Recent advances of Pin1 inhibitors as potential anticancer agents.

Pin1 (proline isomerase peptidyl-prolyl isomerase NIMA-interacting-1), as a member of PPIase family, catalyzes cis-trans isomerization of pThr/Ser-Pro amide bonds of its substrate proteins, further regulating cell proliferation, division, apoptosis, and transformation. Pin1 is overexpressed in various cancers and is positively correlated with tumor initiation and progression. Pin1 inhibition can effectively reduce tumor growth and cancer stem cell expansion, block metastatic spread, and restore chemosensitivity, suggesting that targeting Pin1 may be an effective strategy for cancer treatment. Considering the promising therapeutic effects of Pin1 inhibitors on cancers, we herein are intended to comprehensively summarize the reported Pin1 inhibitors, mainly highlighting their structures, biological functions and binding modes, in hope of providing a reference for the future drug discovery.

Molecular docking, 3D-QASR and molecular dynamics simulations of benzimidazole Pin1 inhibitors.

Pin1 (protein interacting with never-in-mitosis akinase-1) is a member of the family of peptidylprolyl cis-trans isomerases (PPIases) that specifically recognize and isomerize substrates containing phosphorylated Ser/Thr-Pro sequences. Pin1 is involved in many cellular processes and plays a key role in the cell cycle, transcriptional regulation, cell metabolism, proliferation and differentiation, and its abnormalities lead to degenerative and neoplastic diseases. Pin1 is highly expressed in human cancers and promotes the development of tumors by activating multiple oncogenes and inactivating multiple tumor suppressor genes, making it an attractive target for cancer therapy. In this study, we investigated the binding mechanism and conformational relationship between benzimidazole Pin1 inhibitors and Pin1 proteins by molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling, binding free energy calculations and decomposition, and molecular dynamics simulations. Molecular docking and molecular dynamics simulations disclosed the most likely binding pose of benzimidazoles with the Pin1 protein. The results of 3D-QSAR modeling indicated that electrostatic fields, hydrophobic fields and hydrogen bonding play important roles in the binding process of inhibitors to proteins. The binding free energy calculations and energy decomposition indicated that Lys63, Arg69, Cys113, Leu122, Met130, and Ser154 may be key residues in the binding of benzimidazole-based inhibitors to the Pin1 protein. This study provides an important theoretical basis for the design and optimization of benzimidazole compounds.

The metabolic crosstalk between PIN1 and the tumour microenvironment.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is a member of a family of peptidyl-prolyl isomerases that specifically recognizes and binds phosphoproteins, catalyzing the rapid cis-trans isomerization of phosphorylated serine/threonine-proline motifs, which leads to changes in the structures and activities of the targeted proteins. Through this complex mechanism, PIN1 regulates many hallmarks of cancer including cell autonomous metabolism and the crosstalk with the cellular microenvironment. Many studies showed that PIN1 is largely overexpressed in cancer turning on a set of oncogenes and abrogating the function of tumor suppressor genes. Among these targets, recent evidence demonstrated that PIN1 is involved in lipid and glucose metabolism and accordingly, in the Warburg effect, a characteristic of tumor cells. As an orchestra master, PIN1 finely tunes the signaling pathways allowing cancer cells to adapt and take advantage from a poorly organized tumor microenvironment. In this review, we highlight the trilogy among PIN1, the tumor microenvironment and the metabolic program rewiring.

The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C θ via a critical Phospho-Thr-Pro motif in the V3 regulatory domain.

Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.

Expression, Purification, Structural and Functional Characterization of Recombinant Human Parvulin 17.

Parvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis-trans isomerization of prolyl bonds in polypeptides, contributing to folding and function regulation of many proteins. Among Parvulins, Par17, exclusively expressed in hominids, is the least examined in terms of structure, catalytic function and cellular activity. Setting the conditions for the preparation of recombinant active Par17 may therefore significantly foster future studies. Here, we comparatively evaluated the impact of several parameters, including host strains, culture media, isopropyl ß-D-1-thiogalactopyranoside concentration, post-induction incubation time and temperature, on the overexpression of Par17 in E. coli cells. A similar approach was also comparatively adopted for the preparation of the recombinant full-length Pin1 protein, the most representative Parvulin, and the catalytic domains of both enzymes. Proteins were efficiently expressed and purified to homogeneity and were subjected to a structural characterization by Size Exclusion Chromatography and Circular Dichroism. Moreover, a single-step homogeneous protease-based fluorimetric assay, potentially scalable in HTS format, has been developed for determining the peptidyl-prolyl cis-trans isomerase activity of recombinant Parvulins. Results obtained show that proteins are folded and active. These new data mark an important milestone for progressing the investigation of Parvulins.

Regulation of eukaryotic protein kinases by Pin1, a peptidyl-prolyl isomerase.

The peptidyl-prolyl isomerase Pin1 cooperates with proline-directed kinases and phosphatases to regulate multiple oncogenic pathways. Pin1 specifically recognizes phosphorylated Ser/Thr-Pro motifs in proteins and catalyzes their cis-trans isomerization. The Pin1-catalyzed conformational changes determine the stability, activity, and subcellular localization of numerous protein substrates. We conducted a survey of eukaryotic protein kinases that are regulated by Pin1 and whose Pin1 binding sites have been identified. Our analyses reveal that Pin1 target sites in kinases do not fall exclusively within the intrinsically disordered regions of these enzymes. Rather, they fall into three groups based on their location: (i) within the catalytic kinase domain, (ii) in the C-terminal kinase region, and (iii) in regulatory domains. Some of the kinases downregulated by Pin1 activity are tumor-suppressing, and all kinases upregulated by Pin1 activity are functionally pro-oncogenic. These findings further reinforce the rationale for developing Pin1-specific inhibitors as attractive pharmaceuticals for cancer therapy.

Inner membrane YfgM-PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation.

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM-Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD-YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon.

Structural features of chloroplast trigger factor determined at 2.6†Šresolution.

The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl cis-trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their bacterial orthologs and their molecular mechanism in plant organelles has been little investigated to date. Here, the crystal structure of the plastidic trigger factor from the green alga Chlamydomonas reinhardtii is presented at 2.6 Šresolution. Due to the high intramolecular flexibility of the protein, diffraction to this resolution was only achieved using a protein that lacked the N-terminal ribosome-binding domain. The eukaryotic trigger factor from C. reinhardtii exhibits a comparable dragon-shaped conformation to its bacterial counterpart. However, the C-terminal chaperone domain displays distinct charge distributions, with altered positioning of the helical arms and a specifically altered charge distribution along the surface responsible for substrate binding. While the PPIase domain shows a highly conserved structure compared with other PPIases, its rather weak activity and an unusual orientation towards the C-terminal domain points to specific adaptations of eukaryotic trigger factor for function in chloroplasts.

Design and Synthesis of Oligopeptidic Parvulin Inhibitors.

Pin1 catalyzes the cis-trans isomerization of pThr-Pro or pSer-Pro amide bonds of various proteins involved in several physio/pathological processes. In this framework, recent research activity is directed toward the identification of new selective Pin1 inhibitors. Here, we developed a set of peptide-based Pin1 inhibitors. Direct-binding experiments allowed the identification of the peptide-based inhibitor 5 k (methylacetyl-l-alanyl-l-histidyl-l-prolyl-l-phenylalaninate) as a potent ligand of Pin1. Notably, 5 k binds Pin1 with higher affinity than Pin4. The comparative analysis of molecular models of Pin1 and Pin4 with the selected compound gave a rational explanation of the biochemical activity and pinpointed the chemical elements that, if opportunely modified, may further improve inhibitory potency, pharmacological properties, and selectivity of future peptide-based parvulin inhibitors. Since 5 k showed limited cell penetration and no antiproliferative activity, it was conjugated to a polyarginine stretch (R8), known to promote cell penetration of peptides, to obtain the R8-5 k derivative, which displayed antiproliferative effects on cancer cell lines over non-tumor cells. The effect of R8 on cell proliferation was also investigated. This work warrants caution about applying the R8 strategy in the development of cell-penetrating antiproliferative peptides, as it is not inert.

Molecular docking, 3D-QASR and molecular dynamics simulations of thiazoles Pin1 inhibitors.

Pin1 (protein interacting with never-in-mitosis akinase-1) is a member of the PPIase (peptidylprolyl cis-trans isomerase) family. It can interact with a variety of carcinogenic or tumor suppressive phosphorylated proteins. The interaction results in the conformational changes of target proteins, and ultimately regulates the activity of these proteins. These activity changes play a key role in tumorigenesis. Pin1 is an attractive target for cancer therapy due to its over-expression and/or activation in various types of cancer and the disorder of Proline directed phosphorylation. In this study, molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR) and molecular dynamics (MD) simulations were performed to investigate the structure-activity relationship and binding mechanism of 45 thiazole-class Pin1 inhibitors. Molecular docking studies predict the binding mode and the interactions between the ligand and the receptor protein. The results of the 3 D-QSAR model show that electrostatic field, hydrophobic field and hydrogen bond play important roles in the binding process of inhibitors to protein. Molecular dynamics simulation results reveal that the complex of the ligand and the receptor protein are stable at 300 K. The binding free energy calculation and energy decomposition results show that His59, Cys113, Ser114, Ser115, Leu122, Met130, Gln131, Phe134, Ser154 and His157 may be the key to the inhibitor binding to Pin1 protein. This study provides an important theoretical basis for further development of the new Pin1 inhibitor design. These results can provide more useful information for our further drug design. Communicated by Ramaswamy H. Sarma.

Common sequence motifs of nascent chains engage the ribosome surface and trigger factor.

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and in vitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol.

Structural insights into the interplay of protein biogenesis factors with the 70S ribosome.

Bacterial co-translational N-terminal methionine excision, an early event of nascent polypeptide chain processing, is mediated by two enzymes: peptide deformylase (PDF) and methionine aminopeptidase (MetAP). Trigger factor (TF), the only ribosome-associated bacterial chaperone, offers co-translational chaperoning assistance. Here, we present two high-resolution cryoelectron microscopy structures of tRNA-bound E. coli ribosome complexes showing simultaneous binding of PDF and TF, in the absence (3.4 Å) and presence of MetAP (4.1 Å). These structures establish molecular details of the interactions of the factors with the ribosome, and thereby reveal the structural basis of nascent chain processing. Our results suggest that simultaneous binding of all three factors is not a functionally favorable mechanism of nascent chain processing. Strikingly, an unusual structural distortion of the 70S ribosome, potentially driven by binding of multiple copies of MetAP, is observed when MetAP is incubated with a pre-formed PDF-TF-bound ribosome complex.

Hit identification against peptidyl-prolyl isomerase of Theileria annulata by combined virtual high-throughput screening and molecular dynamics simulation approach.

Theileria annulata secretes peptidyl prolyl isomerase enzyme (TaPIN1) to manipulate the host cell oncogenic signaling pathway by disrupting the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7) protein level leading to an increased level of c-Jun proto-oncogene. Buparvaquone is a hydroxynaphthoquinone anti-theilerial drug and has been used to treat theileriosis. However, TaPIN1 contains the A53 P mutation that causes drug resistance. In this study, potential TaPIN1 inhibitors were investigated using a library of naphthoquinone derivatives. Comparative models of mutant (m) and wild type (wt) TaPIN1 were predicted and energy minimization was followed by structure validation. A naphthoquinone (hydroxynaphthalene-1,2-dione, hydroxynaphthalene-1,4-dione) and hydroxynaphthalene-2,3-dione library was screened by Schrödinger Glide HTVS, SP and XP docking methodologies and the docked compounds were ranked by the Glide XP scoring function. The two highest ranked docked compounds Compound 1 (4-hydroxy-3-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxynaphthalene-1,2-dione) and Compound 2 (6-acetyl-1,4,5,7,8-pentahydroxynaphthalene-2,3-dione) were used for further molecular dynamics (MD) simulation studies. The MD results showed that ligand Compound 1 was located in the active site of both mTaPIN1 and wtTaPIN1 and could be proposed as a potential inhibitor by acting as a substrate antagonist. However, ligand Compound 2 was displaced away from the binding pocket of wtTaPIN1 but was located near the active site binding pocket of mTaPIN1 suggesting that could be selectively evaluated as a potential inhibitor against the mTaPIN1. Compound 1 and Compound 2 ligands are potential inhibitors but Compound 2 is suggested as a better inhibitor for mTaPIN1. These ligands could also further evaluated as potential inhibitors against human peptidyl prolyl isomerase which causes cancer in humans by using the same mechanism as TaPIN1.

Auxin-transporting ABC transporters are defined by a conserved D/E-P motif regulated by a prolylisomerase.

The plant hormone auxin must be transported throughout plants in a cell-to-cell manner to affect its various physiological functions. ABCB transporters are critical for this polar auxin distribution, but the regulatory mechanisms controlling their function is not fully understood. The auxin transport activity of ABCB1 was suggested to be regulated by a physical interaction with FKBP42/Twisted Dwarf1 (TWD1), a peptidylprolyl cis-trans isomerase (PPIase), but all attempts to demonstrate such a PPIase activity by TWD1 have failed so far. By using a structure-based approach, we identified several surface-exposed proline residues in the nucleotide binding domain and linker of Arabidopsis ABCB1, mutations of which do not alter ABCB1 protein stability or location but do affect its transport activity. P1008 is part of a conserved signature D/E-P motif that seems to be specific for auxin-transporting ABCBs, which we now refer to as ATAs. Mutation of the acidic residue also abolishes auxin transport activity by ABCB1. All higher plant ABCBs for which auxin transport has been conclusively proven carry this conserved motif, underlining its predictive potential. Introduction of this D/E-P motif into malate importer, ABCB14, increases both its malate and its background auxin transport activity, suggesting that this motif has an impact on transport capacity. The D/E-P1008 motif is also important for ABCB1-TWD1 interactions and activation of ABCB1-mediated auxin transport by TWD1. In summary, our data imply a new function for TWD1 acting as a putative activator of ABCB-mediated auxin transport by cis-trans isomerization of peptidyl-prolyl bonds.

Cyclophilin A Inhibitor Debio-025 Targets Crk, Reduces Metastasis, and Induces Tumor Immunogenicity in Breast Cancer.

The Crk adaptor protein, a critical modifier of multiple signaling pathways, is overexpressed in many cancers where it contributes to tumor progression and metastasis. Recently, we have shown that Crk interacts with the peptidyl prolyl cis-trans isomerase, Cyclophilin A (CypA; PP1A) via a G219P220Y221 (GPY) motif in the carboxyl-terminal linker region of Crk, thereby delaying pY221 phosphorylation and preventing downregulation of Crk signaling. Here, we investigate the physiologic significance of the CypA/Crk interaction and query whether CypA inhibition affects Crk signaling in vitro and in vivo. We show that CypA, when induced under conditions of hypoxia, regulates Crk pY221 phosphorylation and signaling in cancer cell lines. Using nuclear magnetic resonance spectroscopy, we show that CypA binds to the Crk GPY motif via the catalytic PPII domain of CypA, and small-molecule nonimmunosuppressive inhibitors of CypA (Debio-025) disrupt the CypA-CrkII interaction and restores phosphorylation of Crk Y221. In cultured cell lines, Debio-025 suppresses cell migration, and when administered in vivo in an orthotopic model of triple-negative breast cancer, Debio-025 showed antitumor efficacy either alone or in combination with anti-PD-1 mAb, reducing both tumor volume and metastatic lung dispersion. Furthermore, when analyzed by NanoString immune profiling, treatment of Debio-025 with anti-PD-1 mAb increased both T-cell signaling and innate immune signaling in tumor microenvironment. IMPLICATIONS: These data suggest that pharmacologic inhibition of CypA may provide a promising and unanticipated consequence in cancer biology, in part by targeting the CypA/CrkII axis that regulates cell migration, tumor metastasis, and host antitumor immune evasion.

Functional Analysis of Peptidyl-prolyl cis-trans Isomerase from Aspergillus flavus.

Aspergillus flavus, a ubiquitous filamentous fungus found in soil, plants and other substrates has been reported not only as a pathogen for plants, but also a carcinogen producing fungus for human. Peptidyl-Prolyl Isomerase (PPIases) plays an important role in cell process such as protein secretion cell cycle control and RNA processing. However, the function of PPIase has not yet been identified in A. flavus. In this study, the PPIases gene from A. flavus named ppci1 was cloned into expression vector and the protein was expressed in prokaryotic expression system. Activity of recombinant ppci1 protein was particularly inhibited by FK506, CsA and rapamycin. 3D-Homology model of ppci1 has been constructed with the template, based on 59.7% amino acid similarity. The homologous recombination method was used to construct the single ppci1 gene deletion strain Δppci1. We found that, the ppci1 gene plays important roles in A. flavus growth, conidiation, and sclerotia formation, all of which showed reduction in Δppci1 and increased in conidiation compared with the wild-type and complementary strains in A. flavus. Furthermore, aflatoxin and peanut seeds infection assays indicated that ppci1 contributes to virulence of A. flavus. Furthermore, we evaluated the effect of PPIase inhibitors on A. flavus growth, whereby these were used to treat wild-type strains. We found that the growths were inhibited under every inhibitor. All, these results may provide valuable information for designing inhibitors in the controlling infections of A. flavus.

The Multiple Roles of Peptidyl Prolyl Isomerases in Brain Cancer.

Peptidyl prolyl isomerases (PPIases) are broadly expressed enzymes that accelerate the cis-trans isomerization of proline peptide bonds. The most extensively studied PPIase family member is protein interacting with never in mitosis A1 (PIN1), which isomerizes phosphorylated serine/threonine⁻proline bonds. By catalyzing this specific cis-trans isomerization, PIN1 can alter the structure of its target proteins and modulate their activities in a number of different ways. Many proteins are targets of proline-directed phosphorylation and thus PIN1-mediated isomerization of proline bonds represents an important step in the regulation of a variety of cellular mechanisms. Numerous other proteins in addition to PIN1 are endowed with PPIase activity. These include other members of the parvulin family to which PIN1 belongs, such as PIN4, as well as several cyclophilins and FK506-binding proteins. Unlike PIN1, however, these other PPIases do not isomerize phosphorylated serine/threonine⁻proline bonds and have different substrate specificities. PIN1 and other PPIases are overexpressed in many types of cancer and have been implicated in various oncogenic processes. This review will discuss studies providing evidence for multiple roles of PIN1 and other PPIases in glioblastoma and medulloblastoma, the most frequent adult and pediatric primary brain tumors.

Structural flexibility in the Helicobacter pylori peptidyl-prolyl cis,trans-isomerase HP0175 is achieved through an extension of the chaperone helices.

Helicobacter pylori infects the gastric epithelium of half the global population, where infections can persist into adenocarcinomas and peptic ulcers. H. pylori secretes several proteins that lend to its pathogenesis and survival including VacA, CagA, γ-glutamyltransferase and HP0175. HP0175, also known as HpCBF2, classified as a peptidyl-prolyl cis,trans-isomerase, has been shown to induce apoptosis through a cascade of mechanisms initiated though its interaction with toll like receptor 4 (TLR4). Here, we report the structure of apo-HP0175 at 2.09 Šwith a single monomer in the asymmetric unit. Chromatographic, light scattering and mass spectrometric analysis of HP0175 in solution indicate that the protein is mainly monomeric under low salt conditions, while increasing ionic interactions facilitates protein dimerization. A comparison of the apo-HP0175 structure to that of the indole-2-carboxylic acid-bound form shows movement of the N- and C-terminal helices upon interaction of the catalytic residues in the binding pocket. Helix extension of the N/C chaperone domains between apo and I2CA-bound HP0175 supports previous findings in parvulin PPIases for their role in protein stabilization (and accommodation of variable protein lengths) of those undergoing catalysis.

Protein Folding Mediated by Trigger Factor and Hsp70: New Insights from Single-Molecule Approaches.

Chaperones assist in protein folding, but what this common phrase means in concrete terms has remained surprisingly poorly understood. We can readily measure chaperone binding to unfolded proteins, but how they bind and affect proteins along folding trajectories has remained obscure. Here we review recent efforts by our labs and others that are beginning to pry into this issue, with a focus on the chaperones trigger factor and Hsp70. Single-molecule methods are central, as they allow the stepwise process of folding to be followed directly. First results have already revealed contrasts with long-standing paradigms: rather than acting only "early" by stabilizing unfolded chain segments, these chaperones can bind and stabilize partially folded structures as they grow to their native state. The findings suggest a fundamental redefinition of the protein folding problem and a more extensive functional repertoire of chaperones than previously assumed.

Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated.