions of FkpA to polypeptide substrates.     

Involvement of the cis/trans Isomerase Cti in Solvent Resistance of Pseudomonas putida DOT-T1E

Pseudomonas putida DOT-T1E is a solvent-resistant strain that is able to grow in the presence of high concentrations of toluene. We have cloned and sequenced the cti gene of this strain, which encodes the cis/trans isomerase, termed Cti, that catalyzes the cis-trans isomerization of esterified fatty acids in phospholipids, mainly cis-oleic acid (C(16:1,9)) and cis-vaccenic acid (C(18:1,11)), in response to solvents. To determine the importance of this cis/trans isomerase for solvent resistance a Cti-null mutant was generated and characterized. This mutant showed a longer lag phase when grown with toluene in the vapor phase; however, after the lag phase the growth rate of the mutant strain was similar to that of the wild type. The mutant also showed a significantly lower survival rate when shocked with 0.08% (vol/vol) toluene. In contrast to the wild-type strain, which grew in liquid culture medium at temperatures up to 38.5 degrees C, the Cti-null mutant strain grew significantly slower at temperatures above 37 degrees C. An in-frame fusion of the Cti protein with the periplasmic alkaline phosphatase suggests that this constitutively expressed enzyme is located in the periplasm. Primer extension studies confirmed the constitutive expression of Cti. Southern blot analysis of total DNA from various pseudomonads showed that the cti gene is present in all the tested P. putida strains, including non-solvent-resistant ones, and in some other Pseudomonas species.     

cis/trans Isomerase of Unsaturated Fatty Acids of Pseudomonas putida P8: Evidence for a Heme Protein of the Cytochrome c Type

From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.     

Solution structure of the single-domain prolyl cis/trans isomerase PIN1At from Arabidopsis thaliana

The 119-amino acid residue prolyl cis/trans isomerase from Arabidopsis thaliana (PIN1At) is similar to the catalytic domain of the human hPIN1. However, PIN1At lacks the N-terminal WW domain that appears to be essential for the hPIN1 function. Here, the solution structure of PIN1At was determined by three-dimensional nuclear magnetic resonance spectroscopy. The PIN1At fold could be superimposed on that of the catalytic domain of hPIN1 and had a 19 residue flexible loop located between strand beta1 and helix alpha1. The dynamical features of this beta1/alpha1-loop, which are characteristic for a region involved in protein-protein interactions, led to exchange broadening in the NMR spectra. When sodium sulfate salt was added to the protein sample, the beta1/alpha1 loop was stabilized and, hence, a complete backbone resonance assignment was obtained. Previously, with a phospho-Cdc25 peptide as substrate, PIN1At had been shown to catalyze the phosphoserine/phosphothreonine prolyl cis/trans isomerization specifically. To map the catalytic site of PIN1At, the phospho-Cdc25 peptide or sodium sulfate salt was added in excess to the protein and chemical shift changes in the backbone amide protons were monitored in the (1)H(N)-(15)N heteronuclear single quantum coherence spectrum. The peptide caused perturbations in the loops between helix alpha4 and strand beta3, between strands beta3 and beta4, in the alpha3 helix, and in the beta1/alpha1 loop. The amide groups of the residues Arg21 and Arg22 showed large chemical shift perturbations upon phospho-Cdc25 peptide or sulfate addition. We conclude that this basic cluster formed by Arg21 and Arg22, both located in the beta1/alpha1 loop, is homologous to that found in the hPIN1 crystal structure (Arg68 and Arg69), which also is involved in sulfate ion binding. We showed that the sulfate group competed for the interaction between PIN1At and the phospho-Cdc25 peptide. In the absence of the WW domain, three hydrophobic residues (Ile33, Ile34, and Leu35) located in the long flexible loop and specific for the plant PIN-type peptidyl prolyl cis/trans isomerases (PPIases) could be an additional interaction site in PIN1At. However, phospho-peptide addition did not affect the resonances of these residues significantly. Electrostatic potential calculations revealed a negatively charged area not found in hPIN1 on the PIN1At molecular surface, which corresponds to the surface shielded by the WW domain in hPIN1. Based on our experimental results and the molecular specificities of the PIN1At enzyme, functional implications of the lack of WW domains in this plant PIN-type PPIase will be discussed.     

Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B

We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.Hepatitis C virus (HCV)⁴ is a small, positive strand, RNA-enveloped virus belonging to the Flaviviridae family and the genus Hepacivirus. With 120–180 million chronically infected individuals worldwide, hepatitis C virus infection represents a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1). The HCV viral genome (∼9.6 kb) codes for a unique polyprotein of ∼3000 amino acids (recently reviewed in Refs. 2–4). Following processing via viral and cellular proteases, this polyprotein gives rise to at least 10 viral proteins, divided into structural (core, E1, and E2 envelope glycoproteins) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). Nonstructural proteins are involved in polyprotein processing and viral replication. The set composed of NS3, NS4A, NS4B, NS5A, and NS5B constitutes the minimal protein component required for viral replication (5).Cyclophilins are cellular proteins that have been identified first as CsA-binding proteins (6). As FK506-binding proteins (FKBP) and parvulins, cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyze the cis/trans isomerization of the peptide linkage preceding a proline (6, 7). Several subtypes of cyclophilins are present in mammalian cells (8). They share a high sequence homology and a well conserved three-dimensional structure but display significant differences in their primary cellular localization and in abundance (9). CypA, the most abundant of the cyclophilins, is primarily cytoplasmic, whereas CypB is directed to the endoplasmic reticulum lumen or the secretory pathway. CypD, on the other hand, is the mitochondrial cyclophilin. Cyclophilins are involved in numerous physiological processes such as protein folding, immune response, and apoptosis and also in the replication cycle of viruses including vaccinia virus, vesicular stomatitis virus, severe acute respiratory syndrome (SARS)-coronavirus, and human immunodeficiency virus (HIV) (for review see Ref. 10). For HIV, CypA has been shown to interact with the capsid domain of the HIV Gag precursor polyprotein (11). CypA thereby competes with capsid domain/TRIM5 interaction, resulting in a loss of the antiviral protective effect of the cellular restriction factor TRIM5α (12, 13). Moreover, it has been shown that CypA catalyzes the cis/trans isomerization of Gly²²¹-Pro²²² in the capsid domain and that it has functional consequences for HIV replication efficiency (14–16). For HCV, Watashi et al. (17) have described a molecular and functional interaction between NS5B, the viral RNA-dependent RNA polymerase (RdRp), and cyclophilin B (CypB). CypB may be a key regulator in HCV replication by modulating the affinity of NS5B for RNA. This regulation is abolished in the presence of cyclosporin A (CsA), an inhibitor of cyclophilins (6). These results provided for the first time a molecular mechanism for the early-on observed anti-HCV activity of CsA (18–20). Although this initial report suggests that only CypB would be involved in the HCV replication process (17), a growing number of studies have recently pointed out a role for other cyclophilins (21–25).In vitro selection of CsA-resistant HCV mutants indicated the importance of two HCV nonstructural proteins, NS5B and NS5A (26), with a preponderant effect for mutations in the C-terminal half of NS5A. NS5A is a large phosphoprotein (49 kDa), indispensable for HCV replication and particle assembly (27–29), but for which the exact function(s) in the HCV replication cycle remain to be elucidated. This nonstructural protein is anchored to the cytoplasmic leaflet of the endoplasmic reticulum membrane via an N-terminal amphipathic α-helix (residues 1–27) (30, 31). Its cytoplasmic sequence can be divided into three domains: D1 (residues 27–213), D2 (residues 250–342), and D3 (residues 356–447), all connected by low complexity sequences (32). D1, a zinc-binding domain, adopts a dimeric claw-shaped structure, which is proposed to interact with RNA (33, 34). NS5A-D2 is essential for HCV replication, whereas NS5A-D3 is a key determinant for virus infectious particle assembly (27, 35). NS5A-D2 and -D3, for which sequence conservation among HCV genotypes is significantly lower than for D1, have been proposed to be natively unfolded domains (28, 32). Molecular and structural characterization of NS5A-D2 from HCV genotype 1a has confirmed the disordered nature of this domain (36, 37).As it is still not clear which cyclophilins are cofactors for HCV replication, and as mutations in HCV NS5A protein have been associated with CsA resistance, we decided to examine the interaction between both CypA and CypB and domain 2 of the HCV NS5A protein. We first characterized, at the molecular level, NS5A-D2 from the HCV JFH1 infectious strain (genotype 2a) and showed by NMR spectroscopy that this natively unfolded domain indeed interacts with both cyclophilin A and cyclophilin B. Our NMR chemical shift mapping experiments indicated that the interaction occurs at the level of the cyclophilin active site, whereas it lacks a precise localization on NS5A-D2. A peptide derived from the only well conserved amino acid motif in NS5A-D2 did interact with cyclophilin A but only with a 10-fold lower affinity than the full domain. We concluded from this that the many proline residues form multiple anchoring points, especially when they adopt the cis conformation. NMR exchange spectroscopy further demonstrated that NS5A-D2 is a substrate for the PPIase activities of both CypA and CypB. Both the NS5A/cyclophilin interaction and the PPIase activity of the cyclophilins on NS5A-D2 were abolished by CsA, underscoring the specificity of the interaction.     

Steric and electronic interactions controlling the cis/trans isomer equilibrium at X‐pro tertiary amide motifs in solution

A systematic understanding of the noncovalent interactions that influence the structures of the cis conformers and the equilibrium between the cis and the trans conformers, of the X‐Pro tertiary amide motifs, is presented based on analyses of ¹H‐, ¹³C‐NMR and FTIR absorption spectra of two sets of homologous peptides, X‐Pro‐Aib‐OMe and X‐Pro‐NH‐Me (where X is acetyl, propionyl, isobutyryl and pivaloyl), in solvents of varying polarities. First, this work shows that the cis conformers of any X‐Pro tertiary amide motif, including Piv‐Pro, are accessible in the new motifs X‐Pro‐Aib‐OMe, in solution. These conformers are uniquely observable by FTIR spectroscopy at ambient temperatures and by NMR spectroscopy from temperatures as high as 273 K. This is made possible by the persistent presence of n_i‐1→π_i* interactions at Aib, which also influence the disappearance of steric effects at these cis X‐Pro rotamers. Second, contrary to conventional understanding, the energy contribution of steric effects to the cis/trans equilibrium at the X‐Pro motifs is found to be nonvariant (0.54 ± 0.02 kcal/mol) with increase in steric bulk on the X group. Third, the current studies provide direct evidence for the weak intramolecular interactions namely the n_i‐1→π_i*, the N_Pro???H_i+1 (C₅a), and the C₇ hydrogen bond that operate and influence the structures, stabilities, and dynamics between different conformational states of X‐Pro tertiary amide motifs. NMR and IR spectral data suggest that the cis conformers of X‐Pro motifs are ensembles of short‐lived rotamers about the C′_X–N_Pro bond. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 66–77, 2014.     

A peptidylprolyl cis/trans isomerase from Xenopus laevis skin: cloning, biochemical characterization and putative role in the secretion

In amphibian skin secretions, a peptidylprolyl cis/trans isomerase activity was detected. A Xenopus laevis skin cDNA coding for this protein was cloned, sequenced and over-expressed in Escherichia coli. The primary structure of the protein shows extensive similarity with members of the cyclophilin A family. Catalytic parameters of the recombinant protein are similar to those of the human enzyme. The enzymatic activity is inhibited by cyclosporin A. Data suggesting that peptidylprolyl isomerization influences the biological activity of antibacterial peptides of amphibian origin are presented, and its putative role in the defence mechanism discussed.     

Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface

H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that binds to H/ACA RNAs specifically. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent from eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of Saccharomyces cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10 and RNA binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs expressed in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA.     

The Structure of Sedoheptulose-7-Phosphate Isomerase from Burkholderia pseudomallei Reveals a Zinc Binding Site at the Heart of the Active Site

Heptoses are found in the surface polysaccharides of most bacteria, contributing to structures that are essential for virulence and antibiotic resistance. Consequently, the biosynthetic enzymes for these sugars are attractive targets for novel antibiotics. The best characterized biosynthetic enzyme is GmhA, which catalyzes the conversion of sedoheptulose-7-phosphate into d-glycero-d-manno-heptopyranose-7-phosphate, the first step in the biosynthesis of heptose. Here, the structure of GmhA from Burkholderia pseudomallei is reported. This enzyme contains a zinc ion at the heart of its active site: this ion stabilizes the active, closed form of the enzyme and presents coordinating side chains as a potential acid and base to drive catalysis. A complex with the product demonstrates that the enzyme retains activity in the crystal and thus suggests that the closed conformation is catalytically relevant and is an excellent target for the development of therapeutics. A revised mechanism for the action of GmhA is postulated on the basis of this structure and the activity of B. pseudomallei GmhA mutants.     

Cell adhesion promoting peptide GVKGDKGNPGWPGAP from the collagen type IV triple helix: cis/trans proline-induced multiple 1H NMR conformations and evidence for a KG/PG multiple turn repeat motif in the all-trans proline state

Peptide GVKGDKGNPGWPGAPY (called peptide IV-H1), derived from the protein sequence of human collagen type IV, triple-helix domain residues 1263-1277, represents an RGD-independent, cell-specific, adhesion, spreading, and motility promoting domain in type IV collagen. In this study, peptide IV-H1 has been investigated by 1H NMR (500 MHz) spectroscopy. Cis-trans proline isomerization at each of the three proline residues gives rise to a number of slowly exchanging (500-MHz NMR time scale) conformation states. At least five such states are observed, for example, for the well-resolved A14 beta H3 group, and K3, which is six residues sequentially removed from the nearest proline, i.e., P9, shows two sets. The presence of more than two sets of resonances for residues sequentially proximal to a proline, e.g., A14-cis-P15 and A14-trans-P15, and more than one set for a residue sequentially well-removed from a proline, e.g., K3, indicates long range conformation interactions and the presence of preferred structure in this short linear peptide. Many resonances belonging to these multiple species have been assigned by using mono-proline-substituted analogues. Conformational (isomer) state-specific 2D 1H NMR assignments for the combination of cis and trans proline states have been made via analysis of COSY-type, HOHAHA, and NOESY spectra. Peptide IV-H1 in the all-trans proline state ttt exists in relatively well-defined conformation populations showing numerous short- and long-range NOEs and long-lived backbone amide protons and reduced backbone NH temperature coefficients, suggesting hydrogen-bonding, and structurally informative 3J alpha N coupling constants. The NMR data indicate significant beta-turn populations centered at K3-G4, K5-G6, P9-G10, and P12-G13, and a C-terminal gamma-turn within the A14-P15-Y16 sequence. These NMR data are supported by circular dichroic studies which indicate the presence of 52% beta-turn, 10% helix, and 38% random coil structural populations. Since equally spaced KG and PG residues are found on both sides of peptide IV-H1 in the native collagen type IV sequence, this multiple turn repeat motif may continue through a longer segment of the protein. Synthetic peptide IV-H1 overlapping sequence "walk throughs" indicate that the primary biological activity is localized in the GNPGWPGAP double beta-turn domain, which contains the backbone constraining proline residues. This proline-domain conformation may suggest a collagen type IV receptor-specific, metastatic cell adhesion promoting binding domain.