Structural plasticity of the cellular prion protein and implications in health and disease.

Two lines of transgenic mice expressing mouse/elk and mouse/horse prion protein (PrP) hybrids, which both form a well-structured ß2-α2 loop in the NMR structures at 20 °C termed rigid-loop cellular prion proteins (RL-PrP(C)), presented with accumulation of the aggregated scrapie form of PrP in brain tissue, and the mouse/elk hybrid has also been shown to develop a spontaneous transmissible spongiform encephalopathy. Independently, there is in vitro evidence for correlations between the amino acid sequence in the ß2-α2 loop and the propensity for conformational transitions to disease-related forms of PrP. To further contribute to the structural basis for these observations, this paper presents a detailed characterization of RL-PrP(C) conformations in solution. A dynamic local conformational polymorphism involving the ß2-α2 loop was found to be evolutionarily preserved among all mammalian species, including those species for which the WT PrP forms an RL-PrP(C). The interconversion between two ensembles of PrP(C) conformers that contain, respectively, a 310-helix turn or a type I ß-turn structure of the ß2-α2 loop, exposes two different surface epitopes, which are analyzed for their possible roles in the still evasive function of PrP(C) in healthy organisms and/or at the onset of a transmissible spongiform encephalopathy.

Cellular prion protein conformation and function.

In the otherwise highly conserved NMR structures of cellular prion proteins (PrP(C)) from different mammals, species variations in a surface epitope that includes a loop linking a ß-strand, ß2, with a helix, α2, are associated with NMR manifestations of a dynamic equilibrium between locally different conformations. Here, it is shown that this local dynamic conformational polymorphism in mouse PrP(C) is eliminated through exchange of Tyr169 by Ala or Gly, but is preserved after exchange of Tyr 169 with Phe. NMR structure determinations of designed variants of mouse PrP(121-231) at 20 °C and of wild-type mPrP(121-231) at 37 °C together with analysis of exchange effects on NMR signals then resulted in the identification of the two limiting structures involved in this local conformational exchange in wild-type mouse PrP(C), and showed that the two exchanging structures present characteristically different solvent-exposed epitopes near the ß2-α2 loop. The structural data presented in this paper provided a platform for currently ongoing, rationally designed experiments with transgenic laboratory animals for renewed attempts to unravel the so far elusive physiological function of the cellular prion protein.

Putative prion protein from Fugu (Takifugu rubripes).

Prion proteins (PrP) of mammals, birds, reptiles and amphibians have been successfully cloned, expressed and purified in sufficient yields to enable 3D structure determination by NMR spectroscopy in solution. More recently, PrP ortholog genes have also been identified in several fish species, based on sequence relationships with tetrapod PrPs. Even though the sequence homology of fish PrPs to tetrapod PrPs is below 25%, structure prediction programs indicate a similar organization of the 3D structure. In this study, we generated recombinant polypeptide constructs that were expected to include the C-terminal folded domain of Fugu-PrP1 and analyzed these proteins using biochemical and biophysical methods. Because soluble expression could not be achieved, and refolding from guanidine-HCl did not result in a properly folded protein, we co-expressed Escherichia coli chaperone proteins in order to obtain the protein in a soluble form. Although CD spectroscopy indicated the presence of some regular secondary structure in the protein thus obtained, there was no evidence for a globular 3D fold in the NMR spectra. We thus conclude that the polypeptide products of the fish genes annotated as corresponding to bona fide prnp genes in non-fish species cannot be prepared for structural studies when using procedures similar to those that were successfully used with PrPs from mammals, birds, reptiles and amphibians.

Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles.

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34. The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.

Alpha-tocopherol influences the lipid membrane affinity of desipramine in a pH-dependent manner.

Phopholipidosis is a lipid storage disorder caused by cationic amphiphilic drugs (CADs) characterized by the lysosomal accumulation of phospholipids and drug. alpha-Tocopherol (alpha-Toc) has a reversible effect on phospholipidosis in rats and cell culture. We studied the influence of alpha-Toc on the partitioning of the CAD desipramine in a liposome/buffer system using equilibrium dialysis with the following lipid compositions: egg phosphatidylcholine (PhC) or wheat germ phosphatidylinositol (PhI) or a combination of PhC, PhI and cholesterol, containing between 1.5 and 20% (mol per mol total lipids) of alpha-Toc, alpha-tocopherol acetate (alpha-TocAc), 2,2,5,7,8-pentamethyl-6-chromanol (PMC) or cholesterol. alpha-Toc (1.5%) enhanced the partition coefficient of neutral desipramine by up to 1.1 log units while it had no influence on the partitioning of the ionized compound. In the PhC liposome system, at pH 7.4 logD increased with increasing alpha-Toc concentrations but was unchanged at pH 4.5. Similar effects were found with PMC while alpha-TocAc or cholesterol, between 1.5 and 20%, had no influence on the partitioning of desipramine. From these results we postulate that in vivo, alpha-Toc could mediate a redistribution of CADs from lysosomal membranes (pH approximately 4.5) to membranes and lipoproteins at physiological pH.

Prion protein mPrP[F175A](121-231): structure and stability in solution.

The three-dimensional structures of prion proteins (PrPs) in the cellular form (PrP(C)) include a stacking interaction between the aromatic rings of the residues Y169 and F175, where F175 is conserved in all but two so far analyzed mammalian PrP sequences and where Y169 is strictly conserved. To investigate the structural role of F175, we characterized the variant mouse prion protein mPrP[F175A](121-231). The NMR solution structure represents a typical PrP(C)-fold, and it contains a 3(10)-helical ß2-α2 loop conformation, which is well defined because all amide group signals in this loop are observed at 20°C. With this "rigid-loop PrP(C)" behavior, mPrP[F175A](121-231) differs from the previously studied mPrP[Y169A](121-231), which contains a type I ß-turn ß2-α2 loop structure. When compared to other rigid-loop variants of mPrP(121-231), mPrP[F175A](121-231) is unique in that the thermal unfolding temperature is lowered by 8°C. These observations enable further refined dissection of the effects of different single-residue exchanges on the PrP(C) conformation and their implications for the PrP(C) physiological function.

Prion protein library of recombinant constructs for structural biology.

A survey of plasmids for 51 prion protein constructs from bank vole, cat, cattle, chicken, dog, elk, ferret, frog, fugu, horse, human, pig, sheep, turtle, and wallaby, and for 113 mouse prion protein constructs and variants thereof, is presented. This includes information on the biochemistry of the recombinant proteins, in particular on successful and unsuccessful expression attempts. The plasmid library was generated during the past 12 years in the context of NMR structure determination and biophysical characterization of prion proteins in our laboratory. The plasmids are now available for general use, and are distributed free of charge to not-for-profit institutions.

Prion protein NMR structure from tammar wallaby (Macropus eugenii) shows that the beta2-alpha2 loop is modulated by long-range sequence effects.

NMR structures are presented for the recombinant construct of residues 121-230 from the tammar wallaby (Macropus eugenii) prion protein (PrP) twPrP(121-230) and for the variant mouse PrPs mPrP[Y225A,Y226A](121-231) and mPrP[V166A](121-231) at 20 degrees C and pH 4.5. All three proteins exhibit the same global architecture as seen in other recombinant PrP(C)s (cellular isoforms of PrP) and shown to prevail in natural bovine PrP(C). Special interest was focused on a loop that connects the beta2-strand with helix alpha2 in the PrP(C) fold, since there are indications from in vivo experiments that this local structural feature affects the susceptibility of transgenic mice to transmissible spongiform encephalopathies. This beta2-alpha2 loop and helix alpha3 form a solvent-accessible contiguous epitope, which has been proposed to be the recognition area for a hypothetical chaperone, the "protein X". This hypothetical chaperone would affect the conversion of PrP(C) into the disease-related scrapie form (PrP(Sc)) by moderating intermolecular interactions related to the transmission barrier of transmissible spongiform encephalopathies between different species. In contrast to mPrP(121-231) and most other mammalian PrP(C)s, the beta2-alpha2 loop is well defined at 20 degrees C in tammar wallaby PrP and in the two aforementioned variants of mPrP, showing that long-range interactions with helix alpha3 can have an overriding influence on the structural definition of the beta2-alpha2 loop. Further NMR studies with two variant mPrPs, mPrP[Y225A](121-231) and mPrP[Y226A](121-231), showed that these interactions are dominantly mediated by close contacts between residues 166 and 225. The results of the present study then lead to the intriguing indication that well-defined long-range intramolecular interactions could act as regulators of the functional specificity of PrP(C).

NMR structure of the bank vole prion protein at 20 degrees C contains a structured loop of residues 165-171.

The recent introduction of bank vole (Clethrionomys glareolus) as an additional laboratory animal for research on prion diseases revealed an important difference when compared to the mouse and the Syrian hamster, since bank voles show a high susceptibility to infection by brain homogenates from a wide range of diseased species such as sheep, goats, and humans. In this context, we determined the NMR structure of the C-terminal globular domain of the recombinant bank vole prion protein (bvPrP) [bvPrP(121-231)] at 20 degrees C. bvPrP(121-231) has the same overall architecture as other mammalian PrPs, with three alpha-helices and an antiparallel beta-sheet, but it differs from PrP of the mouse and most other mammalian species in that the loop connecting the second beta-strand and helix alpha2 is precisely defined at 20 degrees C. This is similar to the previously described structures of elk PrP and the designed mouse PrP (mPrP) variant mPrP[S170N,N174T](121-231), whereas Syrian hamster PrP displays a structure that is in-between these limiting cases. Studies with the newly designed variant mPrP[S170N](121-231), which contains the same loop sequence as bvPrP, now also showed that the single-amino-acid substitution S170N in mPrP is sufficient for obtaining a well-defined loop, thus providing the rationale for this local structural feature in bvPrP.

Prion protein NMR structures of cats, dogs, pigs, and sheep.

The NMR structures of the recombinant cellular form of the prion proteins (PrPC) of the cat (Felis catus), dog (Canis familiaris), and pig (Sus scrofa), and of two polymorphic forms of the prion protein from sheep (Ovis aries) are presented. In all of these species, PrPC consists of an N-terminal flexibly extended tail with approximately 100 amino acid residues and a C-terminal globular domain of approximately 100 residues with three alpha-helices and a short antiparallel beta-sheet. Although this global architecture coincides with the previously reported murine, Syrian hamster, bovine, and human PrPC structures, there are local differences between the globular domains of the different species. Because the five newly determined PrPC structures originate from species with widely different transmissible spongiform encephalopathy records, the present data indicate previously uncharacterized possible correlations between local features in PrPC three-dimensional structures and susceptibility of different mammalian species to transmissible spongiform encephalopathies.