Prion Nucleation Site Unmasked by Transient Interaction with Phospholipid Cofactor
Infectious mammalian prions can be formed de novo from purified recombinant prion protein (PrP) substrate through a pathway that requires the sequential addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and RNA cofactor molecules. Recent studies show that the initial interaction between PrP and POPG causes widespread and persistent conformational changes to form an insoluble intermediate species, termed PrPInt1. Here, we characterize the mechanism and functional consequences of the interaction between POPG and PrP. Negative-stain electron microscopy of PrPInt1 revealed the presence of amorphous aggregates. Pull-down and photoaffinity label experiments indicate that POPG induces the formation of a PrPC polybasic-domain-binding neoepitope within PrPInt1. The ongoing presence of POPG is not required to maintain PrPInt1 structure, as indicated by the absence of stoichiometric levels of POPG in solid-state NMR measurements of PrPInt1. Together, these results show that a transient interaction with POPG cofactor unmasks a PrPC binding site, leading to PrPInt1 aggregation.
<!>Expression and Purification of Recombinant MoPrP and of AviTag PrP<!>Biotinylation of AviTag PrP<!>Preparation of PrPInt1 and PrPC<!>Polybasic-Domain Peptide Pull-Down Assay<!>Photoaffinity Labeling<!>Amylase and Amyloglucosidase Treatment of recPrPSc<!>Electron Microscopy<!>NMR Spectroscopy<!>Cofactor Induces PrP Aggregation<!>Cofactor Unmasks Binding Site for PrPC N-Terminal Polybasic Domain<!>Photoaffinity Labeling of PrPInt1 with Polybasic-Domain Peptide<!>PrPInt1 Does Not Structurally Incorporate Phospholipid<!>Cofactor Induces Helical Transition of N-Terminus<!>DISCUSSION
<p>The mechanism of prion diseases such as Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD), and scrapie involves the conformational change of the host-encoded prion protein (PrPC) into a misfolded, aggregated, infectious conformer (PrPSc).1 Once formed, PrPSc can seed the conversion of additional PrPC molecules in an exponential-growth process responsible for the pathogenesis and transmission of disease. PrP oligomerization and aggregation appear to be critical steps in prion formation2 and toxicity.3,4 In recent years, various experimental approaches have provided valuable insights about the process by which PrPC molecules interact with PrPSc molecules and undergo induced conformational change into infectious prions.</p><p>In one line of investigation, studies using motif-grafted antibodies and tagged PrP peptides identified two linear polybasic domains on PrPC (residues 23–33 and 100–110) as consensus PrPSc-binding epitopes.5–7 Moreover, the functional importance of the N-terminal 23–33 polybasic domain was confirmed by showing that a N-truncated (Δ23–28) PrP mutant was unable to bind PrPSc or to undergo templated conformational change efficiently.8 Together, these studies argue that the N-terminal polybasic domain of PrPC interacts with the prion nucleation site of PrPSc.</p><p>Much insight into the process of prion formation has also been gained through the development of protocols that enable in vitro PrPSc formation and propagation. A series of seminal studies showed that PrPSc molecules9–11 and infectious prions12 could be formed in vitro, allowing the conversion process to be studied by using a reductionist approach. Using a reconstitution system, Deleault et al. showed that infectious prions could be formed de novo by subjecting a substrate mixture of purified PrPC (containing stoichiometric amounts of an unidentified copurified lipid but no other proteins or nucleic acids) and synthetic homopolymeric RNA molecules to serial protein-misfolding cyclic amplification (sPMCA).13 Critically, no PrPSc molecules or prion infectivity could be formed using PrPC alone, showing that cofactor molecules are necessary for efficient prion propagation in vitro. Wang et al. were also able to produce infectious prions de novo using bacterially expressed, refolded, recombinant (rec)PrP substrate combined with the synthetic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and RNA.14</p><p>To study the mechanism by which cofactor molecules facilitate the formation of infectious PrPSc molecules de novo, we recently conducted a deuterium-exchange mass spectrometry (DXMS) study to characterize structural changes induced during prion formation in vitro with recPrP and POPG.15 One important insight provided by this study is that the initial interaction between POPG and recPrP induces major protein conformational changes, some of which appear to persist in the final PrPSc structure. Here, we investigate the functional consequences and mechanism of this critical interaction using a combination of biophysical and biochemical approaches</p><!><p>The AviTag PrP sequence was constructed by mutagenesis of a pET-22b(+) expression plasmid encoding the mouse PrP 23–230 sequence originally extracted from pCOMBO3(MoPrP) (Mike Scott, UCSF).16 The AviTag sequence was added to the C-terminus of the PrP sequence by PCR amplification using primers that included an NdeI restriction enzyme site at the 5′ end of the sequence and a 15 amino acid AviTag sequence added to Ser230 followed by a stop codon and an XhoI site at the 3′ end (5′ AAAAAA-CATATGAAAAAGCGGCCAAAGCCTGGAGGGT 3′ and 5′ AAAACTCGAGTCATTATTCGTGCCATTCGATT-TTCTGAGCCTCGAAGATGTCGTTCAGACCGCC-ACCGGATCTTCTCCCGTCGTAATAG 3′). The PCR product was ligated into the pCR4-TOPO (Invitrogen, San Diego, CA) expression vector, and its sequence was confirmed. The product was digested with NdeI and XhoI and ligated into the pET-22b(+) expression vector for subsequent transformation into Escherichia coli Rosetta cells.</p><p>Double-isotope-labeled protein for NMR analysis was prepared by growing E. coli Rosetta cells transformed with the MoPrP plasmid with DM minimal media containing 1× Mo salt stock (50 mM KH2PO4, 30 mM Sodium Citrate, Na3C6H5O7·2H2O, 0.7 mMCaCl2·2H2O, and 10 mM MgSO4·7H2O), 1× DM trace metals stock (100 mM FeCl3·6H2O, 9 mM ZnCl2, 12 mM CoCl2·6H2O, 7.5 mM CuCl2·2H2O,6.8 mM CaCl2·2H2O, 7.45 mM H3BO3, and 12 mM HCl), 1× BME vitamins (Sigma, St. Louis, MO), 3 mL of 400× thiamine (Sigma, St. Louis, MO) and 3 g/L of 15NH4Cl and 4 g/L of D-U-C6-glucose (Cambridge Isotope Laboratories Inc., Tewksbury, MA). The protein was subsequently purified using reverse-phase HPLC,17 and isotope incorporation was verified using MALDI mass spectrometry.</p><!><p>AviTag PrP was biotinylated according to the manufacturer's protocol (Avidity, Aurora, CO). Briefly, Biomix-A and Biomix-B were diluted from their 10× stock concentrations to 1× final concentrations by adding them to AviTag PrP. For 3.2 μg of total protein, 5 μg of BirA enzyme biotin ligase was added to each reaction, and samples were incubated in a thermomixer (Thermo Fisher Scientific, Rockford, IL) at a speed of 750 rpm overnight or for several hours. Five milliliters of 7 kDa zeba spin desalting columns (Thermo Fisher Scientific, Rockford, IL) was used to remove excess biotin. Two-hundred microliters of sample was diluted to a total volume of 500 μL using Assay Buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% Triton, and 1% Tween) before loading onto the column.</p><!><p>PrPInt1 was prepared by mixing 180 μL of recombinant PrP protein at a concentration of 0.25 mg/mL resuspended in Milli-Q H20 with 16 μL of 1-palmitoyl-2-oleoyl-sn-glycero-3- [phospho-rac-(1-glycerol)] (POPG, Avanti Polar Lipids, Alabaster, AL) at a concentration of 2.5 mg/mL, as previously described.14 POPG was prepared by resuspending lyophilized powder in chloroform, drying under a stream of nitrogen, and resuspending in 20 mM Tris-HCl buffer, pH 7.4, at which point the solution was sonicated at 4 °C in a bath sonicator until it was fully hydrated. The mixture of protein and lipid was incubated for 10 min at room temperature; 7359 μL of MQ H2O, 500 μL of 5% Triton X-100, and 900 μL of 10× TN Buffer (1.5 M NaCl and 100 mM Tris-HCL, pH 7.5) were added, and the solution was end-over-end rotated for 5 min. Addition of RNA was omitted from the original protocol, and 45 μL of MQ H2O was added in its place. The sample was centrifuged for 1 h at 100 000g in 1 mL aliquots. The supernatant was removed, retaining approximately 70 μL to conserve the integrity of the pellet. The pellet was washed twice with 1 mL of 1% n-octyl-β-D-glucopyrano-side, spinning at 100 000g for 1 h after each wash. The resulting pellet was resuspended in 200 μL of assay buffer and sonicated using a Misonix s3000 programmable microplate horn sonicator filled with 350 mL of H2O heated at 37 °C. The sonicator was set to a power level of 70, and three 15 s bursts were used followed by thorough vortexing for complete resuspension of pellet.</p><p>PrPC was purified from normal mouse brain as previously described.13</p><!><p>Biotinylated PrP peptide-capture assays were performed using a modified version of a previously described protocol.5 Streptavidin-coated magnetic beads (Dynabeads M280-Streptavidin, Invitrogen, San Diego, CA) were rinsed four times with assay buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% Triton, and 1% Tween) and diluted 10-fold. The beads were then incubated with 10 μM PBD (KKRPKPGGWNTK-Biotin), PBD* (KKRPKPGGWNTQQSRYPGQGK-Biotin), or CTL (PGQGSPGGNRYK-Biotin) peptide (Invitrogen, Carlsbad, CA) for 1 h at 37 °C while end-over-end rotating and subsequently washed four times with assay buffer before resuspension.</p><p>Twenty microliters of PrPInt1 was added to 100 μL of resuspended, peptide-conjugated beads, and 80 μL of assay buffer was added so that the total reaction volume was 200 μL. Samples were end-over-end rotated for 1 h at 37 °C. Reactions were then washed four times with 100 μL of assay buffer. Supernatant and wash fractions were retained, and each sample was resuspended in 100 μL of assay buffer. For analysis, equivalent fractions of supernatant, wash, and pellet fractions were loaded on SDS-PAGE gels and transferred to PVDF for immunodetection with PrP-specific antibody 27–33 as previously described.8</p><!><p>Photoaffinity peptide (PA-PBD; C-terminally labeled KKRPKPGGWNTK-(KLCBiotin)-(KAzidoBz)-amide, C35H54N11O6S) was incubated with PrPInt1 or control PrP samples (at concentrations of 100 μM peptide and 25 ng/μL protein unless otherwise indicated) in 100 μL reaction volumes. Reactions were incubated for 1 h at 37 °C using end-over-end rotation. Samples were then aliquoted into a white opaque shallow-well ProxiPlate-96 microplate (PerkinElmer, Waltham, MA), minimizing exposure to ambient light. Samples were photoaffinity labeled in the 96–well plates using a UV Stratalinker 2400 (Stratagene, La Jolla, CA) equipped with 312 nm bulbs in which samples were placed approximately 2.5 cm from the light source. The Stratalinker was utilized in the time mode, and samples were exposed to ultraviolet (UV) light for 0, 1, 3, or 5 min. The resulting photoaffinity-labeled molecules were run on SDS-PAGE, transferred to PVDF, blocked with a 2.5% solution of bovine serum albumin (Fisher Scientific, Pittsburgh, PA), and incubated with streptavidin-conjugated HRP (ThermoFisher Scientific, Rockford, IL) at a 1:10 000 dilution before being washed with TBST and developed with SuperSignal West Femto maximum sensitivity substrate (ThermoFisher Scientific, Rockford, IL).</p><!><p>Recombinant PrPSc for analysis by electron microscopy was generated, RNase- and protease-digested, purified, and concentrated as previously described.18 An identical volume of the substrate cocktail (containing recPrP, POPG, and total RNA) not subjected to PMCA was processed in parallel (including protease digestion) to serve as a negative control for the analysis. A mixture of 980 μL of deionized H2O, 10 μL of α-amylase (Sigma A7095), and 10 μL of amyloglucosidase (Sigma A8220) was filtered through a 0.22 μm filter. The filtered enzyme mixture or 1 mL of H2O (for mock treatment) was transferred to a new tube containing 20 μL of either processed recPrPSc or control substrate cocktail. Digestion proceeded for 60 min at 37 °C with shaking at 600 rpm. In parallel, digested and mock-treated samples were centrifuged at 100 000g for 1 h, and pellets were sequentially washed once with 1 mL 1% n-octylglucoside, 8.3 mM Tris, pH 7.5, and 150 mM NaCl and twice with 1 mL of H2O prior to spotting on grids for electron microscopy. Enzymatic activity under these experimental conditions was confirmed by digestion of GlycoBlue (Invitrogen, Carlsbad, CA).</p><!><p>PrPInt1 resuspended in molecular grade H2O was spotted onto a Formvar-coated 300 mesh copper grid and allowed to absorb for 1 min, after which two wash steps were performed with H2O. Control samples of PrPInt1 with either no PrP protein or, alternatively, with no POPG lipid were prepared according to the sample protocol, replacing either the protein or lipid portion with molecular grade H2O. The recPrPSc samples were digested with 5 μg/mL (final concentration) of soluble trypsin (Sigma, St. Louis, MO) overnight at room temperature and then inhibited with 2 mM PMSF (Sigma, St. Louis, MO). The digested recPrPSc was then spun at 100 000g for 1 h and resuspended in molecular grade H2O. Samples were stained with 2% uranyl acetate applied for 30 s and then allowed to dry overnight before imaging. Transmission electron microscopy was done at 100 kV using a JEM-1010TEM (JEOL Ltd., Tokyo, Japan) microscope with an integrated AMT digital camera (Advanced Microscopy Techniques, Woburn, MA) to capture images.</p><!><p>A sample of uniformly 13C- and 15N-enriched PrPInt1 prepared as described in the previous section was centrifuged into a 2.5 mm zirconia magic-angle spinning (MAS) rotor. The sample was held in place with micro-O-ring-sealed sample spacers to ensure that the sample maintained a constant state of hydration.19 Both 13C and 15N cross-polarization MAS (CPMAS) spectra were obtained from room temperature to −100 °C using a probe of our own construction20 with pulse sequences described elsewhere.21 31P spectra were acquired at −10 °C using a double-resonance 2.5 mm Agilent MAS probe. Two-dimensional 13C–13C correlation spectra and 15N–13C correlation spectra were also acquired at a temperature of −10 °C. An external-field frequency lock22 was employed to compensate for field drift. All NMR experiments were performed using a Varian VNMRS instrument operating at a 1H frequency of 799.8 MHz. The magic-angle spinning rate was typically 20 kHz, and 1H decoupling was applied at 110 kHz using either TPPM or XiX modulation. 13C chemical shifts were referenced to tetramethylsilane (TMS) using adamantane as an external reference.23</p><!><p>We recently characterized the incremental changes in recPrP conformation as it interacts with cofactors during the process of de novo prion formation.15 An intermediate species (PrPInt1) formed by the rapid interaction between recPrP with POPG at room temperature displays widespread and durable changes in protein conformation15 and can be purified by ultracentrifugation. We examined the ultrastructure of PrPInt1 by negative-stain transmission electron microscopy. Interestingly, POPG appears to induce the formation of irregularly shaped PrPInt1 aggregates. At higher magnification, these aggregates appear to be composed of variably stained, smooth-contoured subunits of ~20–30 nm in diameter (Figure 1). Aggregates were not observed on grids containing samples without POPG or PrP, confirming that interaction between protein and cofactor is necessary to produce aggregates (Figure S1). These results suggest that POPG is able to induce the formation of PrPInt1 aggregates. As previously described, the final product of the prion formation protocol (recPrPSc) also contains aggregates18 (Figure 1). These aggregates are not glycogen granules, which can be detected in some preparations of total liver RNA,24 because no protease-resistant aggregates could be seen on grids containing unsonicated substrate cocktail (which includes total RNA) processed in parallel (Figure S2) and also because treatment with amylase did not degrade recPrPSc aggregates (Figure S2).</p><!><p>Binding between PrPC and PrPSc molecules is an important step in the conversion of cellular PrPC into the PrPSc conformer. The polybasic domains of PrPC and, in particular, residues 23–33 at the extreme N-terminus have been identified as the primary PrPSc-binding epitopes.5–7 Given that POPG is able to induce widespread PrP conformational changes15 and aggregation (Figure 1), we hypothesized that PrPInt1 might contain a binding site for the PrPC polybasic domain. We used a biotinylated peptide representing the N-terminal 23–33 polybasic domain (PBD), which has been previously shown to capture PrPSc selectively,5 in streptavidin-based capture assays to measure binding between this domain and PrPInt1. The results show that PBD pulls down ~70% of total PrPInt1, compared to <10% of input α-helical recPrP (Figure 2A). PrPInt1 is also successfully captured by a longer peptide containing the polybasic domain (PBD*) but not by a control peptide (CTL) corresponding to a region of PrPC (residues 39–49) that does not bind PrPSc5,7 (Figure 2B). Therefore, the interaction between PrP and POPG appears to unmask a PrPC N-terminal polybasic-domain-binding site, demonstrating how cofactor-induced structural changes can promote intermolecular interactions.</p><!><p>To confirm the results of the pull-down assays, we designed a photoaffinity peptide composed of PrP polybasic domain residues 23–33 conjugated at the C-terminus to biotin and an aryl azide moiety (PA-PBD), and we used UV light to covalently attach this peptide to the PrPInt1 conformer (Figure 3A). The photoaffinity-labeled products, representing PA-PBD cross-linked to PrPInt1, can be visualized following SDS-PAGE and transfer to PVDF membranes using streptavidin-conjugated horseradish peroxidase (HRP) to probe for the biotinylated conjugates. The results show that PA-PBD peptide covalently labels PrPInt1 more efficiently than either PrPC (Figure 3A) or α-helical recPrP (Figure 3B). Lengthening the duration of UV light exposure increases the signal intensity of the photoaffinity-labeled band on the blot, whereas no photolabeled bands are seen in the absence of UV light (Figure 3A). Photoaffinity labeling of PrPInt1is also enhanced in proportion to increasing concentrations of PA-PBD peptide (Figure 3C). We measured the efficiency of PA-PBD photoaffinity labeling of PrPInt1 by using AviTag-PrP, a recombinant PrP fusion protein engineered to contain exactly one biotin adduct per molecule, as a reference standard. By comparing a known amount of photoaffinity-labeled PrPInt1 (PA-PrPInt1) to a standard curve representing known concentrations of biotinylated AviTag-PrP, we were able to estimate the stoichiometry of the photoaffinity-labeling reaction of PA-PBD to PrPInt1 to be ~1:2000 (Figure 3D). Potential explanations for the substoichiometric yield of labeled PrPInt1 include the presence of inaccessible subunits within PrPInt1 aggregates and/or relatively low chemical reactivity of photo-activated pheny azide photoreactive groups.</p><!><p>Given the results described above as well as the presence of lipid cofactors in protocols capable of producing high-titer infectious PrPSc,13,14,16 we sought to determine if POPG is incorporated into the PrPInt1 macromolecular structure by analyzing the insoluble conformer with solid-state nuclear magnetic resonance (ssNMR) spectroscopy. Using a sample of uniformly 13C- and 15N-enriched PrPInt1, 31P MAS spectra were acquired using both cross-polarization and single-pulse excitation. Regardless of the conditions employed, no 31P signals were in fact observed. Representative 13C, 15N, and 31P spectra obtained at −10 °C for this sample are shown (Figure 4A). Although the 13C and 15N spectra are easily observed in a few scans, no 31P resonances can be found even with an overnight acquisition (Figure 4A). This quantitative conclusion is based on comparison to the 31P signals obtained on a standard sample of ammonium dihydrogen phosphate (ADP). The 6 μL MAS rotor sample volume accommodates 9.4 × 10−5 mol of ADP. Assuming a typical protein density of 1.4 g/cm3, the same sample cell will hold 3.1 × 10−7 mol of PrPInt1. If there is only one molecule of POPG per PrPInt1, then the 31P NMR signal would have an integrated intensity ~1/300 of that for the ADP standard sample. The ADP spectrum itself has a relatively narrow line of 0.5 ppm full width at half-maximum and in a single scan has a peak signal 210 times as large as the peak-to-peak noise for the 31P window (Figure 4A). If the POPG were as narrow as the ADP standard, then the 10 000 scan spectrum in the 31P spectrum would have a peak 70 times the noise observed. Even if the line shape were as broad as 5 ppm, the raw peak signal to peak-to-peak noise ratio would still be over 7 and quite easily observed. Because the 31P shift range for phospholipids is fairly narrow and MAS line shapes for POPG are quite generally found to be substantially narrower than 5 ppm, the simplest conclusion consistent with these data is that POPG cannot be present in PrPInt1 in stoichiometric amounts.</p><!><p>The ssNMR analysis of the sample also allowed us to look at the overall structural features of PrPInt1. Analysis of the 13C chemical shifts and spectral intensities demonstrates that PrPInt1 is a highly structured macromolecular complex with little molecular mobility and identifiable helical content (Figure 4B). CPMAS 13C spectra detect relatively rigid components in solid samples, whereas Bloch decay MAS spectra will preferentially observe motionally narrowed segments. Regardless of the temperature, the 13C CPMAS and Bloch decay spectra of PrPInt1 have essentially identical intensity profiles. This is in stark contrast to results obtained for huPrP23–144 amyloid (no CPMAS signals observed for 23–111)25 or on Syrian hamster PrP23–231 amyloid (weak or no CPMAS signals observed for 23–129).26 PrPInt1, therefore, appears to be well-structured, with no evidence for extended highly dynamic segments.</p><p>Although the spectra are not well-resolved enough to make much headway on site-specific assignments, several unique patterns for different amino acids are nevertheless readily recognized.27 Thr, Pro, Ala, and Val Cα to Cβ cross peaks are easily picked out as indicated with arrows (Figure 4B). Gly Cα carbon resonances characteristically appear in the 42–46 ppm window with respect to TMS. The uniquely positioned Gly Cα to CO cross peaks are also quite evident, making this assignment possible, and have an intensity consistent with 37 Gly residues. Because 37 of the 38 Gly residues are in the 23–127 segment, observation of these peaks alone points to the absence of dynamic disorder in the N-terminus.</p><p>The chemical shifts observed also provide insight into the elements of secondary structure present. Cα to Cβ cross peaks in many instances can be used to assign secondary structure to particular amino acids.28 Of the peaks observed, those for Ala, Val, and Gly provide the best discrimination between α-helical, β-sheet, and random-coil motifs. The average Cα, Cβ, and CO 13C chemical shifts for these three amino acid types are indicated as color-coded ellipses in Figure 4B, where the axes of the ellipses drawn indicate the standard deviations reported.28 The majority of the Gly residues observed here in PrPInt1 contribute to the Cα/CO cross peak at 45.4/173.2 ppm from TMS. These coordinates are both approximately 2 ppm higher than those for either β-sheet (43.3/170.6 ppm) or random coil (43.5/171.9 ppm) structures,28 clearly demonstrating that the observed Gly residues must be present in α-helices. Because 37 of the 38 Gly residues are contained in the N-terminus, it follows that the N-terminus overall has adopted an α-helical conformation in PrPInt1. The Ala residues, six of eight of which are in the N-terminal segment, also have chemical shifts that are most consistent with an α-helical structure of this region.</p><!><p>Evidence from reconstitution studies indicates that cofactor molecules play a critical role in facilitating the formation of infectious mammalian prions in vitro.13,29 High-titer infectious prions can be formed de novo from substrate cocktails containing only purified PrPC and cofactor molecules in the absence of infectious seed,13 whereas various preparations of PrPSc and misfolded PrP lacking cofactors display at least 105-fold lower levels of specific infectivity.30–33 One protocol that can be used to produce infectious prions de novo involves exposing recPrP sequentially to POPG and RNA.14 We recently showed that the interaction between recPrP and POPG causes dramatic changes in recPrP conformation (measured by solvent accessibility by DXMS), some of which are durable (i.e., persist in the final infectious PrPSc conformer).15 Previous studies showed that POPG induces β-sheet structure34–36 and protease resistance.36 These changes are likely mediated through an interaction between the phospholipid and the central hydrophobic region of PrP encompassing the polymorphic residue 129.37 Here, we further characterize the mechanism and consequences of recPrP/POPG interaction to help elucidate the role of cofactor molecules in prion formation.</p><p>Ultrastructural analysis by electron microscopy revealed that PrPInt1, the intermediate conformation formed by the interaction of recPrP and POPG, appears to be aggregated. The induction of protein aggregation is likely to be a key event in prion formation because infectious prions are invariably insoluble and aggregated. Because POPG is a relatively small molecule (unlike RNA), it likely does not cause aggregation simply by acting as a scaffold between PrP monomers. PrPInt1 aggregates differed morphologically from the ~40–50 nm wormlike structures formed when PrP is mixed with POPG for extended periods of time at 37 °C.36 It is not known whether such wormlike structures remain eligible for prion formation (i.e., whether addition of RNA and PMCA would produce infectious prions) or whether they represent an off-pathway product.</p><p>We performed a series of PrP polybasic-domain peptide-binding experiments to investigate the mechanism by which POPG induces PrP aggregation. Previous studies using a series of overlapping biotinylated peptides or motif-grafted antibodies to walk the linear polypeptide sequence of PrP identified the two PrP polybasic binding domains (residues 23–33 and 100–110)5–7 as consensus epitopes on PrPC that bind PrPSc, and removal of residues 23–88 completely inhibits the ability of PrPC to bind PrPSc.8 Thus, conjugated PrP23–33 peptides can be used as specific probes to determine whether a PrP conformer is able to bind PrPC. Using this approach, we found that POPG unmasks a PrPC polybasic-domain-binding site in PrPInt1 in both capture and photoaffinity experiments. Presumably, the POPG-induced conformational unmasking of this neoepitope facilitates binding of recPrP to PrPInt1 and promotes subsequent aggregation. Interestingly, the association between protein aggregates and the N-terminus of PrPC may be a general phenomenon. Resenberger et al.38 showed that the N-terminus of PrPC also binds and mediates the toxicity of Aβ oligomers39 and yeast prions40 in mammalian cells.</p><p>To investigate in greater detail the mechanism by which POPG induces structural and functional changes in PrP, we analyzed PrPInt1 chemical composition with ssNMR. The lack of phosphorus atoms in recPrP allowed us to determine whether POPG (which contains phosphorus in its headgroup) remains bound to PrP after formation of the PrPInt1 conformation or whether the physical interaction between the protein and phospholipid molecules is transient. The results show that POPG is not present in stoichiometric amounts in PrPInt1 and therefore we can conclude that POPG induces the conformational changes in PrP through a transient interaction. Once formed, neither PrPInt1 structure nor its ability to bind the PrPC polybasic domain depends upon continued POPG occupancy. These results are consistent with those of a previous study in which in situ degradation of an incorporated photolabile nucleic acid cofactor did not alter the strain properties or titer of purified hamster prions produced in vitro41 and raise the possibility that cofactor molecules may be able to induce the specific structural changes required for infectivity and strain properties in PrPSc without physically forming a complex with PrP. However, to confirm this important mechanistic point, it will be necessary to perform similar chemical composition determinations of infectious PrPSc molecules formed with only a single cofactor, such as phosphatidylethanolamine.16,29 The conformational change of PrP induced by transient interaction with lipid molecules is remarkable but not unprecedented; for example, conformational changes were also observed for the Bax protein exposed to membrane lipids.42 In the case of PrP aggregation caused by transient interaction with POPG, the structure of the induced aggregation-prone conformer may be stabilized by intermolecular interactions.</p><p>Additional analyses of the ssNMR spectra also provided information about certain aspects of PrPInt1 conformation. In particular, we were able to deduce that POPG induces the N-terminus, which is unstructured in PrPC, to form a helical structure. These results are consistent with recent DXMS data showing that POPG causes a moderate level of protection from solvent accessibility in the N-terminus of PrP15 as well as other experiments examining the effects of another anionic phospholipid, lysophosphatidylserine, on recPrP secondary structure.43 Previous NMR studies show that both α-helical PrP and PrP amyloid fibers possess disordered N-termini25,26,44,45 and therefore the presence of helical elements in the N-terminus of PrPInt1 is unusual. The significance of this finding is unclear, especially because the subsequent addition of RNA during the prion formation process appears to re-expose the N-terminus fully.15 Previous studies described the ability of the N-terminal PrP region to acquire poly(L-proline) type II helix conformation under several solvent conditions including phospholipids.46,47 Another study revealed that the N-terminal polybasic region 23–230 controls the physical properties of alpha-recPrP and its aggregation pathways.48</p><p>Two recent studies showed that noninfectious PrPSc conformers with a smaller protease-resistant core than infectious PrPSc could also be formed in a stochastic fashion when recPrP is subjected to sPMCA in the presence of POPG and RNA.24,49 These conformers are reminiscent of the noninfectious protein-only PrPSc conformer, which is formed in a stochastic manner upon cofactor withdrawal and also possesses a smaller protease-resistant core than infectious PrPSc. It would be interesting to perform similar cofactor-withdrawal experiments to determine whether the noninfectious PrPSc conformers produced in the presence of POPG and RNA are also protein-only conformers formed as a result of suboptimal interactions between the cofactors and recPrP during sPMCA or are truly cofactor-dependent.</p><p>The observation that lipid and polyanionic cofactors are required for the formation of highly infectious prions in vitro is consistent with the important role played by lipid rafts for prion formation in living cells50 as well as the observation that membrane environment influences PrP conformation and stability.43,51 Raft domains may harbor high concentrations of specific phsopholipid and other cofactor molecules that enable prion formation. Although POPG can be used to facilitate prion formation in vitro,14 phosphatidylglycerol itself is not likely to be an endogenous prion conversion cofactor in neurons because it is not generally found in the extracellular raft domains where PrPC normally resides.</p><p>In conclusion, using a combination of biophysical and biochemical techniques, we have determined that the prion propagation cofactor POPG unmasks a PrPC polybasic-domain-binding site in PrP, inducing PrP aggregation. However, these effects do not appear to require the continued presence of the POPG cofactor in the PrP folding intermediate.</p>