Two-rung model of a left-handed beta-helix for prions explains species barrier and strain variation in transmissible spongiform encephalopathies

In this study, a new beta-helical model is proposed that explains the species barrier and strain variation in transmissible spongiform encephalopathies. The left-handed beta-helix serves as a structural model that can explain the seeded growth characteristics of beta-sheet structure in PrP(Sc) fibrils. Molecular dynamics simulations demonstrate that the left-handed beta-helix is structurally more stable than the right-handed beta-helix, with a higher beta-sheet content during the simulation and a better distributed network of inter-strand backbone-backbone hydrogen bonds between parallel beta-strands of different rungs. Multiple sequence alignments and homology modelling of prion sequences with different rungs of left-handed beta-helices illustrate that the PrP region with the highest beta-helical propensity (residues 105-143) can fold in just two rungs of a left-handed beta-helix. Even if no other flanking sequence participates in the beta-helix, the two rungs of a beta-helix can give the growing fibril enough elevation to accommodate the rest of the PrP protein in a tight packing at the periphery of a trimeric beta-helix. The folding of beta-helices is driven by backbone-backbone hydrogen bonding and stacking of side-chains in adjacent rungs. The sequence and structure of the last rung at the fibril end with unprotected beta-sheet edges selects the sequence of a complementary rung and dictates the folding of the new rung with optimal backbone hydrogen bonding and side-chain stacking. An important side-chain stack that facilitates the beta-helical folding is between methionine residues 109 and 129, which explains their importance in the species barrier of prions. Because the PrP sequence is not evolutionarily optimised to fold in a beta-helix, and because the beta-helical fold shows very little sequence preference, alternative alignments are possible that result in a different rung able to select for an alternative complementary rung. A different top rung results in a new strain with different growth characteristics. Hence, in the present model, sequence variation and alternative alignments clarify the basis of the species barrier and strain specificity in PrP-based diseases.     

Molecular cloning and sequence analysis of prion protein gene in Xiji donkey in China

Prion diseases are a group of human and animal neurodegenerative disorders caused by the deposition of an abnormal isoform prion protein (PrP^Sc) encoded by a single copy prion protein gene (PRNP). Prion disease has been reported in many herbivores but not in Equus and the species barrier might be playing a role in resistance of these species to the disease. Therefore, analysis of genotype of prion protein (PrP) in these species may help understand the transmission of the disease. Xiji donkey is a rare species of Equus not widely reared in Ningxia, China, for service, food and medicine, but its PRNP has not been studied. Based on the reported PrP sequence in GenBank we designed primers and amplified, cloned and sequenced the PRNP of Xiji donkey. The sequence analysis showed that the Xiji donkey PRNP was consisted of an open reading frame of 768 nucleotides encoding 256 amino acids. Amino acid residues unique to donkey as compared with some Equus animals, mink, cow, sheep, human, dog, sika deer, rabbit and hamster were identified. The results showed that the amino acid sequence of Xiji donkey PrP starts with the consensus sequence MVKSH, with almost identical amino acid sequence to the PrP of other Equus species in this study. Amino acid sequence analysis showed high identity within species and close relation to the PRNP of sika deer, sheep, dog, camel, cow, mink, rabbit and hamster with 83.1–99.7% identity. The results provided the PRNP data for an additional Equus species, which should be useful to the study of the prion disease pathogenesis, resistance and cross species transmission.     

Osmolyte trimethylamine N-oxide converts recombinant alpha-helical prion protein to its soluble beta-structured form at high temperature

The thermal unfolding of full-length human recombinant alpha-helical prion protein (alpha-PrP) in neutral pH is reversible, whereas, in the presence of the osmolyte N-trimethylamine oxide (TMAO), the protein acquires a beta-sheet structure at higher temperatures and the thermal unfolding of the protein is irreversible. Lysozyme, an amyloidogenic protein similar to prion protein, regains alpha-helical structure on cooling from its thermally unfolded form in buffer and in TMAO solutions. The thermal stability of alpha-PrP decreases, whereas that of lysozyme increases in TMAO solution. Light-scattering and turbidity values indicate that beta-sheet prion protein exists as soluble oligomers that increase thioflavin T fluorescence and bind to 1-anilino 8-naphthalene sulfonic acid (ANS). The oligomers are resistant to proteinase K digestion and during incubation for long periods they form linear amyloids>5 microm long. The comparable fluorescence polarization of the tryptophan groups and their accessibility to acrylamide in alpha-PrP and oligomers indicate that the unstructured N-terminal segments of the protein, which contain the tryptophan groups, do not associate among themselves during oligomerization. Partial unfolding of alpha-helical prion protein in TMAO solution leads to its structural conversion to misfolded beta-sheet form. The formation of the misfolded prion protein oligomers and their polymerization to amyloids in TMAO are unusual, since the osmolyte generally induces denatured protein to fold to a native-like state and protects proteins from thermal denaturation and aggregation.     

Spatial and temporal down-regulation of transgene expression using the TRSID-silencer in mice: application to Prnp

Spatial and temporal control of ovine prion protein (Prnp) gene expression was achieved in mice using two transgenes: a Prnp minigene with tet-operator sequences inserted 5' to exon 1 and a mouse neurofilament genomic clone carrying the chimeric-repressor TRSID cDNA. In bi-transgenic mice, ovine PrP(C) expression could be reversibly controlled in neuronal cells by doxycycline treatment whereas it remains constant in other cell types. Overall, this model opens opportunities to assess the involvement of cell types in prion diseases and PrP physiological function. It demonstrates the potentiality of the TRSID-silencer to precisely control temporal and spatial gene expression in vivo.     

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.     

Cloning and polymorphism analysis of prion protein gene in domestic bactrian camel in China

Up to now, little is known about the prion protein gene (PRNP) of domestic bactrian camels, and no polymorphisms of the bactrian camel PRNP have been analyzed or reported. In this study, we cloned and analyzed the PRNP sequences of 89 domestic bactrian camels. The results showed that the amino acid sequence of bactrian camel PrP starts with the consensus sequence MVKSH, with almost identical amino acid sequence to the PrP of dromedary camels. A four octapeptide PHGGGWGQ repeat region follows a nonapeptide (PQGGGGWGQ) in the N-terminal of deduced amino acid sequence from residues 54 to 95. Polymorphisms of PRNP in both species of camels were observed in codons 16(A → V), 17(M → T), 120(N → S), 176(R → K), 215(I → V), 234(S → Y), 237(Y → S), and 239(Q → G) by comparing with other ruminants. The PrP gene nucleotide sequence alignments of bactrian camels (HQ204566.1 and HQ204567.1) showed high identity with dromedary camel (99.2%, 99.1%), sheep (91.9%, 91.8%) and cattle (91.8%, 91.6%). This study provides valuable data for future research on susceptibility or resistance of camels to prion diseases.     

Tyrosine kinase activity of a Ca2+/calmodulin-dependent protein kinase II catalytic fragment

A 30-kDa fragment of Ca²⁺/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX₄₀-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca²⁺/calmodulin-dependent protein kinase I (CX₄₀) were fused to the N-terminal end of 30K-CaMKII. Although CX₄₀-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX₄₀ region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.     

The Unfolded State of the Murine Prion Protein and Properties of Single-point Mutants Related to Human Prion Diseases

The prion protein can exist both in a normal cellular isoform and in a pathogenic conformational isoform. The latter is responsible for the development of different neurodegenerative diseases, for example Creutzfeldt-Jakob disease or fatal familial insomnia. To convert the native benign state of the protein into a highly ordered fibrillar aggregate, large-scale rearrangements of the tertiary structure are necessary during the conversion process and intermediates that are at least partially unfolded are present during fibril formation. In addition to the sporadic conversion into the pathogenic isoform, more than 20 familial diseases are known that are caused by single point mutations increasing the probability of aggregation and neurodegeneration. Here, we demonstrate that the chemically denatured states of the mouse and human prion proteins have very similar structural and dynamic characteristics. Initial studies on the single point mutants E196K, F198S, V203I and R208H of the oxidized mouse construct, which are related to human prion diseases, reveal significant differences in the rate of aggregation. Aggregation for mutants V203I and R208H is slower than it is for the wild type, and the constructs E196K and F198S show accelerated aggregation. These differences in aggregation behaviour are not correlated with the thermal stability of the mutants, indicating different mechanisms promoting the conformational conversion process.     

An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity

Prevention of abnormal misfolding and aggregation of α synuclein (syn) protein in vulnerable neurons should be viable therapeutic strategies for reducing pathogenesis in Parkinson's disease. The nonamyloid component (NAC) region of α-syn shows strong tendencies to form β-sheet structures, and deletion of this region has been shown to reduce aggregation and toxicity in vitro and in vivo. The binding of a molecular species to this region may mimic the effects of such deletions. Single-chain variable fragment (scFv) antibodies retain the binding specificity of antibodies and, when genetically manipulated to create high-diversity libraries, allow in vitro selection against peptides. Accordingly, we used a yeast surface display library of an entire naïve repertoire of human scFv antibodies to select for binding to a NAC peptide. Candidate scFv antibodies (after transfer to mammalian expression vectors) were screened for viability in a neuronal cell line by transient cotransfection with A53T mutant α-syn. This provided a ranking of the protective efficacies of the initial panel of intracellular antibodies (intrabodies). High steady-state expression levels and apparent conformational epitope binding appeared more important than in vitro affinity in these assays. None of the scFv antibodies selected matched the sequences of previously reported anti-α-syn scFv antibodies. A stable cell line expressing the most effective intrabody, NAC32, showed highly significant reductions in abnormal aggregation in two separate models. Recently, intrabodies have shown promising antiaggregation and neuroprotective effects against misfolded mutant huntingtin protein. The NAC32 study extends such work significantly by utilizing information about the pathogenic capacity of a specific α-syn region to offer a new generation of in vitro-derived antibody fragments, both for further engineering as direct therapeutics and as a tool for rational drug design for Parkinson's disease.     

Structure of Fab fragment of malaria transmission blocking antibody 2A8 against P. vivax P25 protein

Understanding the structural basis of recognition between antigen and antibody requires the structural comparison of free and complexed components. Previously, we have reported the crystal structure of the complex between Fab fragment of murine monoclonal antibody 2A8 (Fab2A8) and Plasmodium vivax P25 protein (Pvs25) at 3.2 Å resolution. We report here the crystallization and X-ray structure of native Fab2A8 at 4.0 Å resolution. The 2A8 antibody generated against Pvs25 prevents the formation of P. vivax oocysts in the mosquito, when assayed in membrane feeding experiment.Comparison of native Fab2A8 structure with antigen bound Fab2A8 structure indicates the significant conformational changes in CDR-H1 and CDR-H3 regions of V_H domain and CDR-L3 region of V_L domain of Fab2A8. Upon complex formation, the relative orientation between V_L and V_H domains of Fab2A8 is conserved, while significant differences are observed in elbow angles of heavy and light chains. The combing site residues of complexed Fab2A8 exhibited the reduced temperature factor compared to native Fab2A8, suggesting a loss of conformational entropy upon antigen binding.     

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 °C. bvPrP(121-231) has the same overall architecture as other mammalian PrPs, with three α-helices and an antiparallel β-sheet, but it differs from PrP of the mouse and most other mammalian species in that the loop connecting the second β-strand and helix α2 is precisely defined at 20 °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.     

Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K_d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K_d, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K_d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.     

Oligomerization of H-pyrophosphatase and its structural and functional consequences

The H⁺-pyrophosphatase (H⁺-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H⁺-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H⁺-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H⁺-PPase is present as an oligomer made up of at least two or three sets of dimers.     

The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics

The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala₁₂₂₇ to Ser₁₂₅₁), which contains a single Trp residue (W₁₂₄₆) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala₁₂₂₇ was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring (∼50% α-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp₁₂₄₆ to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including λ_max, lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.     

Molecular cloning and polymorphism analysis of the prion protein gene in Tan sheep of Ningxia, China

The resistance or susceptibility of sheep to scrapie is associated with polymorphisms of the prion protein gene (PRNP), particularly, single nucleotide polymorphisms (SNPs) in amino acid positions 136, 154 and 171. The prion protein (PrP) gene sequence and the deduced amino acid alignment of prion protein in Tan sheep, a local Chinese sheep breed traditionally raised in Ningxia, northwestern China, were determined and variability of the PrP amino acids sequence was analyzed in this study. The PrP nucleic acids and amino acids sequences of 112 Tan sheep were highly homogenous, although polymorphism of the PrP gene was detected at several sites, particularly codons 106, 154, and 171. The analysis of both sequences revealed that the most predominant allele at codons 136, 154 and 171 in Tan sheep was ARQ, which was known to be associated with high susceptibility to scrapie in sheep. The result suggests that Tan sheep is potentially susceptible to scrapie. Our findings provide valuable information for future breeding projects to scrapie resistance in Tan sheep.     

Horse Prion Protein NMR Structure and Comparisons with Related Variants of the Mouse Prion Protein

The NMR structure of the horse (Equus caballus) cellular prion protein at 25 °C exhibits the typical PrP^C [cellular form of prion protein (PrP)] global architecture, but in contrast to most other mammalian PrP^Cs, it contains a well-structured loop connecting the β2 strand with the α2 helix. Comparison with designed variants of the mouse prion protein resulted in the identification of a single amino acid exchange within the loop, D167S, which correlates with the high structural order of this loop in the solution structure at 25 °C and is unique to the PrP sequences of equine species. The β2-α2 loop and the α3 helix form a protein surface epitope that has been proposed to be the recognition area for a hypothetical chaperone, “protein X,” which would promote conversion of PrP^C into the disease-related scrapie form and thus mediate intermolecular interactions related to the transmission barrier for transmissible spongiform encephalopathies (TSEs) between different species. The present results are evaluated in light of recent indications from in vivo experiments that the local β2-α2 loop structure affects the susceptibility of transgenic mice to TSEs and the fact that there are no reports on TSE in horses.     

Scrapie prion protein structural constraints obtained by limited proteolysis and mass spectrometry

Elucidation of the structure of scrapie prion protein (PrP^Sc), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrP^Sc. Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in β-strands. We treated PrP^Sc isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrP^Sc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and limited proteolysis after partial unfolding of PrP^Sc confirmed that only the mentioned cleavage sites at the N-terminal side of the PrP^Sc are susceptible to PK. Our results indicate that besides the “classic” amino-terminal PK cleavage points, PrP^Sc contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short β-sheet stretches connected by loops and turns.     

Electrostatic contributions to the kinetics and thermodynamics of protein assembly

The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D(9k) reconstitution was measured in the presence of Ca(2+) using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K(A) = 1.3 x 10(10) M(-1); K(D) = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.     

A Gly/Ala switch contributes to high affinity binding of benzoxazinone-based non-peptide oxytocin receptor antagonists

Non-peptide antagonists of the oxytocin receptor (OTR) have been developed to prevent pre-term labour. The benzoxazinone-based antagonists L-371,257 and L-372,662 display pronounced species-dependent pharmacology with respect to selectivity for the OTR over the V(1a) vasopressin receptor. Examination of receptor sequences from different species identified Ala(318) in helix 7 of the human OTR as a candidate discriminator required for high affinity binding. The mutant receptor [A318G]OTR was engineered and characterised using ligands representing many different chemical classes. Of all the ligands investigated, only the benzoxazinone-based antagonists had decreased affinity for [A318G]OTR. Molecular modelling revealed that Ala(318) provides a direct hydrophobic contact with a methoxy group of L-371,257 and L-372,662.     

Structure of the Flexible Amino-Terminal Domain of Prion Protein Bound to a Sulfated Glycan

The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence, PHGGGWGQ, that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear (¹H, ¹³C, ¹⁵N) NMR (nuclear magnetic resonance), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23-106 (PrP 23-106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23-106 remain disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The β-like turns within the repeats are corroborated by CD data demonstrating that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan side chains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23-106 when bound to PPS, consistent with the alignment of tryptophan side chains. Chemical-shift mapping identified binding sites on PrP 23-106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations.