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Biophys J. 2015 Nov 17; 109(10): 2090–2100.
Published online 2015 Nov 17. doi: 10.1016/j.bpj.2015.10.009
PMCID: PMC4656879
PMID: 26588568

Insight into Early-Stage Unfolding of GPI-Anchored Human Prion Protein

Emilia L. Wu,1 Yifei Qi,1 Soohyung Park,1 Sairam S. Mallajosyula,2,3 Alexander D. MacKerell, Jr.,3 Jeffery B. Klauda,4 and Wonpil Im1,∗
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Associated Data

Supplementary Materials

Abstract

Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrPSc) converted from a normal host cellular prion protein (PrPC). Experimental studies suggest that PrPC is enriched with α-helical structure, whereas PrPSc contains a high proportion of β-sheet. In this study, we report the impact of N-glycosylation and the membrane on the secondary structure stability utilizing extensive microsecond molecular dynamics simulations. Our results reveal that the HB (residues 173 to 194) C-terminal fragment undergoes conformational changes and helix unfolding in the absence of membrane environments because of the competition between protein backbone intramolecular and protein-water intermolecular hydrogen bonds as well as its intrinsic instability originated from the amino acid sequence. This initiation of the unfolding process of PrPC leads to a subsequent increase in the length of the HB-HC loop (residues 195 to 199) that may trigger larger rigid body motions or further unfolding around this region. Continuous interactions between prion protein and the membrane not only constrain the protein conformation but also decrease the solvent accessibility of the backbone atoms, thereby stabilizing the secondary structure, which is enhanced by N-glycosylation via additional interactions between the N-glycans and the membrane surface.

Introduction

Prion diseases are an unusual class of fatal mammalian neurodegenerative disorders, which include scrapie in sheep, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Sträussler-Scheinker syndrome, and Kuru in humans (1, 2). These diseases affect the central nervous system by sporadic, genetic, or infectious means (3, 4, 5, 6). The fundamental events in the pathogenic process of the prion diseases, according to the widely accepted protein-only hypothesis, involve conversion of the normal cellular form of prion protein (PrPC) to misfolded scrapie isoforms (PrPSc), followed by the aggregation of PrPSc into amyloid fibers and plaques in the brain (7, 8, 9, 10, 11, 12, 13). PrPSc and PrPC share the same primary sequence, and they are chemically indistinguishable (6, 14); however, their secondary structures are remarkably distinct (15, 16, 17, 18, 19, 20). Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic studies reveal that PrPC adopts an α-helix-rich fold (42%) with little β-sheet structures (3%) (19), whereas PrPSc contains a significant β-sheet content (43% to 54% depending on the molecular length used for measurements) and less helical structures (17% to 30%) (19, 21).

PrPC is expressed in a variety of different tissues, including the brain with particularly high levels, circulating lymphocytes, heart, and skeletal muscle (22). Despite intensive studies, the precise physiological function of PrPC is elusive, although there is evidence that PrPC is involved in the regulation of copper metabolism, signal transduction, and neuron protection from oxidative stress (23, 24, 25). The solution structures of human, mouse, hamster, and bovine PrPC fragments have been determined by NMR spectroscopy (26, 27, 28, 29, 30). In contrast, the structural properties of PrPSc aggregates still remain unresolved by traditional high-resolution techniques, such as x-ray diffraction and solution NMR spectroscopy, because of its insoluble nature (21, 31). Human PrPC (HuPrP) 90-230 fragment, generated by N-terminal truncation through digestion with proteinase K, is considered to be the minimal unit for prion infectivity (8, 32). This fragment is composed of a large unstructured domain (residues 90 to 124) and a globular domain (residues 125 to 230) (Fig. 1 A) with three α-helices (HA: residues 144 to 154, HB: residues 173 to 194, and HC: residues 200 to 228), a short two-stranded antiparallel β-sheet (S1: residues 128 to 131 and S2: residues 161 to 164), and a single disulfide bond (Cys179-Cys214) connecting HB and HC.

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Snapshots of four PrPC simulation systems: (A) PrP-only, (B) PrP-nglycan, (C) PrP-memb, and (D) PrP-nglycan-memb. PrPC, N-glycan1, N-glycan2, GPI anchor, CHOL, POPE, and PSM are represented in cartoon (HA, orange; HB, blue; HC, red), green surface, blue surface, magenta sticks, light green spheres, light blue spheres, and light yellow spheres, respectively. Water molecules and KCl ions are omitted for clarity. To see this figure in color, go online.

HuPrP is a glycoprotein with two N-glycosylation sites at Asn181 and Asn197 (Figs. 1 B and ​and2),2), and the glycosylation sites are variably occupied. The unglycosylated (5%) and monoglycosylated (25%) isoforms are minor cell-surface components, whereas the diglycosylated (70%) form is dominant (34, 35). The flexible nature and conformational dynamics of these oligosaccharides make the characterization of their structures very challenging by NMR experiments (36). Mutation experiments to omit each N-glycosylation suggest that PrPC has an intrinsic tendency to acquire some PrPSc-like properties, and the N-glycan oligosaccharide chains can modulate the efficiency of this conversion process (37).

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Sequence and linkage information for HuPrP N-glycan and GPI anchor oligosaccharide chains (33). To see this figure in color, go online.

The mature form of PrPC is anchored to the cell membrane via a glycosylphospatidylinositol (GPI) anchor (Fig. 1, C and D), which is a complex glycophospholipid present in eukaryotic cells (38). The most fundamental function of GPI anchors is to provide the efficient and stable association of protein with the lipid bilayer (39) and also allow the anchored proteins to have increased mobility compared to transmembrane proteins (40). Although its exact function is not clear, abnormalities in GPI synthesis have been implicated in some diseases such as paroxysmal nocturnal hemoglobinuria and embryonic lethality (38, 40, 41, 42). GPI anchors have diverse structures but share a conserved core structure: phosphoethanolamine (EtNP)-Man-α(1→2)-Man-α(1→6)-Man-α(1→4)-GlcN-α(1→6)-myo-inositol phospholipid (38), where the EtNP unit is covalently bound to the protein C-terminus (Fig. 2). After attachment of the GPI anchor to HuPrP Ser230, this diglycosylated protein is targeted to lipid rafts on the outer leaflet of the plasma membrane (7, 39). Lipid rafts are nano- or microscale membrane microdomains enriched in cholesterol (CHOL) and sphingomyelin (PSM) (43, 44, 45). They are postulated to play an important role in membrane trafficking and cell signaling (46, 47, 48).

Exploring the structural and dynamical properties of membrane-bound PrPC in its native membrane environment is an important, but challenging task. In this study, we have performed extensive molecular dynamics (MD) simulations to model glycosylated PrPC in a lipid mixture (mimicking a lipid raft) to investigate the influence of N-glycosylation and interactions with the membrane on its conformational stability at the atomic level and to provide insight into the possible mechanism of early-stage unfolding of PrPC.

Materials and Methods

System setup

A HuPrP model with two N-glycans and GPI anchor was constructed by generating each part of the molecule and linking them together with proper patches using CHARMM (49). The NMR structure of HuPrP (residues 125 to 228) was obtained from PDB: 1QLX (26) and the two C-terminal missing residues (Gly229 and Ser230) were added based on CHARMM (49) internal coordinate (IC) information. The missing unstructured N-terminal residues were ignored. The protonation states were based on pH value of 7 (i.e., neutral His residues and charged acidic and basic residues), which corresponds to the extracellular environment (50). Cys179 and Cys214 were patched together to form a disulfide bond. The N-glycan oligosaccharide chains (Fig. 2) were generated by connecting all the sugars residues together and then attaching them to Asn181 and Asn197, respectively. The identical glycoform was used for both glycosylation sites. For modeling and simulation of the GPI anchor (Fig. 2), we constructed several additional CHARMM residues (AGLCNP for GlcN, ETH and ETHN for EtNP, and DSPI for distearoylphosphatidylinositol) and patches (GPIP1 linkage between DSPI and GlcN, GPIP2 and GPIP4 linkages between Man and EtNP, and GPIP3 linkage between EtNP and Ser230 of HuPrP). The force field parameters of these new residues and patches were transferred by analogy based on the CHARMM36 (C36) lipid (51) and carbohydrate (52, 53, 54) force field (available at http://mackerell.umaryland.edu/charmm_ff.shtml) except for the dihedral angle C5-C6-OP1-P1 (in GPIP2 and GPIP4 linkages), which was explicitly parameterized as part of this study (see the details in the Supporting Material). The sequence and linkage information for N-glycan and GPI anchor are based on the study of DeMarco et al. (33). The initial coordinates of the N-glycans and GPI anchor were generated with CHARMM IC functionality and the glycosidic torsion angles were further refined using the top cluster conformation searched from Glycan Fragment DB (GFDB: http://www.glycanstructure.org) (55).

Following the general procedure to build a protein/membrane complex structure in Membrane Builder (56, 57, 58) in CHARMM-GUI (59), the HuPrP model with the N-glycans and GPI-anchor were preorientated with respect to the membrane normal (i.e., the z axis), the DSPI phosphate group was initially positioned around z = 20 Å, and the orientation of the inositol ring with respect to the membrane normal was adjusted to 60° according to our recent study about inositol ring orientations in membranes (60). The entire model was then inserted into a CHOL/POPE/PSM lipid mixture (number of lipids: 145/149/135) to mimic a raft model. Phosphatidylethanolamine (PE) lipids were chosen because they are more prevalent than phosphatidylcholine (PC) for neurological myelin sheath membranes. Spingomyelin (SM) and PE lipids constitute 70% of phospholipids (in equal proportion) with PC only being 8% (61, 62). A free GPI anchor was inserted into the other leaflet to prevent an area mismatch in both leaflets and also to examine the conformation of GPI that is not connected to protein. The rest of the system building followed the same steps in Membrane Builder (56, 57, 58), i.e., building a bulk water box, addition of K+ and Cl− ions (150 mM), and the assembly of each component (Fig. 1 D).

In addition to the HuPrP system with the N-glycans and GPI anchor in a CHOL/POPE/PSM membrane (denoted as PrP-nglycan-memb, see Table S1 for system naming in this study), we also constructed three other systems to explore the impact of the N-glycans and membrane on the structural stability of PrPC. These include 1) HuPrP in solution (PrP-only: Fig. 1 A) to represent the recombinant PrP primarily used in in vitro experiments (33), 2) HuPrP with two N-glycans in solution (PrP-nglycan: Fig. 1 B), and 3) HuPrP only with the GPI anchor in a CHOL/POPE/PSM membrane (PrP-memb: Fig. 1 C). All molecular graphics was generated by PyMOL (63).

Simulation details

About 4.5-ns equilibration simulation was first performed using CHARMM (49) with the C36 force fields for the protein (64), lipid (51, 65), carbohydrate (52, 53, 54), and TIP3P for water (66). Based on the equilibration protocol used in CHARMM-GUI Membrane Builder, various restraints were applied to the protein, lipid, and water molecules to ensure gradual equilibration (56). The restraint forces were gradually reduced during the equilibration. Additional dihedral angle restraints were applied to restrain the sugar and inositol rings (of the N-glycans and GPI anchor) to the pertinent chair conformation. The NVT (constant particle number, volume, and temperature) dynamics was used first and followed by the NPT (constant particle number, pressure, and temperature) dynamics. Then, an additional 50-ns NPT equilibration run was performed with NAMD (67) without any restraints. All equilibration simulations were performed using the following protocol. We used a 2-fs time step with the SHAKE algorithm (68). The van der Waals interactions were smoothly switched off at 10 to 12 Å by a force-switching function (69) and the long-range electrostatic interactions were calculated using the particle-mesh Ewald method (70). The temperature and pressure were held at 300 K and 1 bar, respectively. In CHARMM simulations, Langevin temperature control was used for NVT dynamics. Temperature and pressure controls were achieved with a Hoover thermostat (71) and Langevin-piston barostat for NPT dynamics (72, 73). For NAMD NPT simulations, Langevin dynamics was used to maintain a constant temperature with a Langevin coupling coefficient set to 1 ps−1, and a Nosé-Hoover Langevin-piston (74, 75) was used to maintain constant pressure with a piston period of 50 fs and a piston decay of 25 fs.

For the production run, each system was simulated for 5 μs on Anton (76) with the CHARMM C36 force fields (51, 52, 53, 54, 64, 65), yielding a total of 20 μs, which is significantly longer than any previous simulation studies of similar systems (33). NVT ensemble was used with the temperature maintained at 300 K using the Nosé-Hoover method (77). Short-range forces and long-range electrostatics were evaluated every 2 and 6 fs, respectively. Long-range electrostatics was calculated using the k-Gaussian split Ewald method (78) with a 64 × 64 × 64 grid. SHAKE (68) was used to constrain all bonds involving hydrogen atoms and the time step was 2 fs. Trajectories were saved every 240 ps. The last 4 μs of each trajectory was analyzed to obtain the average structural properties.

Results and Discussion

Membrane structural properties

Various membrane structural properties were calculated to evaluate the appropriateness of using the CHOL/POPE/PSM lipid mixture to mimic a lipid raft. Fig. S1 shows the time series of the membrane hydrophobic thickness of PrP-nglycan-memb and PrP-memb systems by measuring the average distance between acyl chain C2 atoms (C4S and C2F for PSM) in the top and bottom leaflets. For both bilayers, the calculated hydrophobic thicknesses (35 to 36 Å) are significantly bigger than regular POPC (28 to 29 Å) and POPE (31 Å) bilayers (60, 79), indicating that the bilayers are in a liquid-ordered state. The 0.5-Å difference between PrP-nglycan-memb and PrP-memb arises from the fact that the bilayer slightly expanded in the xy dimension because of the interaction between the lipids and N-glycans, which will be discussed below. Consistent with the hydrophobic thickness, lipid chain order parameters of POPE and PSM (Fig. S2) are much higher than those in a lipid-disordered state (generally well below 0.3) (60), suggesting that the current lipid mixture is reasonable in terms of mimicking an ordered lipid raft.

The heavy atom density profiles along the bilayer normal (z axis) for water, lipid components, prion, and N-glycans are shown in Fig. 3. Lipid carbon tail and headgroup distributions are identical for PrP-nglycan-memb and PrP-memb systems. As expected from the hydrophobic thickness and lipid deuterium order parameters, the high chain order is also reflected with bigger plateau and sharp narrow trough in the lipid tail distribution. Prion and N-glycans are very flexible (in terms of their orientations with respect to the membrane) as shown in their broad distributions, which overlap with lipid headgroups.

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Density profiles of water (blue), lipid headgroup (orange), lipid carbon tail (green), prion protein (gray), N-glycan1 (magenta), and N-glycan2 (cyan) for PrP-nglycan-memb (solid lines) and PrP-memb (dotted lines). To aid viewing, the distributions of prion protein, N-glycan1, and N-glycan2 are scaled by a factor of ten. To see this figure in color, go online.

To investigate the domain formation in the membrane and characterize the nature of the neighbors of the GPI anchor, a hidden Markov model (HMM) was used to map the two-dimensional (xy plane) lipid distributions and their order-states (see Supporting Material). As shown in Fig. S3, there is no clear domain formation for both leaflets of PrP-nglycan-memb and PrP-memb, possibly because of small system size. The number of the nearest neighbors around each lipid indicates that there is a slight tendency for POPE aggregation, whereas it is opposite for CHOL (Fig. S4). Considering that POPE is in a relatively low order-state (Fig. S3) among these three lipids, the GPI anchor does not show a preference of any lipid types in its neighbors given that the number of high order-state lipid (sum of PSM and CHOL) and the number of low order-state lipid (POPE) are similar (Fig. S4, A and B). Free GPI prefers higher order-state lipids (PSM and CHOL) (Fig. S4, C and D). The lipid chain orders of both GPI anchor and free GPI show that they are in the liquid-ordered state (Fig. S5).

GPI conformations

The density profiles of the inositol rings and the specific residues in the terminus of each oligosaccharide branches of both GPI anchor and free GPI are shown in Fig. 4. Compared to the distributions of the GPI anchors (Fig. 4 A), the free GPIs are more flexible with broader distributions (Fig. 4 B), indicating that the conformations of the GPI anchor are more confined. The distributions of the free GPIs are very similar for PrP-nglycan-memb and PrP-memb, indicating good sampling over the simulations. For the GPI anchors, the distributions of mannose (blue) and phosphoethanolamine (green), which are either close or directly connected to HuPrP, apparently shift away from the membrane with larger z values in PrP-memb (Fig. 4 A). This is likely a reflection of less interactions with the membrane when the two N-glycans are absent, which will be discussed below.

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Density profiles of some specific residues in (A) GPI anchor and (B) free GPI for PrP-nglycan-memb (solid lines) and PrP-memb (dotted lines). The corresponding residues are shown on the top with the same color scheme. To see this figure in color, go online.

HuPrP conformational variations in different environments

To analyze the overall conformational stability of the protein, the root-mean-square deviation (RMSD) time series of HuPrP backbone heavy atoms from the starting structure were calculated (Fig. 5). The HuPrP conformations of PrP-nglycan and PrP-only exhibit larger overall RMSD, but the structures anchored in the membrane (PrP-nglycan-memb and PrP-memb) tend to be more stable with lower RMSD. The RMSD jumps at ∼2 μs in PrP-nglycan and at ∼1.5 μs in PrP-only mainly arise from the loop between HB and HC (circled in black dash line in Fig. S6) being swung around.

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Time series of the root-mean-square deviation (RMSD) of HuPrP backbone heavy atoms from the starting structure. The structural alignment was based on the most stable helical portion (HA: residues 144 to 154, HB: residues 173 to 187, and HC: residues 200 to 224). To see this figure in color, go online.

PrP secondary structure stability

PrPC can misfold spontaneously in vivo without mutations or exposure to infected material (80). As shown in Fig. S7, the helicity of PrP-only and PrP-nglycan drop quickly from the beginning of the simulations. In contrast, the two membrane-containing systems (PrP-nglycan-memb and PrP-memb) can maintain the integrity of the secondary structure over the 5-μs simulations.

To better understand the α-helical propensity of HuPrP amino acid sequence, we investigated the secondary structure time series for each residue (Fig. 6). For PrP-only and PrP-nglycan, HB and HC start to unfold and the length of helices become shorter at the C-terminus early in the simulations, and the secondary structure of HA fluctuates between helix and coil. For PrP-nglycan-memb and PrP-memb, HB maintains the helix structure throughout the simulations with longer HB length in PrP-nglycan-memb. Residues localized on both HA and HC C-termini unfold and fold back multiple times during the simulations. Furthermore, all four systems exhibit either elongated or additional β-sheet structure formation transiently. Thus, most helices unfold to different extents over the simulations in all four systems, with the most unfolding occurring at the C-termini of the helices. Interestingly, the HB C-terminus (residues 188 to 194) presents distinct behaviors between systems with and without the membrane. Additionally, the secondary structures of prion protein in PrP-nglycan-memb and PrP-memb are better preserved throughout the simulations than those in PrP-only and PrP-nglycan, which is in good accordance with the aforementioned RMSD analysis.

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Time series of HuPrP secondary structure (α-helix, red; coil, green; β-sheet, black) of each residue: (A) PrP-only, (B) PrP-nglycan, (C) PrP-memb, and (D) PrP-nglycan-memb. The secondary structures are defined based on DSSP algorithm (81, 82). To see this figure in color, go online.

Causes of distinct unfolding behaviors of HB in different environments

To understand the reason why a pronounced variation of helical stability of HB exists in different environments, it is vital to explore how the helix unfolds during the simulation. The HB C-terminus (residues 188 to 194), where distinct helix unwinding features are found between systems with and without the membrane, is a fragment with highly conserved threonine-rich sequence (TVTTTTK). Here, one threonine residue (Thr190) in PrP-nglycan is selected as an example to illustrate the unfolding process at the HB C-terminus. Fig. 7 is a series of snapshots showing the hydrogen bond network around Thr190 at ∼60 ns, when helix unfolding occurs. In the initial structure, the amide NH of Thr190 is hydrogen-bonded to the backbone carbonyl oxygen of Gln186 and the helix is intact (Fig. 7 A). At 62.4 ns, an additional hydrogen bond is formed between Gln186 and the hydroxyl oxygen of the Thr190 side chain (Fig. 7 B). This leads to disruption of the backbone NH…O hydrogen bond, while the one with the side chain hydroxyl still remains, so that the helix is distorted (Fig. 7 C). At 63.6 ns, the remaining hydrogen bond between Thr190 and Gln186 is broken, and the corresponding hydroxyl oxygen atom forms hydrogen bonds with water molecules (Fig. 7 D) leading to further distortion of the helix. Right after that, the helix structure at Thr190 unfolds (Fig. 7 E). It is therefore likely that the competition between hydrogen bonding of the sidechain hydroxyl with the backbone and solvent plays a crucial role in the helix unfolding mechanism.

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Hydrogen bond network of HuPrP Thr190 in PrP-nglycan: (A) initial structure, (B) at 62.4 ns, (C) at 63.36 ns, (D) at 63.6 ns, and (E) at 64.32 ns. Helices are shown in cartoon representation, Thr190 and Gln186 in sticks representation, and other residues including water molecules in line representation. Hydrogen bonds are shown in black dash lines. To see this figure in color, go online.

To further examine the quantitative contribution of solvation to helix stability, the time series of HuPrP backbone atoms’ solvent-accessible surface area (SASA) of each residue were analyzed (Fig. 8). As expected, a good correlation between the PrP secondary structure (Fig. 6) and its SASA value was observed. For example, when the SASA of a certain residue increases, the corresponding residue has a higher tendency to unfold, indicating that solvation contributes to helix unfolding. For PrP-only and PrP-nglycan, the increased exposure of the prion protein to water results in increased helix unwinding. However, for PrP-memb and PrP-nglycan-memb, the interactions between prion protein (and N-glycans) and the membrane effectively shield certain residues from the solvent. This likely explains the different helix stability in solvent environments (PrP-only and PrP-nglycan) and in membrane environments (PrP-memb and PrP-nglycan-memb).

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Time series of HuPrP backbone atoms’ solvent-accessible surface area (Å2) of each residue: (A) PrP-only, (B) PrP-nglycan, (C) PrP-memb, and (D) PrP-nglycan-memb. To see this figure in color, go online.

Interactions of the prion protein with the membrane are responsible for lowering protein-solvent interactions, which increase the helix stability in PrP-memb and PrP-nglycan-memb. Then, the question is: What is the role of the two N-glycans? To answer this question, it is of interest to examine the interaction pattern of each protein residue (and N-glycan sugar residues) with lipid molecules or water in PrP-memb and PrP-nglycan-memb. A cutoff distance of 5 Å was used to define a contact in this analysis, and the results are presented in Fig. 9. Compared to PrP-nglycan-memb, the C-terminal regions of HA and HC form more contacts with the membrane in PrP-memb. On the other hand, more HB C-terminal residues interact with the membrane in PrP-nglycan-memb. In addition, the N-terminus loop and the HA-HB loop also show more contacts with the membrane. Both N-glycans form strong and consistent interactions with the membrane, and some sugar residues penetrate through the lipid headgroup region and interact with lipid aliphatic tails. N-glycan2 exhibits slightly more interactions with the membrane (66% of the overall time period) compared to N-glycan1 (62%). It is worth noting that N-glycan2 is in the vicinity of the HB C-terminus spatially, so the interaction between N-glycan2 and the membrane could effectively protect residues in HB from interacting with the solvent and thus unfolding as well.

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Interaction patterns of HuPrP residues (and N-glycan sugar residues) with the environment components in (A) PrP-memb and (B) PrP-nglycan-memb. The graph shows, for each residue, the frequency of contact occurrence of a water molecule (blue), a headgroup (orange), and a lipid tail (green). A contact is first counted when the distance between the heavy atoms of a residue and those of its interacting partner is within 5 Å, and normalized for each interacting partner. The bar below each set of patterns indicates protein helices (coral) and two N-glycans (green and blue). To see this figure in color, go online.

Based on the interaction pattern comparison between PrP-memb and PrP-nglycan-memb, it is evident that the prion protein interacts with the membrane in two different modes depending on existence of the N-glycans. Two representative snapshots are shown in Fig. 10. In PrP-memb, the HA C-terminal side of the protein points down and interacts with the membrane (Fig. 10 A), whereas in PrP-nglycan-memb, the HB C-terminal or N-glycan2 side mainly interacts with the membrane (Fig. 10 B). These interactions between the prion protein (and N-glycans) and the membrane can decrease its exposure to solvent and also provide effective constraints for the protein conformation, which is essential for stabilizing the protein secondary structure. The presence of the N-glycans enhances the interaction of HB with the membrane, which could potentially facilitate retention of the helical conformation in PrP-nglycan-memb.

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Representative snapshots of HuPrP (and N-glycans) on the membrane surface: (A) PrP-memb and (B) PrP-nglycan-memb. The color scheme is the same as in Fig. 1: HA, orange; HB, blue; HC, red. To see this figure in color, go online.

To illustrate the relative position of the prion protein with respect to the membrane, the time series of the z coordinate of the center of mass (ZCOM) of the HB C-terminal fragment (residues 188 to 194), which is unfolded in solution environments (PrP-only and PrP-nglycan), are shown in Fig. 11. It is particularly evident that the prion protein’s orientation with respect to the membrane undergoes considerable fluctuations considering its rigid conformation (Fig. 5). Prion protein can tilt over with the HB C-terminus close to the membrane surface, or it can stay upright with the HB C-terminus far from the membrane. In general, the protein displays a rocking motion with the GPI anchor as a hinge (see Figs. 1 and ​and1010 for different orientations of prion protein with respect to the membrane). For 79% of the overall time period, ZCOM is less than 40 Å from the membrane for PrP-nglycan-memb, whereas it is 61% for PrP-memb, indicating that the N-glycans effectively enhance the prion-membrane interactions: e.g., the head group peak occurs at z = 21 Å in Fig. 3. Without the N-glycans, the HB C-terminal residues occasionally detach from the membrane as evidenced by higher ZCOM between 2 to 4 μs. Although the HB C-terminal residues remain folded over the 5-μs simulation for PrP-memb (Fig. 6 C), the loss of contact of HB with the membrane could potentially lead to unfolding of the HB C-terminus, leading to the HB C-terminus in PrP-memb having a higher tendency to unfold compared to PrP-nglycan-memb. Thus, the prion-membrane interactions are likely required for the maintenance of the protein secondary structure. Once interactions with the membrane are lost, helix unfolding could happen because of the backbone and solvent hydrogen bonding competition, as discussed above (Fig. 7).

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Time series of z coordinates of the center of mass of the HB C-terminus fragment (residues 188 to 194) in PrP-nglycan-memb (red) and PrP-memb (blue). To see this figure in color, go online.

Conclusions

The conversion of PrPC to PrPSc is the central event in prion disease transmission. Despite intense efforts, the conversion process remains poorly understood. Improving our understanding of the early-stage unfolding mechanism of PrPC could be of great importance for investigation of the subsequent misfolding, propagation, and even the treatment of prion diseases.

In our study, we have concentrated on exploring the impact of the N-glycosylation and the membrane on prion secondary structure stability to shed light on the nature of the conformational instability that drives PrPC to PrPSc via multiple microsecond MD simulations. Our results demonstrate that various parts of the globular domain undergo conformational changes and helices show unfolding to different extents in different environments. The HB C-terminus (residues 188 to 194), which includes a highly conserved threonine-rich fragment, exhibits significant instability in the absence of the membrane but remains helical in the presence of the membrane. The dominant factor responsible for instability of the HB C-terminus is competition between the protein backbone intramolecular i−i+4 hydrogen bonds with both the solvent and side chains, with the latter implicating the role of the intrinsic amino acid sequence on stability of this fragment. It was experimentally revealed that this HB peptide fragment has an inherently high propensity for β-strands and tends to form β-sheet-rich amyloid-like fibrils (8, 83). Surewicz’s lab also showed that the conformational conversion of PrPC involves major refolding of the C-terminal α-helical region, and the HB-HC subdomain has been shown to be part of the β-sheet core by site-directed spin labeling EPR spectroscopy and hydrogen/deuterium exchange (84, 85). Although it will be challenging to observe folding from a coiled conformation to a β-sheet in accessible simulation times, it is tempting to speculate that unfolding of the HB C-terminus and the subsequently increased flexibility in the HB-HC loop in our simulations represent the starting point that triggers the PrPC → PrPSc pathological conversion pathway. Moreover, CD and NMR studies by Ziegler et al. suggested that HA has a high intrinsic helical propensity and is unlikely to be involved in the initial steps of the pathogenic conformational change (86), which also agrees with the relative stability of HA compared to HB in our solution simulations.

The interactions between protein and membrane can provide effective constraints on the protein conformation and decrease the solvent accessibility of the protein backbone, thus stabilizing the protein secondary structure. In the case of HuPrP, the two N-glycans can enhance such interactions by forming more contacts with the membrane surface, thereby stabilizing protein-membrane interactions. Although the helical structure of the HB C-terminus is retained in both PrP-nglycan-memb and PrP-memb on the timescale of our current simulations, its position remains close to the membrane surface (<40 Å) for longer period of time when both N-glycans are present. More frequent detachment of HB from the membrane surface without N-glycans could result in increased exposure to solvent and potentially acquiring PrPSc-like conformation compared to PrP-nglycan-memb.

Author Contributions

E.L.W. and W.I. designed research; E.L.W., Y.Q., S.P., and S.S.M. performed research; E.L.W., Y.Q., S.P., and S.S.M. contributed analytic tools; E.L.W., Y.Q., S.P., S.S.M., A.D.M., J.B.K., and W.I. analyzed data; and E.L.W., S.P., S.S.M., A.D.M., J.B.K., and W.I. wrote the article.

Acknowledgments

This research was supported by grants from NSF MCB-1157677 (W.I.), NSF DBI1145987 (W.I.), NIH U54 GM087519 (W.I.), XSEDE MCB070009 (W.I.), NSF DBI-1145652 (J.B.K.), NSF MCB-1149187 (J.B.K.), and NIH GM070855 (A.D.M.). Anton computer time was provided by the National Center for Multiscale Modeling of Biological Systems (MMBioS) through Grant P41GM103712-S1 from the NIH and the Pittsburgh Supercomputing Center (P.S.C.). The Anton machine at PSC was generously made available by D.E. Shaw Research.

Notes

Editor: Scott Feller

Footnotes

Supporting Materials and Methods, twelve figures, and eight tables are available at http://www.biophysj.org/biophysj/supplemental/S0006-3495(15)01048-6.

Supporting Material

Document S1. Supporting Materials and Methods, twelve figures, and eight tables:
Document S2. Article plus Supporting Material:

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