Myr-Arf1 conformational flexibility at the membrane surface sheds light on the interactions with ArfGAP ASAP1.

ADP-ribosylation factor 1 (Arf1) interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. Defining the dynamic conformational landscape of Arf1 in its active form, when bound to the membrane, is of high functional relevance and key to understanding how Arf1 can alter diverse cellular processes. Through concerted application of nuclear magnetic resonance (NMR), neutron reflectometry (NR) and molecular dynamics (MD) simulations, we show that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. These configurational dynamics expose different interfaces for interaction with effectors. Interaction with the Pleckstrin homology domain of ASAP1, an Arf-GTPase activating protein (ArfGAP), restricts motions of the G domain to lock it in what seems to be a conformation exposing functionally relevant regions.

A conserved motif promotes HpaB-regulated export of type III effectors from Xanthomonas.

The type III secretion (T3S) system, an essential pathogenicity factor in most Gram-negative plant-pathogenic bacteria, injects bacterial effector proteins directly into the plant cell cytosol. Here, the type III effectors (T3Es) manipulate host cell processes to suppress defence and establish appropriate conditions for bacterial multiplication in the intercellular spaces of the plant tissue. T3E export depends on a secretion signal which is also present in 'non-effectors'. The latter are secreted extracellular components of the T3S apparatus, but are not translocated into the plant cell. How the T3S system discriminates between T3Es and non-effectors is still enigmatic. Previously, we have identified a putative translocation motif (TrM) in several T3Es from Xanthomonas campestris pv. vesicatoria (Xcv). Here, we analysed the TrM of the Xcv effector XopB in detail. Mutation studies showed that the proline/arginine-rich motif is required for efficient type III-dependent secretion and translocation of XopB and determines the dependence of XopB transport on the general T3S chaperone HpaB. Similar results were obtained for other effectors from Xcv. As the arginine residues of the TrM mediate specific binding of XopB to cardiolipin, one of the major lipid components in Xanthomonas membranes, we assume that the association of T3Es to the bacterial membrane prior to secretion supports type III-dependent export.

Influence of thylakoid membrane lipids on the structure of aggregated light-harvesting complexes of the diatom Thalassiosira pseudonana and the green alga Mantoniella squamata.

The study investigated the effect of the thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure of two algal light-harvesting complexes (LHCs). In contrast to higher plants whose thylakoid membranes are characterized by an enrichment of the neutral galactolipids MGDG and DGDG, both the green alga Mantoniella squamata and the centric diatom Thalassiosira pseudonana contain membranes with a high content of the negatively charged lipids SQDG and PG. The algal thylakoids do not show the typical grana-stroma differentiation of higher plants but a regular arrangement. To analyze the effect of the membrane lipids, the fucoxanthin chlorophyll protein (FCP) complex of T. pseudonana and the LHC of M. squamata (MLHC) were prepared by successive cation precipitation using Triton X-100 as detergent. With this method, it is possible to isolate LHCs with a reduced amount of associated lipids in an aggregated state. The results from 77 K fluorescence and photon correlation spectroscopy show that neither the neutral galactolipids nor the negatively charged lipids are able to significantly alter the aggregation state of the FCP or the MLHC. This is in contrast to higher plants where SQDG and PG lead to a strong disaggregation of the LHCII whereas MGDG and DGDG induce the formation of large macroaggregates. The results indicate that LHCs which are integrated into thylakoid membranes with a high amount of negatively charged lipids and a regular arrangement are less sensitive to lipid-induced structural alterations than their counterparts in membranes enriched in neutral lipids with a grana-stroma differentiation.

Binding of the GTPase Sar1 to a Lipid Membrane Monolayer: Insertion and Orientation Studied by Infrared Reflection⁻Absorption Spectroscopy.

Membrane-interacting proteins are polyphilic polymers that engage in dynamic protein⁻protein and protein⁻lipid interactions while undergoing changes in conformation, orientation and binding interfaces. Predicting the sites of interactions between such polypeptides and phospholipid membranes is still a challenge. One example is the small eukaryotic GTPase Sar1, which functions in phospholipid bilayer remodeling and vesicle formation as part of the multimeric coat protein complex (COPII). The membrane interaction of Sar1 is strongly dependent on its N-terminal 23 amino acids. By monolayer adsorption experiments and infrared reflection-absorption spectroscopy (IRRAS), we elucidate the role of lipids in inducing the amphipathicity of this N-terminal stretch, which inserts into the monolayer as an amphipathic helix (AH). The AH inserting angle is determined and is consistent with the philicities and spatial distribution of the amino acid monomers. Using an advanced method of IRRAS data evaluation, the orientation of Sar1 with respect to the lipid layer prior to the recruitment of further COPII proteins is determined. The result indicates that only a slight reorientation of the membrane-bound Sar1 is needed to allow coat assembly. The time-course of the IRRAS analysis corroborates a role of slow GTP hydrolysis in Sar1 desorption from the membrane.

Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation.

As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum. Here we have revisited these model systems, to address the influence of lipid composition, GTP hydrolyzing conditions and mechanical perturbation on the experimental outcome. We observed that the lipid-dependence of COPII-induced membrane remodeling differs from that predicted based on the lipid-dependence of COPII membrane binding. Under GTP non-hydrolyzing conditions, a structured coat was seen while GTP-hydrolyzing conditions yielded uncoated membranes as well as membranes coated by a thick protein coat of rather unstructured appearance. Detailed up-to-date protocols for purifications of Saccharomyces cerevisiae COPII proteins and for reconstituted reactions using these proteins with giant liposomes are also provided.

The structure of the COPII transport-vesicle coat assembled on membranes.

Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available. We have used cryo-electron tomography and subtomogram averaging to determine the structure of the complete, membrane-assembled COPII coat. We describe a novel arrangement of the outer coat and find that the inner coat can assemble into regular lattices. The data reveal how coat subunits interact with one another and with the membrane, suggesting how coordinated assembly of inner and outer coats can mediate and regulate packaging of vesicles ranging from small spheres to large tubular carriers. DOI:http://dx.doi.org/10.7554/eLife.00951.001.

Death-associated protein kinase 1 phosphorylates Pin1 and inhibits its prolyl isomerase activity and cellular function.

Pin1 is a phospho-specific prolyl isomerase that regulates numerous key signaling molecules and whose deregulation contributes to disease notably cancer. However, since prolyl isomerases are often believed to be constitutively active, little is known whether and how Pin1 catalytic activity is regulated. Here, we identify death-associated protein kinase 1 (DAPK1), a known tumor suppressor, as a kinase responsible for phosphorylation of Pin1 on Ser71 in the catalytic active site. Such phosphorylation fully inactivates Pin1 catalytic activity and inhibits its nuclear location. Moreover, DAPK1 inhibits the ability of Pin1 to induce centrosome amplification and cell transformation. Finally, Pin1 pSer71 levels are positively correlated with DAPK1 levels and negatively with centrosome amplification in human breast cancer. Thus, phosphorylation of Pin1 Ser71 by DAPK1 inhibits its catalytic activity and cellular function, providing strong evidence for an essential role of the Pin1 enzymatic activity for its cellular function.

Multibudded tubules formed by COPII on artificial liposomes.

COPII-coated vesicles form at the endoplasmic reticulum for cargo transport to the Golgi apparatus. We used in vitro reconstitution to examine the roles of the COPII scaffold in remodeling the shape of a lipid bilayer. Giant Unilamellar Vesicles were examined using fast confocal fluorescence and cryo-electron microscopy in order to avoid separation steps and minimize mechanical manipulation. COPII showed a preference for high curvature structures, but also sufficient flexibility for binding to low curvatures. The COPII proteins induced beads-on-a-string-like constricted tubules, similar to those previously observed in cells. We speculate about a mechanical pathway for vesicle fission from these multibudded COPII-coated tubules, considering the possibility that withdrawal of the Sar1 amphipathic helix upon GTP hydrolysis leads to lipid bilayer destabilization resulting in fission.

Identification of an atypical peptidyl-prolyl cis/trans isomerase from trypanosomatids.

The parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases) catalyzes the cis/trans isomerization of the peptide bonds preceding Pro residues. Eukaryotic parvulin-type PPIases have been shown to be involved in cell proliferation and cell cycle progression. Here we present the biochemical and molecular characterization of a novel multi-domain parvulin-type PPIase from the human pathogenic Trypanosoma cruzi, annotated as TcPar45. Like most other parvulins, Par45 has an N-terminal extension, but, in contrast to human Pin1, it contains a forkhead-associated domain (FHA) instead of a WW domain at the N-terminal end. Par45 shows a strong preference for a substrate with the basic Arg residue preceding Pro (Suc-Ala-Arg-Pro-Phe-NH-Np: k(cat)/K(M)=97.1 /M/s), like that found for human Par14. In contrast to human Pin1, but similarly to Par14, Par45 does not accelerate the cis/trans interconversion of acidic substrates containing Glu-Pro bonds. It is preferentially located in the parasite nucleus. Single RNA interference (RNAi)-mediated knock-down showed that there was a growth inhibition in procyclic Trypanosoma brucei cells. These results identify Par45 as a phosphorylation-independent parvulin required for normal cell proliferation in a unicellular eukaryotic cell.

Isoform-specific inhibition of cyclophilins.

Cyclophilins belong to the enzyme class of peptidyl prolyl cis-trans isomerases which catalyze the cis-trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family, CypA and CypB, exhibit specific cellular functions in several inflammatory diseases, cancer development, and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4(+) T cells by the inhibitor provided biological proof of discrimination in vivo.

On the benefit of bivalency in peptide ligand/pin1 interactions.

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO(3)H(2))/Thr(PO(3)H(2))-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.

Structural basis for high-affinity peptide inhibition of human Pin1.

Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors. The structures of the bound high-affinity peptides identify a type-I beta-turn conformation for Pin1 prolyl peptide isomerase domain-peptide binding and an extensive molecular interface for high-affinity recognition. Moreover, these structures suggest chemical elements that may further improve the affinity and pharmacological properties of future peptide-based Pin inhibitors. Finally, an intramolecular hydrogen bond observed in both peptide complexes mimics the cyclic conformation of FK506 and rapamycin. Both FK506 and rapamycin are clinically important inhibitors of other peptidyl-prolyl cis-trans isomerases. This comparative discovery suggests that a cyclic peptide polyketide bridge, like that found in FK506 and rapamycin or a similar linkage, may significantly improve the binding affinity of structure-based Pin1 inhibitors.

The Trypanosoma cruzi PIN1 gene encodes a parvulin peptidyl-prolyl cis/trans isomerase able to replace the essential ESS1 in Saccharomyces cerevisiae.

Parvulins are a conserved group of peptidyl-prolyl cis/trans isomerases (PPIases) that catalyze the cis/trans isomerization of proline-preceding peptide bonds. Parvulin-class PPIases are structurally unrelated to cyclophilins and FK506-binding proteins that are defined as receptors for immunosuppressive drugs. In Trypanosoma cruzi we identified parvulin TcPIN1 as a homolog of the human hPin1 PPIase. The 117 amino acids of the TcPIN1 display 40% identity with the catalytic core of hPin1 and exhibit prolyl cis/trans isomerase activity. TcPIN1 lacks the WW domain at the N-terminus, and is able to rescue the temperature-sensitive phenotype on a mutation in the Saccharomyces cerevisiae hPin1 homolog, ESS1/PTF1. Western blot analysis revealed that the enzyme was present both in dividing and non-dividing forms of T. cruzi. In epimastigote cells neither cell growth kinetics nor cell morphology was affected by the overexpression of the small parvulin TcPIN1. These results suggest the occurrence of a supplementary conserved level of post-translational control in trypanosomatids.

Thermodynamics of phosphopeptide binding to the human peptidyl prolyl cis/trans isomerase Pin1.

Proteins containing phosphorylated Ser/Thr-Pro motifs play key roles in numerous regulatory processes in the cell. The peptidyl prolyl cis/trans isomerase Pin1 specifically catalyzes the conformational transition of phosphorylated Ser/Thr-Pro motifs. Here we report the direct analysis of the thermodynamic properties of the interaction of the PPIase Pin1 with its substrate-analogue inhibitor Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2 specifically targeted to the PPIase active site based on the combination of isothermal titration calorimetry and studies on inhibition of enzymatic activity of wt Pin1 and active site variants. Determination of the thermodynamic parameters revealed an enthalpically and entropically favored interaction characterized by binding enthalpy deltaH(ITC) of -6.3 +/- 0.1 kcal mol(-1) and a TdeltaS(ITC) of 4.1 +/- 0.1 kcal mol(-1). The resulting dissociation constant KD for binding of the peptidic inhibitor with 1.8 x 10(-8) M resembles the dissociation constant of a Pin1 substrate in the transition state, suggesting a transition state analogue conformation of the bound inhibitor. The strongly decreased affinity of Pin1 for ligand at increasing ionic strength implicates that the potential of bidentate binding of a substrate protein by the PPIase and the WW domain of Pin1 may be required to deploy improved efficiency and specificity of Pin1 under conditions of physiological ionic strength.

Polo-like kinase 1-mediated phosphorylation stabilizes Pin1 by inhibiting its ubiquitination in human cells.

The Polo-like kinase 1 (Plk1) is a key regulator of mitosis. It is reported that the human peptidyl-prolyl cis/trans-isomerase Pin1 binds to Plk1 from mitotic cell extracts in vitro. Here we demonstrate that Ser-65 in Pin1 is the major site for Plk1-specific phosphorylation, and the polo-box domain of Plk1 is required for this phosphorylation. Interestingly, the phosphorylation of Pin1 by Plk1 does not affect its isomerase activity but rather is linked to its protein stability. Pin1 is ubiquitinated in HeLa S3 cells, and substitution of Glu for Ser-65 reduces the ubiquitination of Pin1. Furthermore, inhibition of Plk1 activity by expression of a dominant negative form of Plk1 or by transfection of small interfering RNA targeted to Plk1 enhances the ubiquitination of Pin1 and subsequently reduces the amount of Pin1 in human cancer cells. Since previous reports suggested that Plk1 is a substrate of Pin1, our work adds a new dimension to this interaction of two important mitotic regulators.

Characterization of the S272A,D site-directed mutations of O-acetylserine sulfhydrylase: involvement of the pyridine ring in the alpha,beta-elimination reaction.

O-Acetylserine sulfhydrylase catalyzes the synthesis of l-cysteine from O-acetyl-l-serine (OAS) and inorganic bisulfide. An anti-E2 mechanism has been proposed for the OASS-catalyzed elimination of acetate from OAS (Tai, C.-H., and Cook, P. F. (2001) Acc. Chem. Res. 34, 49-59). Site-directed mutagenesis was used to change S272 to alanine, which would be expected to eliminate the hydrogen bond to N1 of PLP or to aspartate, which would be expected to enhance the hydrogen-bonding interaction. Both mutant enzymes catalyze the overall reaction and are in fact still good enzymes, consistent with the proposed anti-E2 mechanism. Data suggest that hydrogen bonding to the pyridine ring does not play a significant role in the alpha,beta-elimination reaction catalyzed by OASS-A. The V/K(OAS), which reflects the first half-reaction, is identical to the wild-type enzyme in the case of the S272D mutant enzyme and is decreased by only a factor of 3 in the case of the S272A mutant enzyme. In the case of the alanine mutation, and to a lesser extent the aspartate mutation, a decrease in the rate of the elimination is compensated by an increase in affinity for OAS, leading to the observed second-order rate constant, V/K. The decrease in the rate of the elimination is proposed to result from a change in the orientation of the bound cofactor, as might be expected since one of the ligands that determines the position of the bound PLP has been changed. Consistent with a change in the orientation of the cofactor are the results from a number of the spectral probes. The visible CD data for the internal Schiff base have a molar ellipticity 50% that of wild-type enzyme, and the alpha-aminoacrylate intermediate has a molar ellipticity 25% that of wild-type enzyme. The alpha-aminoacrylate intermediate can be formed from l-cysteine and l-serine with the S272A,D mutant enzymes, but not with the wild-type enzyme, and taken together with the increased K(d) for the serine external Schiff base is consistent with a change in cofactor orientation in the active site. The long wavelength fluorescence emission for the S272A mutant enzyme, attributed to Förster resonance energy transfer (McClure, G. D., Jr., and Cook, P. F. (1994) Biochemistry 33, 1647-1683) has an intensity near zero, as compared to significant fluorescence for the wild-type enzyme.