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.

Amylose-defective mutants were selected after UV mutagenesis of Chlamydomonas reinhardtii cells. Two recessive nuclear alleles of the ST-2 gene led to the disappearance not only of amylose but also of a fraction of the amylopectin. Granule-bound starch synthase activities were markedly reduced in strains carrying either st-2-1 or st-2-2, as is the case for amylose-deficient (waxy) endosperm mutants of higher plants. The main 76-kDa protein associated with the starch granule was either missing or greatly diminished in both mutants, while st-2-1-carrying strains displayed a novel 56-kDa major protein. Methylation and nuclear magnetic resonance analysis of wild-type algal storage polysaccharide revealed a structure identical to that of higher-plant starch, while amylose-defective mutants retained a modified amylopectin fraction. We thus propose that the waxy gene product conditions not only the synthesis of amylose from endosperm storage tissue in higher-plant amyloplasts but also that of amylose and a fraction of amylopectin in all starch-accumulating plastids. The nature of the ST-2 (waxy) gene product with respect to the granule-bound starch synthase activities is discussed.Our knowledge of starch synthesis and degradation, while having developed mostly from observations made in higherplant storage tissues, has benefited from investigations performed on a number of model microbial systems. Chlorella pyrenoidosa, for instance, has been the subject of several of the pioneer studies dealing with the enzymology of starch anabolism (16,20). Prokaryotic organisms such as Escherichia coli have yielded a number of relevant genetic and biochemical studies, mostly because of the parallel that can be drawn between the regulation of plant and bacterial ADP-pyrophosphorylases (reviewed in reference 18). However, the very nature of the storage polysaccharide (glycogen) has prevented the use of model bacterial systems to investigate the biogenesis of the starch granule itself. Thus, our knowledge of the intricate pathways of amylose and amylopectin biosyntheses stems solely from those elegant genetic investigations performed on pea or cereal mutations which express themselves only in the endosperm (reviewed in references 15 and 22). Even in that case, we do not yet know precisely which of the starch synthases and starch branching enzymes are responsible for amylose or amylopectin biosynthesis, let alone how they act coordinately to produce a complex structure such as the starch granule. One of the best and most studied endosperm mutants, waxy maize, seemed until very recently to shed at least some light on the biosynthesis of amylose. Waxy mutations have been identified in most cereals (22, 32) and more recently in storage tissues of dicots such as potato (9) or in the perisperm of the amaranth (11). They all lead to the decrease or absence of both amylose and granule-bound starch synthase. While there is no doubt that one of the main proteins associated with the starch granule is the product of the waxy * Corresponding author...

The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions.

Trp-Trp (WW 1 ) domains are compact modules of 38 -40 amino acids long, folded into a three-stranded ␤ sheet, and found in single or tandem repeats in over 25 unrelated cellsignaling proteins (1, 2). Although their binding to prolinebased ligands is now well described (3, 4), little is known about their precise biological function. WW domains form a new family of protein-protein interaction modules targeted to proline residues, analogous to the Src homology (SH) 3 domains (5).Based on their proline-rich sequence binding specificities, WW domains are classified into five distinct groups (6). The N-terminal WW domain of the peptidyl-prolyl isomerase Pin1, an essential regulator at mitotic entry, is grouped among class IV domains, which bind peptides containing a proline residue preceded by a phosphoserine or a phosphothreonine (pSer/ pThr-Pro motif) (7). This latter motif is found in several mitotic Pin1-binding phosphoproteins (8), including the mitotic phosphatase Cdc25 (9, 10) and the microtubule-associated tau () protein (11).Site-directed mutagenesis experiments indicate that Pin1 binds phosphoproteins through its N-terminal WW domain and that the binding site mainly implicates the conserved residues Tyr 18 and Trp 29 (7, 11), which constitute a nearly flat hydrophobic area on the molecular surface of the WW domain. WW domains interacting with the core sequence PPXY, like Yesassociated protein, use the same hydrophobic surface for molecular recognition (3,4,12). However, this hydrophobic binding site alone is not likely to explain the phospho-dependent character of the ligand binding to the Pin1 WW domain.Recently the x-ray crystal structure of the full Pin1 protein bound to a doubly phosphorylated peptide (YpSPTpSPS) from the C-terminal domain (CTD) of RNA polymerase II was reported (13). The protein-peptide interactions are essentially limited to two regions on the WW domain surface. First, a phosphate binding pocket, encompassing the side chains of Ser 11 , Arg 12 , and the backbone amide of Arg 12 , anchors the interacting phosphate moiety via several hydrogen bonds. Second, the aromatic pair Tyr 18 -Trp 29 forms a molecular clamp that constrains the proline at position ϩ1 of the interacting phosphoserine. The peptide ligand binds to the Pin1 WW domain in a N-to C-terminal orientation, in contrast to the C-to N-terminal orientation that is found for two other proline-rich peptides in complex with a group I WW domain (3,14). Other proline recognition domains, such as the SH3 domain of the Caenorhabditis elegans signaling adaptor protein Sem5, can * This project was partly executed in the framework of the Génopole de Lille. The 600-MHz NMR facility used in this study was funded by the European community, the Région Nord-Pas de Calais, CNRS, and the Institut Pasteur de Lille. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The ab...

A simple modification to the WATERGATE water suppression scheme [Piotto, M., Saudek, V. and Sklenář, V. (1992) J. Biomol. NMR, 2, 661-665] is proposed. Radiation damping is used as an active element during the mixing time of a NOESY experiment, in order to obtain a reproducable state of the water magnetization at the end of the mixing time. Through the use of a water flip-back pulse and a gradient-tailored excitation scheme, we obtain both an excellent water suppression and a water magnetization close to equilibrium at the beginning of the acquisition time. We show experimentally that this modification results in a 20% gain in intensity for all signals when using a relaxation delay of 1.5 s, and also that avoiding a semisaturated state for the water magnetization allows the amide protons as well as other proton resonances to relax to equilibrium with their proper relaxation time.

The mechanism of pI258 arsenate reductase (ArsC) catalyzed arsenate reduction, involving its P-loop structural motif and three redox active cysteines, has been unraveled. All essential intermediates are visualized with x-ray crystallography, and NMR is used to map dynamic regions in a key disulfide intermediate. Steadystate kinetics of ArsC mutants gives a view of the crucial residues for catalysis. ArsC combines a phosphatase-like nucleophilic displacement reaction with a unique intramolecular disulfide bond cascade. Within this cascade, the formation of a disulfide bond triggers a reversible ''conformational switch'' that transfers the oxidative equivalents to the surface of the protein, while releasing the reduced substrate.

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an ␣-helix. NMR analysis identifies two putative ␣-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA. Hepatitis C virus (HCV),3 a small enveloped virus from the Flaviviridae family, is a major cause of chronic liver disease that may lead to steatosis, liver cirrhosis, and hepatocellular carcinoma. Given about 180 million chronically infected individuals worldwide, HCV is an important health challenge (1). Current therapy is based on a combination of pegylated interferon-␣ and ribavirin but is not fully satisfying because numerous patients are not responding or suffer from serious side effects caused by this treatment. The development of new drugs to treat HCV infections requires a better understanding of the structural and functional features of the viral proteins and their relationships with host cell factors. The HCV genome (ϳ9.6 kb) encodes for a single polyprotein precursor (ϳ3,000 aa) that is co-and post-translationally processed by cellular and viral proteases to yield 10 mature proteins (2, 3). They are classified into structural proteins (Core, E1, and E2), which constitute the viral particle, and nonstructural proteins, of which two, p7 and nonstructural protein 2 (NS2), are required for virus assembly. The remainder of the nonstructural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) are involved in HCV RNA replication (reviewed in Refs. 2 and 3).Cyclophilins are host cell factors that in addition to viral proteins, are equally essential for HCV replication. The human genome encodes up to 16 different cyclophilins (4) that, despite differences in their tissue distributio...

Whereas the interaction between Tau and the microtubules has been studied in great detail both by macroscopic techniques (cosedimentation, cryo-electron microscopy, and fluorescence spectroscopy) using the full-length protein or by peptide mapping assays, no detailed view at the level of individual amino acids has been presented when using the full-length protein. Here, we present a nuclear magnetic resonance (NMR) study of the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). As signal disappearance in the heteronuclear 1H-15N correlation spectra of isotope-labeled Tau in complex with MTs is due to direct association of the corresponding residue with the solid-like MT wall, we can map directly the fragment in interaction with the MT surface, and obtain a molecular picture of the precise interaction zones. The N-terminal region projects from the microtubule surface, and the lack of chemical shift variations when compared with free Tau proves that this region can regulate microtubular separation without adopting a stable conformation. Amino acids in the four microtubule binding repeats (MTBRs) lose all of their intensity, underscoring their immobilization upon binding to the MTs. The same loss of NMR intensity was observed for the proline-rich region starting at Ser214, underscoring its importance in the Tau:MT interaction. Fluorescence resonance energy transfer (FRET) experiments were used to obtain thermodynamic binding parameters, and led to the conclusion that the NMR defined fragment indeed is the major player in the interaction. When the same Ser214 is phosphorylated by the PKA kinase, the Tau:MT interaction strength decreases by 2 orders of magnitude, but the proline-rich region including the phospho-Ser214 does not gain sufficient mobility in the complex to make it observable by NMR spectroscopy. The presence of an intramolecular disulfide bridge, on the contrary, does lead to a partial detachment of the C-terminus of Tau, and decreases significantly the overloading of Tau on the MT surface.