Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia
Received on December 16, 1999; revised on April 11, 2000; accepted on April 18, 2000.
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Abstract |
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Key words: human prion protein/N-linked glycan/GPI anchor/molecular dynamics/conformation
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Introduction |
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The mature form of PrPC, made up of residues 23231, is anchored to the cell membrane via a glycosyl-phosphatidyl-inositol anchor (GPI anchor) at its C terminus (Stahl et al., 1987, 1992; Baldwin et al., 1993
), and has one disulfide bridge (Cys179-Cys214; human numbering). In addition, mammalian PrP contains two consensus sites for N-linked glycosylation, at Asn181 and Asn197 of human PrP (HuPrP) and Syrian hamster PrP (ShPrP), and at Asn180 and Asn196 in murine PrP (MoPrP). Studies of the N-linked glycans of MoPrP (Stimson et al., 1999
) and ShPrP (Endo et al., 1989
; Rudd et al., 1999a
) have shown that both sites are occupied and that they contain up to 60 different but overlapping sets of sugars, including charged sialyl-Lewisx (SiaLex) epitopes, which are suggested to serve as cell-surface recognition molecules (Eggens et al., 1989
; Bevilacqua et al., 1991
).
NMR structures of the full-length and N-terminally truncated forms of recombinant MoPrP (Riek et al., 1996; Billeter et al., 1997
; Riek et al., 1997
; Riek et al., 1998
), ShPrP (Donne et al., 1997
; James et al., 1997
; Liu et al., 1999
), and HuPrP (Hosszu et al., 1999
; Zahn et al., 2000
) have revealed that the whole N-terminal segment PrP23126 is flexibly disordered and that only the C-terminal part PrP127231 possesses a defined 3-D structure. The structurally well-defined part of PrP consists of three
-helices and a small two-stranded antiparallel ß-sheet. All NMR studies could identify the three
-helices, although with slightly different lengths, but showed differences for the antiparallel ß-sheet, which was not always as well defined (Donne et al., 1997
; James et al., 1997
; Hosszu et al., 1999
).
The biological roles of oligosaccharides of glycoproteins are as diverse as their structures, and include cell surface signaling, cell recognition, intracellular trafficking, secretion, stabilizing and protecting tertiary protein structure, regulating activity of enzymes, and clearance and turnover of the glycoproteins (Varki, 1993). In addition, oligosaccharides are so flexible in their 3-D structures that even NMR experiments can rarely define their structural and dynamical properties unambiguously (Peters and Pinto, 1996
). Recent reports indicate combining NMR and x-ray crystallography can help to identify the structures of oligosaccharides on proteins, at least for the first five to six sugar residues (Rudd et al., 1999b
). The great variety of possible functions combined with the structurally flexible nature of oligosaccharides makes elucidation of structureactivity relationships very difficult, especially given the lack of experimental tools to characterize the structural and dynamical properties of oligosaccharides linked to proteins. Molecular dynamics (MD) simulations alone (Barboni et al., 1995
; Woods et al., 1995
; Naidoo et al., 1997
), or in combination with NMR (Agrawal et al., 1999
; Rudd et al., 1999c
), have proven to be a valuable tool for assessing the flexibility of oligosaccharides. However, all NMR studies with PrP were done with protein lacking both N-linked glycans and GPI anchor. Hence, in order to identify a possible role for the glycosylation of mammalian PrP, we have undertaken MD simulations and analyzed the structure and flexibility of the PrP glycans, and their influence on the protein structure.
We have recently undertaken MD simulations with nonglycosylated models of ShPrP90231 and HuPrP90230 (Zuegg and Gready, 1999). These revealed that correct treatment of the long-range electrostatic interactions was necessary to get stable trajectories. This requirement was attributed to the relatively high number of charged residues on the surface of PrP and the fact that the protein has different regions with distinct electrostatic properties. PrP90230 consists of an N-terminal part with only positively charged residues (PrP90112), a hydrophobic part with no charged residues at all (PrP113133), and a C-terminal part with a large number of positively and negatively charged residues (PrP134230). Only by considering all long-range electrostatic interactions using the particle mesh Ewald (PME) method (Darden et al., 1993
), could trajectories with low root mean square deviation (RMSD) and with stable secondary structure be generated. However, the simulations showed some flexibility in the structured part of PrP, PrP127230, leading to shortened
-helical structure and in some cases even to a split in the third
-helix (Helix-C). In this paper we investigate the influence on the structure of PrP of glycosylation of the two N-glycosylation sites, by comparing the results of MD simulations of the HuPrP homology model with results of simulations of HuPrP with both N-glycans attached. Also, in order to define possible orientations of PrP with respect to the membrane, the oligosaccharide part of the GPI anchor bonded to the C-terminal Ser230 was included in the model. To identify a possible distance between PrP and the membrane, the results of these MD simulations of the fully glycosylated HuPrP model (glyc-HuPrP) were combined with simulations of a membrane model which included just the GPI anchor. Finally, we have compared the dynamic and structural behavior of the glycans when bound to PrP with results of simulations of their free, solvated forms. This allows assessment of any restrictions on the flexibility of the glycans by the protein environment.
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Results |
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Protein structure comparison between HuPrP and glyc-HuPrP
Both HuPrP models, with (glyc-HuPrP) and without the oligosaccharides (HuPrP), were simulated with the same parameters and for the same time of 2 ns (see Table I). Comparison of the RMSDs of the backbone atoms of both models from their common starting conformation shows that even though the RMSD for the whole protein (PrP90-231) is higher for the glycosylated model, the RMSDs of the structured part of the protein PrP127227 are the same in both simulations (see Figure 2). The higher RMSD of the glycosylated model is due to different conformations of the N-terminal part of the protein (PrP90-126) only. The RMSDs of the oligosaccharides in glyc-HuPrP are between 3.7 and 5.1 Å, i.e., higher than the RMSD of the protein backbone of the structured part (2.1Å) but still considerably lower than the RMSD of the flexible N-terminal part (11.8 Å) (see Table I). Of the three oligosaccharides, NGlyc197 shows the highest RMSD (5.1 Å). Secondary structure analysis, shown in Figure 3, indicates increased stability of the
-helical structure for the glycosylated model. The two C-terminal
-helices, B and C, show increased lengths which resemble more the NMR structure of ShPrP (Liu et al., 1999
), although Helix-C is still shorter by 3 residues at its C-terminal end. In addition, no splitting of Helix-C could be seen in the glycosylated model in contrast to the HuPrP model (Zuegg and Gready, 1999
). The flexibility of the backbone, shown in Figure 3 as standard deviation (SD) of the
and
backbone torsion angles and in Figure 4 as the radius of the coil tracing the positions of the C
atoms, decreases significantly for the residues around the N197 glycosylation site in Turn-D, but remain similar throughout the other parts of the protein. However, the Turn-C region, PrP165170, in the glyc-HuPrP model shows a slightly increased flexibility. The average structures for both simulations are also very similar (see Figure 4), even though the RSMD of the backbone atoms between both structures is calculated to be 2.5 Å. The main differences between the two structures are of slightly different conformations for turns A, C, and D. Turn-D is at the N197 glycosylation site and, therefore, influenced by NGlyc197, but the other two turns are not directly influenced by the glycans. The intra-protein interactions are also similar for both models. The three major salt bridges, Glu146
Arg208, Arg164
Asp178 and Arg156
Glu196 (Zuegg and Gready, 1999
), are present in both simulations for more than 90% of the time (see Table II). The exception is the salt bridge Arg156
Glu196, between Helix-A and Turn-D, for which the occupancy drops in the glyc-HuPrP simulation to 78.4 %, due to the proximity of the N197 glycan. The salt bridges in the Helix-A region on the other hand, Asp144
His140, Asp144
Arg148, Asp147
Arg151 and Glu152
Arg148, which involve only neighboring residues (e.g., ± 4), are somewhat reorganized, with some increasing and others decreasing their occupancies. Differences in the occupancies between this work and previous work (Zuegg and Gready, 1999
) are due only to a longer simulation time (2300 ps compared with 720 ps). The H-bond network is also similar between the two simulations with similar overall number of H-bonds (see Table III).
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Structure and dynamics of glycans
As may be seen in Figure 4, the simulation of the glycosylated HuPrP model showed no unique orientation for either NGlyc chain. With a RMSD of the heavy atoms of NGlyc181 and NGlyc197 from the starting conformation of 4.9 and 5.1 Å, respectively, and an average SD of the glycosidic linkage torsion angles ( and
, and
in the case of a 16 linkage) of 17.2° and 21.3°, respectively, both N-glycans exhibit high flexibility. These SD values for the torsion angles are similar to the value of 20.1° from the simulation of the free NGlyc model in solution (see Table I). Nevertheless, the N-linked glycans seem to be slightly restricted by the protein, as the core of the glycan, formed by ManVI-(ManVII-)ManIV-GlcNAcIII-GlcNAcI-Asn181/197, seems to have reduced flexibility compared with the simulation of the solution structure, especially for the usually highly flexible 1
6 linkage of ManVI-ManIV (see Figure 5). Compared with the core structure, the four antennas of the NGlyc models have high flexibility, especially both SiaLex groups of the N197-linked glycan show high flexibility compared with those in the NGlyc181 glycan. Generally, NGlyc197 has higher flexibility than NGlyc181, as can be seen in the higher SD of the torsion angles.
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GPI anchor in membrane
To investigate a possible orientation and, especially, a possible distance of PrP with respect to the membrane, additional simulations were carried out involving a monolayer membrane with an embedded GPI anchor. For the membrane two different models were used, one with ethanolamine (eap-GPI) and one with choline (cho-GPI) as the membrane-head group. Both simulations showed the GPI-anchor glycan to be as flexible as in the simulations of the free GPI glycan in solution or the GPI glycan in the glyc-HuPrP model. The average SD values of the linkage torsion angles are 31.2° and 27.7° (Table I) for the cho-GPI and the eap-GPI simulations, respectively, values similar to the other simulations. In both simulations, the GPI-anchor glycan has reduced flexibility in the first residues from the membrane linkage, with a unique conformation for the membrane-PO2I-mInoII-GlcNIII-ManIV residues (see Figure 8; compare with Figure 5), but with an average distance between mInoII-H6GlcNIII-H1 similar to the other simulations and NMR experiments (see Table IV). The GlcNIII-ManIV linkage, on the other hand, shows a different conformation compared with the other simulations or NMR experiments. For both simulations, the remaining part of the GPI-anchor glycan has high flexibility, especially in the -PO2VII-EaVIII part which is linked to PrP in the glyc-HuPrP model. The main difference between the two models is the distance of this ethanolamine group from the surface of the membrane. For the cho-GPI model, the distance ranges from 5.5 to 13.0 Å with an average of 8.8 Å, whereas for the eap-GPI model it ranges from 9.5 to 14.8 Å with an average of 12.3 Å. All simulations were carried out by fixing the position of the fatty acids, including that to which the GPI glycan is attached.
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Discussion |
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Detailed chemical analysis of the carbohydrate chains of PrPSc purified from scrapie-infected hamster brain showed that both N-glycosylation sites of PrP are occupied, and that a mixture of bi-, tri-, and tetra-antennary complex-type oligosaccharide chains is present, with about 70% of the terminal galactose residues linked to sialic acid (Endo et al., 1989). More detailed analysis of PrPSc from scrapie-infected mouse showed similar oligosaccharide structures as for hamster PrP (Rudd et al., 1999a
; Stimson et al., 1999
). In addition, the analysis revealed that the termini of the oligosaccharides consist of Lewisx (Lex) and sialyl Lewisx groups (SiaLex), Galß(14)(Fuc
(13))GlcNAc and NeuNAc
(23)Galß(14)(Fuc
(13))GlcNAc, respectively. The structures of all these different oligosaccharides, 60 altogether, seem to derive from one tetra-antennary oligosaccharide complex by partial degradation. Both N-glycosylation sites are found to have the same oligosaccharide complex, although with slightly different degradation stages. Even though all detailed structural studies of the oligosaccharides of PrP have been made on PrPSc, the cellular form PrPC is suggested to have a similar type of oligosaccharide composition (Haraguchi et al., 1989
). A recent, more detailed study indicated that PrPC and PrPSc contain the same set of oligosaccharides, but with different relative proportions of individual glycans (Rudd et al., 1999a
).
In this work we investigated the possible influence of the N-linked oligosaccharides and the GPI anchor on the protein structure of human PrP (HuPrP). To achieve this, we compared the trajectories from a molecular dynamics (MD) simulation of HuPrP with all oligosaccharides (glyc-HuPrP) with those from a simulation of HuPrP without any of the glycans (HuPrP). Previous MD simulations (Zuegg and Gready, 1999) showed that the structures of a homology model of HuPrP and the NMR structure of ShPrP are highly sensitive to the electrostatic environment, and that only correct treatment of the long-range electrostatic interactions resulted in a stable structure. As both N-glycans can carry up to three negatively charged SiaLex groups, which would change the electrostatic environment of HuPrP significantly, investigating their effect on the protein structure appeared necessary. For the fully glycosylated HuPrP model, a N-glycan model was generated, which can be considered as the glycan from which all the oligosaccharides found experimentally (Endo et al., 1989
; Rudd et al., 1999a
; Stimson et al., 1999
) could be derived by partial degradation. However, as the exact branching structure could not be determined in the experiments the model has some uncertainties, for example the distribution of the SiaLex groups among the branches. Similarly, the GPI-anchor model used also has some uncertainty, as experiments could neither identify the type of glycosidic linkage nor the anomeric type in one branch (Stahl et al., 1987
, 1992; Baldwin et al., 1993
).
In the MD simulation of the glyc-HuPrP model, both C-terminal -helices, Helix-B and Helix-C, not only have increased length compared with the simulation of non-glycosylated HuPrP, but also the split in Helix-C observed in the HuPrP simulation does not occur. In addition, residues in Turn-D, between Helix-B and C, showed reduced variation in the backbone torsion angles. On the other hand, residues in Turn-C, between Strand-B and Helix-B, showed increased variation of the backbone torsion angles during the simulation, as did the residues in the N-terminal part, PrP90126. Taken together, the results suggest that most of the structured part of the protein, HuPrP127227, experiences a stabilizing effect from addition of the glycans. The perturbation to Turn-C by the presence of the glycan is not associated with any direct interaction between Turn-C and NGlyc181 or with the GPI anchor. NMR experiments of MoPrP121231 (Billeter et al., 1997
), ShPrP90231 (James et al., 1997
), and HuPrP (Hosszu et al., 1999
) show variations in the lengths of all three
-helices, similar to the differences found between our glycosylated and nonglycosylated HuPrP models. Although the C-terminal part of PrP forms a defined structure very quickly (Wildegger et al., 1999
), the differences found in the NMR experiments may be attributed to the lack of glycans. As absence of the glycans results in higher flexibility of the protein backbone, different sequences might adjust to this potential instability differently, some in Turn-D and some in Helix-C. Our simulation of the glycosylated HuPrP model suggests that both N-glycans are able to stabilize PrP in these regions.
The N-terminal part of HuPrP, HuPrP90127, changes its dynamical behavior to more flexible on glycosylation. However, this may be due to the fact that its starting conformation was randomly chosen as NMR experiments show it is disordered and, thus no structural data are available. Hence, using a different starting conformation might result in different structural and dynamical behavior of the N-terminal part. Despite these uncertainties, the simulations suggest that glycans have some structural influence on the N-terminal part of HuPrP, especially on the residues immediately before the first strand, HuPrP120130, which are close to NGlyc181. It is possible that this part would form a defined structure under the influence of the glycans, but unfortunately the simulation time of 2 ns is far too short to see development of any significant secondary structure.
Interestingly, the influence of the glycans on the protein appears not to be due to specific direct interactions, such as H bonds or salt bridges. Indeed, each glycan has only one to two H-bond interactions with the protein. The influence of the glycans seems to be more indirect, by reducing the mobility of the surrounding water molecules. Throughout the simulation, some parts of the glycans in both NGlyc chains are not more then one water molecule away from the protein. Thus, both N-glycans together cover all residues in Turn-D and all surface-facing residues of Helix-B. This includes Lys194, which is reported to be a cleavage point for trypsin only when the glycan at the Asn197 site is small (Stimson et al., 1999). In addition, NGlyc197 covers one residue in Helix-A and NGlyc181 some residues just before the first strand. The extent of the area covered by the N-glycans depends, of course, on their conformations. The current simulations can be considered only as an estimate for the nature and extent of the glycan cover, due to uncertainties in the glycan models used and the fact that a full investigation of the conformational space of the glycans would require simulations 100 times longer (Peters and Pinto, 1996
). However, as the main feature of the N-glycan cover is its negative electrostatic potential, produced by the charged SiaLex groups, its extent is less sensitive to conformational details of the glycans. The negative electrostatic field extends over the whole surface of Helix-B and Helix-C onto the opposite side of PrP to Helix-A. Even though the N-glycans do not sterically cover Helix-C, the negative field extends to the C-terminal end of Helix-C because of negatively charged residues on the surface of Helix-C and because of the PO2 group in the GPI anchor. The field has a positive counterpart at the N-terminal part of the HuPrP model, which has several positively charged residues. The orientation of the dipole moment depends entirely on the position of the N-terminal part, which in all simulations and NMR experiments shows no defined structure. In between the two dominant fields is Helix-A, which has a quite unusual nature. Helix-A, PrP144152, with an amino acid sequence of DYEDRYYRE, is entirely hydrophilic and is stabilized by the salt bridges Glu146
Arg208, between Helix-A and Turn-D, and Arg156
Glu196, between the neighboring Turn-B and Turn-D. Interestingly, Arg208 is a residue whose mutation into His is known to be associated with inherited forms of human disease (Ironside, 1998
). The charged surface residues of Helix-A makes it a good candidate for aggregation in either a parallel or anti parallel orientation, forming in each case two salt bridges (Morrissey and Shakhnovich, 1999
). As Helix-A is not covered by either of the dominant electrostatic fields, it is, therefore, electrostatically accessible. An antiparallel aggregation would not disrupt its structure, and would lead to the HuPrP dimer as shown in Figure 9B, with an extended cover by the N-glycans on top and on the side and with the N-terminal parts sticking out in opposite directions from the middle. A PrP dimer seems to occur naturally (Priola et al., 1995
) or, at least, be the starting point for the conversion from PrPC to PrPSc (Prusiner, 1991
; Warwicker, 1997
). By contrast, a parallel aggregation of Helix-A would disrupt the helical structure and lead to ß-nucleation, which has been suggested to form the mechanism for the conversion of PrPC to PrPSc (Morrissey and Shakhnovich, 1999
). Interestingly, a mutation of Arg208 would disrupt the salt bridge between Helix-A and Turn-D and might, thereby, facilitate the transformation of Helix-A into ß-sheet conformation, necessary for the parallel aggregation. On the other hand, our model suggests that the N-glycans are not favorable for such an orientation, even though such aggregation could include major rearrangements in the N-terminal part of the structured portion of PrP, and, thus, would lead to different locations and relative orientations of the residues of the former Helix-A region, such as opposite Helix-B and Helix-C as in a proposed model of PrPSc (Huang et al., 1996
). However, in addition to inferences from our model, experiments showed that binding of antibody specific for the Helix-A region is not able to inhibit the first step in the formation and aggregation of PrPSc, namely the binding of PrPSc to PrPC (Horiuchi and Caughey, 1999
).
The antibody-binding experiments also showed that antibody specific for the region PrP218231 is able to inhibit the binding of PrPSc to PrPC (Horiuchi and Caughey, 1999). This region is in the vicinity of the protein X binding region (Telling et al., 1995
; Glu168, Gln172, Ile215, and Glu219), where protein X is suggested to play a role during PrPSc propagation (Kaneko et al., 1997a
). Even though this region, involving Turn-C and the end of Helix-C, is not entirely covered by the N-glycans the proximity of the negative electrostatic potential would not favor an aggregation in this region. As both N-glycans and the GPI anchor do not influence the formation of the PrPSc-PrPC complex (Kaneko et al., 1997b
) and PrP lacking Turn-C is still able to generate a PrPSc-like form (Muramoto et al., 1996
), the region around Strand-A, PrP119138, has been suggested to form the potential binding site in PrPSc-PrPC complex formation (Horiuchi and Caughey, 1999
). In addition, this region includes some of the residues important for the species barrier for disease transmission (Kocisko et al., 1995
; Schätz et al., 1995
), and was used as the primary binding site for a dimer model (Warwicker, 1997
). In our solvated model glyc-HuPrP (Figure 4), this region is not influenced by the oligosaccharides and is exposed to the solvent. The monomer model of the membrane-bound HuPrP (Figure 9A) shows this region is close to the membrane. On the other hand, the dimer model (Figure 9B) has part of this proposed PrPSc-PrPC binding region, PrP135141 next to each other. The earlier proposed dimer model (Warwicker, 1997
) used the PrP130136 region as the primary binding site, but this resulted in a different orientation, in which both C-terminal ends were pointing in opposite directions, thus making it impossible for both monomers to be bound to the membrane by GPI anchors. Our dimer model can be seen as a possible model not only for a reported naturally occurring dimer (Priola et al., 1995
), but also as a possible dimer starting point in PrPSc propagation. The monomer model shows the N-terminal part of PrP closer to the membrane, more in agreement with experiments suggesting that this part makes strong interactions with the membrane (Morillas et al., 1999
), but due to the uncertainty of the structure and its high flexibility, an orientation of this N-terminal part towards the membrane is not excluded in the dimer model.
The mutation experiments which deactivated each N-glycosylation site (Lehmann and Harris, 1997), showed that the Asn181 site seems to be associated more with a cell-trafficking role, whereas the Asn197 site seems to stabilize the PrP structure. Structures from the glyc-HuPrP simulation show NGlyc181 affecting an area which already has a stable secondary structure, whereas NGlyc197 affects an otherwise more flexible area of a turn by reducing its flexibility. This is consistent with Asn181 glycosylation having a more functional role. A similar suggestion has been made in comparing PrP with the PrP-like protein doppel (Dpl), where only the site analogous to Asn181 is conserved in the Dpl sequence (Asn111; Moore et al., 1999
). Note, however, that NGlyc181 contributes more to the negative electrostatic potential covering of HuPrP, as NGlyc197 is more solvent exposed. The second N-glycosylation site of Dpl at Asn99 would be in Turn-C which, assuming a similar 3-D structure, would result in an N-glycan oriented on the same side of the protein as Asn111 but closer to the C-terminal part of the protein. In addition, this site is closer to Asn111 than Asn191 is to Asn181 in PrP, thus restricting the available space for oligosaccharides, and suggesting that the N-glycans of Dpl might be a different size compared with the PrP N-glycans, and cover Helices B and C at the C-terminal part of the protein more than in the Turn-D region as in PrP.
The model for the N-glycans used in this work represents the largest and the most charged glycan possible. In addition, both N-glycosylation sites were considered to be occupied by the same glycan. Partial degradation of the N-glycans would, of course, change their properties, especially cleavage of the charged NeuNAc residues would reduce the negative electrostatic potential. Detailed analysis of the N-glycans of PrP showed substantial heterogeneity in their structure (Endo et al., 1989; Rudd et al., 1999a
; Stimson et al., 1999
). It has also been reported that PrPSc strains, encoding distinct disease phenotypes, are associated with different patterns of glycosylation (Collinge et al., 1996
; Mastrianni et al., 1999
; Somerville, 1999
). However, our work showed that even the most charged N-glycans do not have a major influence on the conformational structure of HuPrP, and that most of the important parts of PrPC with respect to proposed mechanism for PrPSc formation (N-terminal part, Strand-A, Turn-A, and Turn-C), are accessible even with the largest possible N-glycans. Recent experiments showed a different glycosylation pattern between PrPC and PrPSc, which correlated with differences in the activity of N-acetylglucosaminyltransferase III and suggested that some cells forming PrPSc undergo changes that diminish the activity of an enzyme in the glycosylation pathway (Rudd et al., 1999a
). Also, different protein conformations can be attributed to different PrPSc strains (Caughey et al., 1989
; Hill et al., 1997
; Safar et al., 1998
), with some conformational differences being correlated with concentrations of Cu2+ and Zn2+ ions (Wadsworth et al., 1999
). Taken together, these results suggest that the structure of PrPSc is more variable than the PrPC structure, and that the structure of PrPSc is more sensitive to particular aspects of the environment, such as the presence of metal ions or glycosylation, or the more general status of the PrPSc forming cell.
The GPI anchor appears to have only a minor influence on the structure of HuPrP, in contrast to the report for the Thy-1 protein (Barboni et al., 1995). Its role seems to consist entirely in attaching PrP to the membrane. Simulations of the GPI anchor in a membrane showed that it keeps the protein at a distance between 9 and 13 Å from the membrane surface, depending on the type of membrane-head group. This distance is enough to maintain several water shells between the membrane surface and the protein and is, thus, enough to guarantee a high degree of freedom for the movement and orientation of PrP. This orientational freedom is assisted by the structurally flexible linkage group, ethanolamine, and by the C-terminal end of the protein which does not have any interactions with the rest of the model and is highly flexible.
In summary, it has been shown that the N-linked glycans do reduce the flexibility of the protein backbone in some parts of PrP, such as residues in the turn between Helix-B and Helix-C and within those helices, but perturb it in other parts to increase the flexibility, such as residues in the turn between Strand-B and Helix-B. But the main influence of the N-glycans appears to come from its negative charges, generating a negative electrostatic field which covers the whole surface of Helix-B and Helix-C. In addition, the simulations show that the GPI anchor has little influence on the structure of PrP. Its flexible structure guarantees a high degree of freedom for the orientation of PrP, but at the same time keeps the protein 913 Å above the membrane surface. A possible orientation of HuPrP could be generated which takes into account the unique nature of Helix-A and the reported PrPSc-PrPC binding regions, allowing HuPrP to form a homodimer on the membrane.
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Materials and methods |
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Model building for glyc-HuPrP
The HuPrP model was generated as described previously (Zuegg and Gready, 1999), and consists of a homology model of human PrP90230 based on the NMR structure of Syrian hamster PrP (James et al., 1997
). The HuPrP model had its ionizable residues set to their solution ionization state at pH 7, as calculated with the program TITRA (Petersen et al., 1997
). The starting models for the N-linked glycan (NGlyc) and GPI-anchor (GPI) oligosaccharides were generated by assuming a 1C4 conformation for Fuc
, a 2C5 conformation for NeuNAc
and a 4C1 conformation for all remaining sugar residues, and changing only the glycosidic linkage torsion angles to produce a model with no steric clashes. Several minimization steps were required in the building process to obtain a low energy conformation of the models. The model oligosaccharides were attached to the HuPrP model, one N-linked glycan model at each N
atom of Asn181 and Asn197, and one GPI model at the backbone C atom of Ser230. The GPI model was terminated at the PO2I group with a CH3 group. The resulting model, glyc-HuPrP, has 3376 atoms and a molecular mass of 25.3 kDa, compared with the HuPrP model with 2180 atoms and a mass of 16.0 kDa. In order to use the particle mesh Ewald (PME) method (Darden et al., 1993
) for the calculation of long-range electrostatic interactions in the simulations, the system has to be neutralized. As in the previous work (Zuegg and Gready, 1999
), this was achieved by adding Na+ and Cl ions to the system. For the glyc-HuPrP model, 20 Na+ and 15 Cl ions were added with the CION program in the AMBER 5 package, compared with 12 Na+ and 13 Cl ions added to the HuPrP model. Both models were immersed in a rectangular box of pre-equilibrated TIP3 water molecules (Jorgensen et al., 1983
), of dimensions of 66 x 63 x 58 Å, containing 5143 water molecules for the HuPrP model, and 68 x 86 x 78 Å with 10321 water molecules for the glyc-HuPrP model.
Model building for solvated glycans
To analyze any structural and dynamical restrictions of the glycans due to the protein, simulations were carried out with free glycan models. The same NGlyc model as used as a starting model for glyc-HuPrP was capped at the first GlcNAcI with a NH-CO-CH3 group, neutralized by adding 3 Na+ ions with the CION program, and immersed in a box of water (solv-NGlyc). Similarly, a solvated, free GPI-anchor glycan model, solv-GPI, was generated by replacing the protein backbone with a CO-CH3 group on one side and the membrane glycerol with a CH3 group on the other side. After adding one Na+ ion to neutralize the model, the system was immersed in a box of water. Both solvated models were simulated with the same MD parameters as the HuPrP and glyc-HuPrP models.
GPI anchor and membrane model
For simulations of the GPI anchor attached to a membrane, two different models were generated differing only in the head group of the membrane monomer. The membrane was generated as a monolayer of a short C5:0 fatty acid using 1,2-divalerianyl-phosphatidyl-ethanolamine (eap-GPI) or 1,2-divalerianyl-phosphatidyl-choline (cho-GPI) as the monomer. The GPI-anchor model was attached to the membrane by replacing one of the head groups with the GPI glycan. The GPI-anchor model was then covered by a cap of water molecules, containing 1244 and 1107 water molecules for eap-GPI and cho-GPI, respectively.
MD simulations
All MD simulations were performed using the SANDER module in the AMBER package. Systems were equilibrated by minimization and short constant pressure simulations as described in the previous work (Zuegg and Gready, 1999). In the simulations, Newtons equations of motion were integrated with a step size of 1 fs, with lengths of all bonds involving hydrogen atoms constrained using the SHAKE algorithm with a relative tolerance of 5 x 106 Å. A pair-list to calculate non-bonded interactions was generated every 50 simulation steps. The temperature of the system was controlled to be 300 K using two independent Berendsen thermostats (Berendsen et al., 1984
), one for the solute and one for the solvent, and with coupling times
Solute = 0.5 ps and
Solvent = 0.75 ps.
For all models in a box of water, HuPrP, glyc-HuPrP, solv-Nglyc, and solv-GPI, the PME method was used to calculate the electrostatic interactions, using grid sizes which produce a grid spacing of ~1 Å. These simulations were carried out using periodic boundary conditions and constant volume, and removing the overall translational and rotational motion of the system every 100 time steps.
For the models with a water cap, cho-GPI and eap-GPI, no PME or periodic boundary conditions could be used. Instead, the electrostatic interactions were calculated by truncating the interaction at a distance of 8 Å, with the water cap being restrained to its cap-like form. In addition, the fatty acids of the membrane model were restrained to their starting positions, in order to maintain the shape of the monolayer.
Analysis
NMR experiments on ShPrP90-231 revealed a highly flexible N-terminal part with only the C-terminal part having a defined secondary structure (James et al., 1997). Therefore, all the root-mean-square deviation (RMSD) analysis of the structure was calculated not only for the complete model (PrP90230), but also for the flexible part (PrP90126) and the structured part (PrP127227) separately. Analysis of molecular trajectories was done with the program CARNAL in AMBER 5, including structural alignment and calculation of the RMSDs of the structures, and also torsion angle analysis. Analysis of the secondary structure was done with the DSSP program (Kabsch and Sander, 1983
). Salt bridges were defined by the distance between the positively and negatively charged heavy atoms. For Arg residues, all three nitrogen atoms of the side chain, N
, N
1,N
2, were used. A salt bridge was deemed present if the distance between the two heavy atoms less the corresponding van der Waals radii was less than 1.5 Å. Calculation of the electrostatic potential was carried out with the DELPHI program (Gilson and Honig, 1987
) in INSIGHTII-98 (1998, Molecular Simulations Inc., San Diego, CA), using default van der Waals radii and the same partial charges as in the simulation with AMBER. For model manipulation and visual analysis, INSIGHTII was used. The pictures were generated using MOLSCRIPT (Kraulis, 1991
), RASTER3D (Merritt and Bacon, 1997
), MOLMOL (Koradi et al., 1996
) and POVRAY (http://www.povray.org). The calculations and analysis were carried out on SGI Power-Challenge (SGI-PC) and Fujitsu VPP300 (VPP) supercomputers, and SGI Indigo2 and Octane workstations.
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Abbreviations |
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References |
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