J Mol Model (2014) 20:2106 DOI 10.1007/s00894-014-2106-y
ORIGINAL PAPER
The role of Cys179–Cys214 disulfide bond in the stability and folding of prion protein: insights from molecular dynamics simulations Lulu Ning & Jingjing Guo & Nengzhi Jin & Huanxiang Liu & Xiaojun Yao
Received: 28 September 2013 / Accepted: 7 December 2013 / Published online: 11 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Prion diseases are associated with misfolding and aggregation of prion protein (PrP). Cellular prion protein contains a disulfide bond linking Cys residues at positions 179 and 214. It has been proposed that this disulfide bond plays an important role in the conversion between cellular (PrPC) and the scrapie form of prion protein (PrPSc). To probe the role of this disulfide bond in the stability and folding of prion protein, we employed molecular dynamics simulations to study the reduced prion protein and a variant of PrP in which the two cysteines were replaced by alanines residues. The simulations highlighted the changes that occurred upon breakage of the disulfide bond. Breakage of the disulfide bond resulted in a shift of H1, elongation of the native β-sheet and perturbation of the hydrophobic core of huPrP. The changes are similar to the conformational transitions of prion protein in low pH, in denaturing conditions or with pathogenic mutations, which indicate that rupture of the disulfide bond may lead to the misfolding of prion protein. Keywords Molecular dynamics simulation . Prion diseases . Disulfide bond . Misfolding L. Ning : J. Guo : H. Liu (*) : X. Yao State Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou University, Lanzhou 730000, China e-mail: [email protected] X. Yao e-mail: [email protected] J. Guo : H. Liu School of Pharmacy, Lanzhou University, Lanzhou 730000, China N. Jin Gansu Computing Center, Lanzhou 730000, China X. Yao State Key Laboratory for Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China
Introduction Transmissible spongiform encephalopathies (TSE) such as Kuru, Creutzfeldt-Jakob disease in human beings and bovine spongiform encephalopathy in animals are characterized by aggregates of the abnormal form of prion protein (PrPSc) [1, 2]. According to the “protein only hypothesis”, the aggregated scrapie form, PrPSc, is the infectious agent of these TSElinked diseases [3, 4]. PrPSc and cellular, non-pathological prion protein (PrPC) share an identical covalent structure but have distinct biophysical and biochemical properties [5]. PrPSc has a high β-sheet content, whereas PrPC is rich in αhelix [6–8]. Furthermore, PrPSc is insoluble in mild detergents and is proteinase K-resistant, whereas PrPC is soluble in mild detergent and highly sensitive to proteinase K [9–11]. PrPC contains a flexible N-terminal region and a globular C-terminal region that consists of two short in-register βsheets and three α-helices with a disulfide bond at position 179 and 214 linking H2 and H3 (Fig. 1) [12, 13]. The structure of PrPSc is still not fully elucidated due to its high propensity to aggregate. Because the conformational conversion of prion protein from its cellular form to its scrapie form is the critical step at the beginning of prion diseases, much effort has been dedicated to understanding the misfolding and aggregation of prion protein [14–26]. However, the molecular mechanisms underlying the conformational transition remain largely unknown currently. It has been proposed that the disulfide bond connecting H2 and H3 is important in the structural conversion. However, the role played by the disulfide bond in the PrPC-PrPSc conversion is still controversial [27–32]. To obtain insights into the role of the disulfide bond in the structural conversion of prion protein, molecular dynamics (MD) simulations were carried out in this study. This strategy has been adopted successfully to study the effects of temperature [33], mutation [34–36] and pH [18, 20, 37–39] on the folding and structural stability of prion protein. MD
2106, Page 2 of 8
J Mol Model (2014) 20:2106
Fig. 1 a Globular domain of human prion protein (huPrP) with the hydrophobic core displayed as a transparent molecular surface. The hydrophobic core contains residues 134, 137 139, 141, 158, 161, 175, 176, 179, 180, 184, 198, 203, 205, 206, 209, 210 and 213–215. Secondary structure elements are labeled (H1, H2, H3 helices; S1, S2 β-sheets) and are colored differently. The disulfide bond between Cys214 and Cys179 is shown in stick form and colored green. b The oneletter amino acid sequence and secondary structure of the globular domain of human prion protein. The letters in magenta represent the residues comprising the hydrophobic core
simulations were performed on wild type, reduced and Cysfree (Cys179 to Ala, Cys214 to Ala) human prion protein. As the effects of the flexible N-terminal region on the structure of the C-terminal core are negligible, we concentrated only on the globular C-terminal domain. Elevated temperature can accelerate protein unfolding without altering the unfolding pathway [40, 41]. MD simulations of prion protein at elevated temperatures have demonstrated the preservation of prion structure [33, 36, 42, 43]. Thus, to observe the possible conformational change in a reasonable time frame, a simulation temperature of 333 K was selected here, i.e., high enough to accelerate the process of conformational change [44] while not high enough to cause non-physiological effects.
Materials and methods This study used the NMR structure of the globular domain of human prion protein (huPrP) with residues 125–228 at pH 7.0 (PDB ID:1HJN) [45]. We modeled the reduced form of prion protein without creating a Cys179–Cys214 disulfide bond and the Cys-free PrP with cysteines mutated to alanines. All
structures were immersed in cubic water boxes with at least 10 Å between protein and the box boundary. Calculations were performed with NAMD version 2.9.b3 [46] in CHARMM22 force field [47]. Water was described with the TIP3P model [48]. All MD simulations were carried out in NPT ensemble at constant pressure of 1 bar at 333 K and pH 7. At pH 7, Lys, Arg and His are positively charged. Glu and Asp are negatively charged. For His, the ε nitrogen was protonated in this study. Langevin dynamics was used for temperature control with the Langevin coupling coefficient set to 5 ps. The Langevin Nosé-Hoover method was applied to keep pressure constant [49, 50]. Electrostatics were treated using the particle mesh Ewald (PME) method [51]. A cutoff of 12 Å was used for Lennard-Jones interaction. An integration step of 2 fs was used together with the SHAKE [52] algorithm to constrain bonds involving hydrogen atoms. The starting structures were first energy minimized for 10,000 steps, then warmed up from 0 K to 333 K. The MDs simulations were then performed for 100 ns. Structures were saved every 5 ps for analysis, resulting in 20,000 conformations for each trajectory. The simulation trajectories were analyzed using VMD 1.9.1 [53].
J Mol Model (2014) 20:2106
Page 3 of 8, 2106
Fig. 2 Overall dynamics of huPrP calculated from simulations. a Cα root mean square deviation (RMSD) of the globular domain from the initial structure as a function of time for all simulations. b Cα root mean square fluctuation (RMSF) of residues
Results and discussion Overall stability of the wild type, Cys-free, and reduced prion protein To monitor main chain stability of the protein, the root mean square deviations (RMSDs) of Cα atoms were calculated for the globular domain (residues 129–224, four N- and C terminal residues are omitted, respectively) from the starting structure. As shown in Fig. 2a, the RMSD of the wild type prion protein stays fairly low, whereas the reduced and C179A/C214A huPrP display larger values of RMSD along the simulation. The changes were most obvious in the simulation with the reduced protein. The Cα RMSD of wild type remains practically steady in the last 60 ns, with values below 2 Å. However, the Cα RMSD values of reduced huPrP reach more than 3.5 Å at the end of simulation. In contrast with WT huPrP, the Cα RMSD of the Cys-free huPrP increases but is still less than that of the reduced huPrP. The Cα RMSDs implied that breakage of the Cys179–Cys214 disulfide bond induced a significant conformational change in the prion protein, especially for the reduced type. Just like the changes in global dynamics, the flexibility of the structure was also affected by removal of the disulfide bond. Cα root mean square fluctuations (RMSFs) from the initial structures were measured throughout the trajectories. As Fig. 2b shows, the flexibility of the reduced huPrP is most obviously enhanced. For the reduced huPrP, the increase in RMSF lies mainly in H1, the loop between S1 and H1 and the loop connecting S2 and H2. Compared with reduced huPrP, the Cα RMSF values of Cys-free huPrP fluctuated in a similar manner except that the loop between S2 and H2 was much more stable. In contrast, simulations of wild type prion protein in 333 K did not highlight any major conformational rearrangement.
donor and the acceptor are within 3 Å and the angle formed by the donor, hydrogen and acceptor is greater than 150°, the hydrogen bond is thought to resist. In the initial structures of prion protein, the native β-sheet contains a βbulge and is connected by four backbone hydrogen bonds: 129 N:163O, 129O:163 N, 131 N:161O and 134 N:159O [35]. In the WT simulation, the four hydrogen bonds are fairly stable (Fig. 3). For the Cys-free and the reduced huPrP, a new hydrogen bond (132 N:161O) formed while the hydrogen bond formed by 134 N:159O broke, which resulted in the disappearance of the β-bulge and the appearance of extended β-sheet. Based on the STRIDE algorithm [54], the β-sheet content averaged over the last 20 ns of the reduced huPrP (6.77 %) and the Cys-free huPrP (6.40 %) increased about 40 % and 30 %, respectively, compared to that of WT huPrP (4.82 %). Our simulation results are consistent with experimentally observed phenomena. It has been reported that upon reduction of the disulfide bond at pH 4.0, the recombinant prion protein is dominated by β-sheet [55]. Recently, Chen et al. [31] reported that the disulfide-reduced recombinant prion protein undergoes a spontaneous α-to-β conformational transition at pH 7.0 without denaturants. The elongation of β-
Stability of local structures Upon rupture of the disulfide bond, elongations of β-sheet are observed for all these proteins. Backbone hydrogen bonds can be used to monitor secondary structure changes. When the
Fig. 3 Main chain hydrogen bonds of β-sheet for three studied prion proteins
2106, Page 4 of 8
J Mol Model (2014) 20:2106
Fig. 4 a Solvent-accessible surface area (SASA) of the hydrophobic core as a function of simulation time. For clarity, the windowed average over a period of 500 ps is shown. b Per-residue SASA of the hydrophobic core averaged over the last 20 ns of simulations for the wild type (WT), C179/C214 and reduced huPrP
sheet, which is correlated closely to the misfolding of prion protein, has been also observed in experiments and many other simulations of prion protein or its mutants in acid environment [35, 56–59]. Fig. 5 Initial misfolding of H1. a Final snapshots of A WT, B C179A/C214A and C reduced huPrP simulations. H1 and the loop connecting H1 are shown in green, the other part of the protein is displayed in gray. Residues M134, P137, I139, F141, M213, V209 and M205, which are involved in hydrophobic interactions between H3 and the H1-S1 loop are shown as transparent spheres. b Number of hydrophobic atom–atom contacts as a function of time for M134M213 (top left), P137-M213 (top right), I139-V209 (bottom left) and P141-M206 (bottom right). For clarity, the windowed average over a period of 500 ps is shown
The tightly packed hydrophobic core (Fig. 1) consisting of 134, 137, 139, 141, 158, 161, 175,176, 179, 180, 184, 198, 203, 205, 206, 209, 210, and 213–215 was also perturbed by rupture of the Cys179–Cys214 disulfide bond [60]. To
J Mol Model (2014) 20:2106
Fig. 6 Evolutions of the salt bridges Glu196–Arg156 and Asp202– Arg156 over time
evaluate the change of the hydrophobic core, its solventaccessible surface area (SASA) was calculated along the simulation time with a probe radius of 0.14 nm. As shown in Fig. 4a, in the last 50 ns, the SASA values of the hydrophobic core of the wild type huPrP maintain constant, with values about 450 Å2, indicating a stable hydrophobic core. As with the Cys-free PrP, significant increases were observed in the last 30 ns, with values of SASA reaching about 550 Å2. The SASA values of the reduced PrP experience an evident increase earlier than 35 ns and remain about 550 Å2 in the last 60 ns. The changes in the SASA of each hydrophobic residue were different for C179A/C214A huPrP and reduced huPrP as shown in Fig. 4b. However, the changes lie primarily in residues Ile139, Phe141, Phe198, Val203 and Val213. As residues Ile139, Phe141 are located in the S1-H1 loop and Fig. 7 Structures of the S2-H2 loop in reduced huPrP and interaction between the S2-H2 loop and the C-terminus of H3. a A Initial structure, B ending structure of S2-H2 loop, C interaction between the loop and the tail of H3 at the end of the simulation. b Hydrogen bonds formed between the S2-H2 loop and the C-terminal of H3 as a function of simulation time
Page 5 of 8, 2106
Phe198 is in the H2-H3 loop, the changes in surface area reflected fluctuation of these regions. Therefore, rupture of the disulfide bond disturbed the global stability of the hydrophobic core. Based on the Cα RMSF, it can be seen that significant changes in the positioning of H1 occurred in C179A/C214A and in reduced huPrP. As shown in Fig. 5a, simulations of Cys-free and reduced PrP featured a downward movement of the N-terminus of H1. The detachment of H1 from the remainder of the protein was accompanied by a substantial loss of hydrophobic interaction as shown in Fig. 5b. In the WT huPrP, Met134, Pro137, Ile139 and Phe141 in the S1-H1 loop interacted with Met213, Val209, and Met205 in H3, respectively. In the Cys-free and reduced huPrP, Pro137 and Ile139 lost contact with Met213 and Val209 almost at the same time (72 ns for Cys-free PrP, 33 ns for reduced PrP). Then, Phe141 underwent a gradual loss of hydrophobic contacts with Met205. The interaction between Met134 and Met213 persisted, which may block the movement of S1. In addition to the loss of hydrophobic interactions, the salt bridge connecting H1 and the plane of H2 and H3 also changed along with detachment of H1. Prior to the loss of hydrophobic contacts between the S1-H1 loop and H3, the Arg156–Glu196 salt bridge broke as shown in Fig. 6. The Arg156–Asp202 salt bridge remains intact throughout the simulation time, causing the C-terminal of H1 to link with H3. Simulations of prion protein without a disulfide bond showed significant conformational changes of H1 similar to those observed in previous research. For example, Daggett et al. [38] reported that H1 and its preceding loop can be rearranged and swing away from H3 at low pH. It was also reported that residues 132–145 detach from the remainder of the protein with denaturant [61]. Our previous study also
2106, Page 6 of 8
suggested that the pathogenic mutations T188K/R/A triggered the movement of H1 [56]. Together, these data indicate that the increased flexibility and displacement of H1 in response to breakage of the disulfide bond may be related to misfolding of prion protein. The S2-H2 loop has been reported to play a vital role in the propagation of prion protein [62, 63]. Simulations of reduced huPrP suggested that flexibility of the S2-H2 loop was amplified (Fig. 7a). As shown in Fig. 7a, the native hydrogen bond of the S2-H2 loop disappears and the loop is straightened out. Interestingly, the S2-H2 loop interacted with the tail of H3. Hydrogen bonds were formed between the residues of the S2H2 loop and residues of the C-terminus of H3, while the native hydrogen bonds of S2-H2 broke (Fig. 7b). The S2-H2 loop of the C179A/C214A huPrP is more stable than that of reduced PrP, possibly due to the smaller volume of the Ala side chain. The steric effects of the side chains of the two cysteine residues push the H3 outward, providing more space for the tail of H3 to curve in and interact with the S2-H2 loop, whereas the side chains of the two Ala residues were not large enough to push H3 away. The perturbation of the S2-H2 loop is not unique to the reduced prion protein, being also found in prion proteins with different mutations [56, 63, 64]. Due to the significance of the loop, we suggest that removal of the disulfide bond by reduction methods may lead to misfolding of prion protein by inducing structural changes of the S2-H2 loop.
Conclusions By performing extensive MD simulations, we investigated the role of the disulfide bond Cys179–Cys214 in the stability and the folding of prion protein. Simulations of reduced huPrP and C179A/C214A huPrP revealed detailed conformational and dynamic changes that may accompany the disappearance of the disulfide bond. Though the disulfide bond was broken in different ways, the conformational changes were similar for the two variants of PrP in that in both cases the native β-sheet was elongated. Furthermore, H1 was detached from the H2H3 core, and the displacement of H1 was related to changes in the S1-H1 loop in which the hydrophobic residues were more exposed to solvent. Furthermore, the S2-H2 loop of the reduced huPrP underwent significant structural transitions. The changes observed in the stability and folding of prion protein in response to rupture of the disulfide bond are similar to the conformational changes of prion protein at low pH, in denaturing conditions or with pathogenic mutations. Overall, we conclude that the disulfide bond stabilizes the structure of prion protein significantly and that deletion of the disulfide bond may lead to misfolding of prion protein.
J Mol Model (2014) 20:2106 Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No: 21103075) and the Natural Science Foundation of Gansu Province, China (Grant No: 1208RJYA034). The authors would also like to thank the Gansu Computing Center for providing computing resources.
References 1. Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, Glenner GG (1983) Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35(2):349–358 2. Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95(23):13363– 13383 3. Legname G, Baskakov IV, Nguyen H-OB, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB (2004) Synthetic mammalian prions. Science 305(5684):673–676 4. Griffith J (1967) Self-replication and scrapie. Nature 215(5105): 1043–1044 5. Stahl N, Baldwin MA, Teplow DB, Hood L, Gibson BW, Burlingame AL, Prusiner SB (1993) Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32(8):1991–2002 6. Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE (1993) Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA 90(23):10962–10966 7. Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS (1991) Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry 30(31): 7672–7680 8. Safar J, Roller PP, Gajdusek DC, Gibbs CJ (1993) Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity. Protein Sci 2(12):2206–2216 9. Stahl N, Prusiner SB (1991) Prions and prion proteins. FASEB J 5(13):2799–2807 10. Meyer RK, McKinley MP, Bowman KA, Braunfeld MB, Barry RA, Prusiner SB (1986) Separation and properties of cellular and scrapie prion proteins. Proc Natl Acad Sci USA 83(8):2310–2314 11. Oesch B, Westaway D, Wälchli M, McKinley MP, Kent S, Aebersold R, Barry RA, Tempst P, Teplow DB, Hood LE (1985) A cellular gene encodes scrapie PrP 27-30 protein. Cell 40(4):735–746 12. Donne DG, Viles JH, Groth D, Mehlhorn I, James TL, Cohen FE, Prusiner SB, Wright PE, Dyson HJ (1997) Structure of the recombinant full-length hamster prion protein PrP (29–231): the N terminus is highly flexible. Proc Natl Acad Sci USA 94(25):13452–13457 13. Riek R, Hornemann S, Wider G, Billeter M, Glockshuber R, Wüthrich K (1996) NMR structure of the mouse prion protein domain PrP (121–231). Nature 382(6587):180–182 14. Zahn R, Liu A, Lührs T, Riek R, Von Schroetter C, García FL, Billeter M, Calzolai L, Wider G, Wüthrich K (2000) NMR solution structure of the human prion protein. Proc Natl Acad Sci USA 97(1): 145–150 15. Knaus KJ, Morillas M, Swietnicki W, Malone M, Surewicz WK, Yee VC (2001) Crystal structure of the human prion protein reveals a mechanism for oligomerization. Nat Struct Mol Biol 8(9):770–774 16. Barducci A, Chelli R, Procacci P, Schettino V (2005) Misfolding pathways of the prion protein probed by molecular dynamics simulations. Biophys J 88(2):1334–1343 17. Chen J, Thirumalai D (2013) Helices 2 and 3 are the initiation sites in the PrP C→ PrP SC transition. Biochemistry 52(2):310–319 18. DeMarco ML, Daggett V (2007) Molecular mechanism for low pH triggered misfolding of the human prion protein. Biochemistry 46(11):3045–3054
J Mol Model (2014) 20:2106 19. DeMarco ML, Daggett V (2009) Characterization of cell‐surface prion protein relative to its recombinant analogue: insights from molecular dynamics simulations of diglycosylated, membrane‐bound human prion protein. J Neurochem 109(1):60–73 20. Garrec J, Tavernelli I, Rothlisberger U (2013) Two misfolding routes for the prion protein around pH 4.5. PLoS Comput Biol 9(5): e1003057 21. Issack BB, Berjanskii M, Wishart DS, Stepanova M (2012) Exploring the essential collective dynamics of interacting proteins: application to prion protein dimers. Proteins Struct Funct Genet 80(7):1847–1865 22. Christen B, Damberger FF, Pérez DR, Hornemann S, Wüthrich K (2013) Structural plasticity of the cellular prion protein and implications in health and disease. Proc Natl Acad Sci USA 110(21):8549– 8554 23. Yu H, Liu X, Neupane K, Gupta AN, Brigley AM, Solanki A, Sosova I, Woodside MT (2012) Direct observation of multiple misfolding pathways in a single prion protein molecule. Proc Natl Acad Sci USA 109(14):5283–5288 24. Lu X, Zeng J, Gao Y, Zhang JZ, Zhang D, Mei Y (2013) The intrinsic helical propensities of the helical fragments in prion protein under neutral and low pH conditions: a replica exchange molecular dynamics study. J Mol Model 19(11):4897–4908 25. Kong Q, Mills JL, Kundu B, Li X, Qing L, Surewicz K, Cali I, Huang S, Zheng M, Swietnicki W (2013) Thermodynamic stabilization of the folded domain of prion protein inhibits prion infection in vivo. Cell Rep 4(2):248–254 26. Schlepckow K, Schwalbe H (2013) Molecular mechanism of prion protein oligomerization at atomic resolution. Angew Chem 125(38): 10186–10189 27. Turk E, Teplow D, Hood LE, Prusiner SB (2005) Purification and properties of the cellular and scrapie hamster prion proteins. Eur J Biochem 176(1):21–30 28. Muramoto T, Scott M, Cohen FE, Prusiner SB (1996) Recombinant scrapie-like prion protein of 106 amino acids is soluble. Proc Natl Acad Sci USA 93(26):15457–15462 29. Herrmann LM, Caughey B (1998) The importance of the disulfide bond in prion protein conversion. Neuroreport 9(11):2457–2461 30. Maiti NR, Surewicz WK (2001) The role of disulfide bridge in the folding and stability of the recombinant human prion protein. J Biol Chem 276(4):2427–2431 31. Sang JC, Lee C-Y, Luh FY, Huang Y-W, Chiang Y-W, Chen RP-Y (2012) Slow spontaneous α-to-β structural conversion in a nondenaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein. Prion 6(5):489–497 32. Eghiaian F, Daubenfeld T, Quenet Y, van Audenhaege M, Bouin A-P, van der Rest G, Grosclaude J, Rezaei H (2007) Diversity in prion protein oligomerization pathways results from domain expansion as revealed by hydrogen/deuterium exchange and disulfide linkage. Proc Natl Acad Sci USA 104(18):7414–7419 33. Gu W, Wang T, Zhu J, Shi Y, Liu H (2003) Molecular dynamics simulation of the unfolding of the human prion protein domain under low pH and high temperature conditions. Biophys Chem 104(1):79– 94 34. van der Kamp MW, Daggett V (2010) Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding. J Mol Biol 404(4):732–748 35. Chen W, van der Kamp MW, Daggett V (2010) Diverse effects on the native β-sheet of the human prion protein due to disease-associated mutations. Biochemistry 49(45):9874–9881 36. Chen X, Duan D, Zhu S, Zhang J (2013) Molecular dynamics simulation of temperature induced unfolding of animal prion protein. J Mol Model 19(10):4433–4441 37. Langella E, Improta R, Barone V (2004) Checking the pH-induced conformational transition of prion protein by molecular dynamics
Page 7 of 8, 2106
38.
39.
40.
41.
42.
43. 44.
45.
46.
47.
48.
49.
50. 51.
52.
53. 54.
55.
56.
57.
58.
simulations: effect of protonation of histidine residues. Biophys J 87(6):3623–3632 Van der Kamp MW, Daggett V (2010) Influence of pH on the human prion protein: insights into the early steps of misfolding. Biophys J 99(7):2289–2298 Vila-Viçosa D, Campos SR, Baptista AM, Machuqueiro M (2012) Reversibility of prion misfolding: insights from constant-pH molecular dynamics simulations. J Phys Chem B 116(30):8812–8821 Day R, Bennion BJ, Ham S, Daggett V (2002) Increasing temperature accelerates protein unfolding without changing the pathway of unfolding. J Mol Biol 322(1):189–203 Daggett V, Levitt M (1993) Protein unfolding pathways explored through molecular dynamics simulations. J Mol Biol 232(2):600– 619 El-Bastawissy E, Knaggs MH, Gilbert IH (2001) Molecular dynamics simulations of wild-type and point mutation human prion protein at normal and elevated temperature. J Mol Graph Model 20(2):145– 154 Zhang J (2011) The structural stability of wild-type horse prion protein. J Biomol Struct Dyn 29(2):369–377 Bruccoleri RE, Karplus M (1990) Conformational sampling using high‐temperature molecular dynamics. Biopolymers 29(14):1847– 1862 Calzolai L, Zahn R (2003) Influence of pH on NMR structure and stability of the human prion protein globular domain. J Biol Chem 278(37):35592–35596 Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L, Schulten K (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26(16):1781–1802 MacKerell AD Jr, Bashford D, Bellott M, Dunbrack RL Jr, Evanseck J, Field M, Fischer S, Gao J, Guo H, Ha S (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102(18):3586–3616 Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79(2):926–935 Feller SE, Zhang Y, Pastor RW, Brooks BR (1995) Constant-pressure molecular-dynamics simulation-the Langevin piston method. J Chem Phys 103(11):4613–4621 Martyna GJ, Tobias DJ, Klein ML (1994) Constant pressure molecular dynamics algorithms. J Chem Phys 101:4177–4189 Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103: 8577–8593 Ryckaert JP, Ciccotti G, Berendsen HJC (1977) Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys 23(3):327–341 Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38 Frishman D, Argos P (2004) Knowledge-based protein secondary structure assignment. Proteins Struct Funct Bioinforma 23(4):566– 579 Jackson G, Hosszu L, Power A, Hill A, Kenney J, Saibil H, Craven C, Waltho J, Clarke A, Collinge J (1999) Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations. Science 283(5409):1935–1937 Guo J, Ning L, Ren H, Liu H, Yao X (2011) Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: insight from molecular dynamics simulations. Biochim Biophys Acta 1820(2):116–123 Santini S, Claude JB, Audic S, Derreumaux P (2003) Impact of the tail and mutations G131V and M129V on prion protein flexibility. Proteins Struct Funct Bioinforma 51(2):258–265 Campos SR, Machuqueiro M, Baptista AM (2010) Constant-pH molecular dynamics simulations reveal a β-rich form of the human prion protein. J Phys Chem B 114(39):12692–12700
2106, Page 8 of 8 59. Alonso DO, An C, Daggett V (2002) Simulations of biomolecules: characterization of the early steps in the pH-induced conformational conversion of the hamster, bovine and human forms of the prion protein. Phil Trans R Soc London A 360(1795):1165–1178 60. Riek R, Wider G, Billeter M, Hornemann S, Glockshuber R, Wüthrich K (1998) Prion protein NMR structure and familial human spongiform encephalopathies. Proc Natl Acad Sci USA 95(20): 11667–11672 61. Hosszu LL, Wells MA, Jackson GS, Jones S, Batchelor M, Clarke AR, Craven CJ, Waltho JP, Collinge J (2005) Definable equilibrium states in the folding of human prion protein. Biochemistry 44(50): 16649–16657
J Mol Model (2014) 20:2106 62. Kaneko K, Zulianello L, Scott M, Cooper CM, Wallace AC, James TL, Cohen FE, Prusiner SB (1997) Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proc Natl Acad Sci USA 94(19):10069– 10074 63. Meli M, Gasset M, Colombo G (2011) Dynamic diagnosis of familial prion diseases supports the β2-α2 loop as a universal interference target. PLoS ONE 6(4):e19093 64. Biljan I, Ilc G, Giachin G, Plavec J, Legname G (2012) Structural rearrangements at physiological pH: nuclear magnetic resonance insights from the V210I human prion protein mutant. Biochemistry 51(38):7465–7474
ORIGINAL PAPER
The role of Cys179–Cys214 disulfide bond in the stability and folding of prion protein: insights from molecular dynamics simulations Lulu Ning & Jingjing Guo & Nengzhi Jin & Huanxiang Liu & Xiaojun Yao
Received: 28 September 2013 / Accepted: 7 December 2013 / Published online: 11 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Prion diseases are associated with misfolding and aggregation of prion protein (PrP). Cellular prion protein contains a disulfide bond linking Cys residues at positions 179 and 214. It has been proposed that this disulfide bond plays an important role in the conversion between cellular (PrPC) and the scrapie form of prion protein (PrPSc). To probe the role of this disulfide bond in the stability and folding of prion protein, we employed molecular dynamics simulations to study the reduced prion protein and a variant of PrP in which the two cysteines were replaced by alanines residues. The simulations highlighted the changes that occurred upon breakage of the disulfide bond. Breakage of the disulfide bond resulted in a shift of H1, elongation of the native β-sheet and perturbation of the hydrophobic core of huPrP. The changes are similar to the conformational transitions of prion protein in low pH, in denaturing conditions or with pathogenic mutations, which indicate that rupture of the disulfide bond may lead to the misfolding of prion protein. Keywords Molecular dynamics simulation . Prion diseases . Disulfide bond . Misfolding L. Ning : J. Guo : H. Liu (*) : X. Yao State Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou University, Lanzhou 730000, China e-mail: [email protected] X. Yao e-mail: [email protected] J. Guo : H. Liu School of Pharmacy, Lanzhou University, Lanzhou 730000, China N. Jin Gansu Computing Center, Lanzhou 730000, China X. Yao State Key Laboratory for Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China
Introduction Transmissible spongiform encephalopathies (TSE) such as Kuru, Creutzfeldt-Jakob disease in human beings and bovine spongiform encephalopathy in animals are characterized by aggregates of the abnormal form of prion protein (PrPSc) [1, 2]. According to the “protein only hypothesis”, the aggregated scrapie form, PrPSc, is the infectious agent of these TSElinked diseases [3, 4]. PrPSc and cellular, non-pathological prion protein (PrPC) share an identical covalent structure but have distinct biophysical and biochemical properties [5]. PrPSc has a high β-sheet content, whereas PrPC is rich in αhelix [6–8]. Furthermore, PrPSc is insoluble in mild detergents and is proteinase K-resistant, whereas PrPC is soluble in mild detergent and highly sensitive to proteinase K [9–11]. PrPC contains a flexible N-terminal region and a globular C-terminal region that consists of two short in-register βsheets and three α-helices with a disulfide bond at position 179 and 214 linking H2 and H3 (Fig. 1) [12, 13]. The structure of PrPSc is still not fully elucidated due to its high propensity to aggregate. Because the conformational conversion of prion protein from its cellular form to its scrapie form is the critical step at the beginning of prion diseases, much effort has been dedicated to understanding the misfolding and aggregation of prion protein [14–26]. However, the molecular mechanisms underlying the conformational transition remain largely unknown currently. It has been proposed that the disulfide bond connecting H2 and H3 is important in the structural conversion. However, the role played by the disulfide bond in the PrPC-PrPSc conversion is still controversial [27–32]. To obtain insights into the role of the disulfide bond in the structural conversion of prion protein, molecular dynamics (MD) simulations were carried out in this study. This strategy has been adopted successfully to study the effects of temperature [33], mutation [34–36] and pH [18, 20, 37–39] on the folding and structural stability of prion protein. MD
2106, Page 2 of 8
J Mol Model (2014) 20:2106
Fig. 1 a Globular domain of human prion protein (huPrP) with the hydrophobic core displayed as a transparent molecular surface. The hydrophobic core contains residues 134, 137 139, 141, 158, 161, 175, 176, 179, 180, 184, 198, 203, 205, 206, 209, 210 and 213–215. Secondary structure elements are labeled (H1, H2, H3 helices; S1, S2 β-sheets) and are colored differently. The disulfide bond between Cys214 and Cys179 is shown in stick form and colored green. b The oneletter amino acid sequence and secondary structure of the globular domain of human prion protein. The letters in magenta represent the residues comprising the hydrophobic core
simulations were performed on wild type, reduced and Cysfree (Cys179 to Ala, Cys214 to Ala) human prion protein. As the effects of the flexible N-terminal region on the structure of the C-terminal core are negligible, we concentrated only on the globular C-terminal domain. Elevated temperature can accelerate protein unfolding without altering the unfolding pathway [40, 41]. MD simulations of prion protein at elevated temperatures have demonstrated the preservation of prion structure [33, 36, 42, 43]. Thus, to observe the possible conformational change in a reasonable time frame, a simulation temperature of 333 K was selected here, i.e., high enough to accelerate the process of conformational change [44] while not high enough to cause non-physiological effects.
Materials and methods This study used the NMR structure of the globular domain of human prion protein (huPrP) with residues 125–228 at pH 7.0 (PDB ID:1HJN) [45]. We modeled the reduced form of prion protein without creating a Cys179–Cys214 disulfide bond and the Cys-free PrP with cysteines mutated to alanines. All
structures were immersed in cubic water boxes with at least 10 Å between protein and the box boundary. Calculations were performed with NAMD version 2.9.b3 [46] in CHARMM22 force field [47]. Water was described with the TIP3P model [48]. All MD simulations were carried out in NPT ensemble at constant pressure of 1 bar at 333 K and pH 7. At pH 7, Lys, Arg and His are positively charged. Glu and Asp are negatively charged. For His, the ε nitrogen was protonated in this study. Langevin dynamics was used for temperature control with the Langevin coupling coefficient set to 5 ps. The Langevin Nosé-Hoover method was applied to keep pressure constant [49, 50]. Electrostatics were treated using the particle mesh Ewald (PME) method [51]. A cutoff of 12 Å was used for Lennard-Jones interaction. An integration step of 2 fs was used together with the SHAKE [52] algorithm to constrain bonds involving hydrogen atoms. The starting structures were first energy minimized for 10,000 steps, then warmed up from 0 K to 333 K. The MDs simulations were then performed for 100 ns. Structures were saved every 5 ps for analysis, resulting in 20,000 conformations for each trajectory. The simulation trajectories were analyzed using VMD 1.9.1 [53].
J Mol Model (2014) 20:2106
Page 3 of 8, 2106
Fig. 2 Overall dynamics of huPrP calculated from simulations. a Cα root mean square deviation (RMSD) of the globular domain from the initial structure as a function of time for all simulations. b Cα root mean square fluctuation (RMSF) of residues
Results and discussion Overall stability of the wild type, Cys-free, and reduced prion protein To monitor main chain stability of the protein, the root mean square deviations (RMSDs) of Cα atoms were calculated for the globular domain (residues 129–224, four N- and C terminal residues are omitted, respectively) from the starting structure. As shown in Fig. 2a, the RMSD of the wild type prion protein stays fairly low, whereas the reduced and C179A/C214A huPrP display larger values of RMSD along the simulation. The changes were most obvious in the simulation with the reduced protein. The Cα RMSD of wild type remains practically steady in the last 60 ns, with values below 2 Å. However, the Cα RMSD values of reduced huPrP reach more than 3.5 Å at the end of simulation. In contrast with WT huPrP, the Cα RMSD of the Cys-free huPrP increases but is still less than that of the reduced huPrP. The Cα RMSDs implied that breakage of the Cys179–Cys214 disulfide bond induced a significant conformational change in the prion protein, especially for the reduced type. Just like the changes in global dynamics, the flexibility of the structure was also affected by removal of the disulfide bond. Cα root mean square fluctuations (RMSFs) from the initial structures were measured throughout the trajectories. As Fig. 2b shows, the flexibility of the reduced huPrP is most obviously enhanced. For the reduced huPrP, the increase in RMSF lies mainly in H1, the loop between S1 and H1 and the loop connecting S2 and H2. Compared with reduced huPrP, the Cα RMSF values of Cys-free huPrP fluctuated in a similar manner except that the loop between S2 and H2 was much more stable. In contrast, simulations of wild type prion protein in 333 K did not highlight any major conformational rearrangement.
donor and the acceptor are within 3 Å and the angle formed by the donor, hydrogen and acceptor is greater than 150°, the hydrogen bond is thought to resist. In the initial structures of prion protein, the native β-sheet contains a βbulge and is connected by four backbone hydrogen bonds: 129 N:163O, 129O:163 N, 131 N:161O and 134 N:159O [35]. In the WT simulation, the four hydrogen bonds are fairly stable (Fig. 3). For the Cys-free and the reduced huPrP, a new hydrogen bond (132 N:161O) formed while the hydrogen bond formed by 134 N:159O broke, which resulted in the disappearance of the β-bulge and the appearance of extended β-sheet. Based on the STRIDE algorithm [54], the β-sheet content averaged over the last 20 ns of the reduced huPrP (6.77 %) and the Cys-free huPrP (6.40 %) increased about 40 % and 30 %, respectively, compared to that of WT huPrP (4.82 %). Our simulation results are consistent with experimentally observed phenomena. It has been reported that upon reduction of the disulfide bond at pH 4.0, the recombinant prion protein is dominated by β-sheet [55]. Recently, Chen et al. [31] reported that the disulfide-reduced recombinant prion protein undergoes a spontaneous α-to-β conformational transition at pH 7.0 without denaturants. The elongation of β-
Stability of local structures Upon rupture of the disulfide bond, elongations of β-sheet are observed for all these proteins. Backbone hydrogen bonds can be used to monitor secondary structure changes. When the
Fig. 3 Main chain hydrogen bonds of β-sheet for three studied prion proteins
2106, Page 4 of 8
J Mol Model (2014) 20:2106
Fig. 4 a Solvent-accessible surface area (SASA) of the hydrophobic core as a function of simulation time. For clarity, the windowed average over a period of 500 ps is shown. b Per-residue SASA of the hydrophobic core averaged over the last 20 ns of simulations for the wild type (WT), C179/C214 and reduced huPrP
sheet, which is correlated closely to the misfolding of prion protein, has been also observed in experiments and many other simulations of prion protein or its mutants in acid environment [35, 56–59]. Fig. 5 Initial misfolding of H1. a Final snapshots of A WT, B C179A/C214A and C reduced huPrP simulations. H1 and the loop connecting H1 are shown in green, the other part of the protein is displayed in gray. Residues M134, P137, I139, F141, M213, V209 and M205, which are involved in hydrophobic interactions between H3 and the H1-S1 loop are shown as transparent spheres. b Number of hydrophobic atom–atom contacts as a function of time for M134M213 (top left), P137-M213 (top right), I139-V209 (bottom left) and P141-M206 (bottom right). For clarity, the windowed average over a period of 500 ps is shown
The tightly packed hydrophobic core (Fig. 1) consisting of 134, 137, 139, 141, 158, 161, 175,176, 179, 180, 184, 198, 203, 205, 206, 209, 210, and 213–215 was also perturbed by rupture of the Cys179–Cys214 disulfide bond [60]. To
J Mol Model (2014) 20:2106
Fig. 6 Evolutions of the salt bridges Glu196–Arg156 and Asp202– Arg156 over time
evaluate the change of the hydrophobic core, its solventaccessible surface area (SASA) was calculated along the simulation time with a probe radius of 0.14 nm. As shown in Fig. 4a, in the last 50 ns, the SASA values of the hydrophobic core of the wild type huPrP maintain constant, with values about 450 Å2, indicating a stable hydrophobic core. As with the Cys-free PrP, significant increases were observed in the last 30 ns, with values of SASA reaching about 550 Å2. The SASA values of the reduced PrP experience an evident increase earlier than 35 ns and remain about 550 Å2 in the last 60 ns. The changes in the SASA of each hydrophobic residue were different for C179A/C214A huPrP and reduced huPrP as shown in Fig. 4b. However, the changes lie primarily in residues Ile139, Phe141, Phe198, Val203 and Val213. As residues Ile139, Phe141 are located in the S1-H1 loop and Fig. 7 Structures of the S2-H2 loop in reduced huPrP and interaction between the S2-H2 loop and the C-terminus of H3. a A Initial structure, B ending structure of S2-H2 loop, C interaction between the loop and the tail of H3 at the end of the simulation. b Hydrogen bonds formed between the S2-H2 loop and the C-terminal of H3 as a function of simulation time
Page 5 of 8, 2106
Phe198 is in the H2-H3 loop, the changes in surface area reflected fluctuation of these regions. Therefore, rupture of the disulfide bond disturbed the global stability of the hydrophobic core. Based on the Cα RMSF, it can be seen that significant changes in the positioning of H1 occurred in C179A/C214A and in reduced huPrP. As shown in Fig. 5a, simulations of Cys-free and reduced PrP featured a downward movement of the N-terminus of H1. The detachment of H1 from the remainder of the protein was accompanied by a substantial loss of hydrophobic interaction as shown in Fig. 5b. In the WT huPrP, Met134, Pro137, Ile139 and Phe141 in the S1-H1 loop interacted with Met213, Val209, and Met205 in H3, respectively. In the Cys-free and reduced huPrP, Pro137 and Ile139 lost contact with Met213 and Val209 almost at the same time (72 ns for Cys-free PrP, 33 ns for reduced PrP). Then, Phe141 underwent a gradual loss of hydrophobic contacts with Met205. The interaction between Met134 and Met213 persisted, which may block the movement of S1. In addition to the loss of hydrophobic interactions, the salt bridge connecting H1 and the plane of H2 and H3 also changed along with detachment of H1. Prior to the loss of hydrophobic contacts between the S1-H1 loop and H3, the Arg156–Glu196 salt bridge broke as shown in Fig. 6. The Arg156–Asp202 salt bridge remains intact throughout the simulation time, causing the C-terminal of H1 to link with H3. Simulations of prion protein without a disulfide bond showed significant conformational changes of H1 similar to those observed in previous research. For example, Daggett et al. [38] reported that H1 and its preceding loop can be rearranged and swing away from H3 at low pH. It was also reported that residues 132–145 detach from the remainder of the protein with denaturant [61]. Our previous study also
2106, Page 6 of 8
suggested that the pathogenic mutations T188K/R/A triggered the movement of H1 [56]. Together, these data indicate that the increased flexibility and displacement of H1 in response to breakage of the disulfide bond may be related to misfolding of prion protein. The S2-H2 loop has been reported to play a vital role in the propagation of prion protein [62, 63]. Simulations of reduced huPrP suggested that flexibility of the S2-H2 loop was amplified (Fig. 7a). As shown in Fig. 7a, the native hydrogen bond of the S2-H2 loop disappears and the loop is straightened out. Interestingly, the S2-H2 loop interacted with the tail of H3. Hydrogen bonds were formed between the residues of the S2H2 loop and residues of the C-terminus of H3, while the native hydrogen bonds of S2-H2 broke (Fig. 7b). The S2-H2 loop of the C179A/C214A huPrP is more stable than that of reduced PrP, possibly due to the smaller volume of the Ala side chain. The steric effects of the side chains of the two cysteine residues push the H3 outward, providing more space for the tail of H3 to curve in and interact with the S2-H2 loop, whereas the side chains of the two Ala residues were not large enough to push H3 away. The perturbation of the S2-H2 loop is not unique to the reduced prion protein, being also found in prion proteins with different mutations [56, 63, 64]. Due to the significance of the loop, we suggest that removal of the disulfide bond by reduction methods may lead to misfolding of prion protein by inducing structural changes of the S2-H2 loop.
Conclusions By performing extensive MD simulations, we investigated the role of the disulfide bond Cys179–Cys214 in the stability and the folding of prion protein. Simulations of reduced huPrP and C179A/C214A huPrP revealed detailed conformational and dynamic changes that may accompany the disappearance of the disulfide bond. Though the disulfide bond was broken in different ways, the conformational changes were similar for the two variants of PrP in that in both cases the native β-sheet was elongated. Furthermore, H1 was detached from the H2H3 core, and the displacement of H1 was related to changes in the S1-H1 loop in which the hydrophobic residues were more exposed to solvent. Furthermore, the S2-H2 loop of the reduced huPrP underwent significant structural transitions. The changes observed in the stability and folding of prion protein in response to rupture of the disulfide bond are similar to the conformational changes of prion protein at low pH, in denaturing conditions or with pathogenic mutations. Overall, we conclude that the disulfide bond stabilizes the structure of prion protein significantly and that deletion of the disulfide bond may lead to misfolding of prion protein.
J Mol Model (2014) 20:2106 Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No: 21103075) and the Natural Science Foundation of Gansu Province, China (Grant No: 1208RJYA034). The authors would also like to thank the Gansu Computing Center for providing computing resources.
References 1. Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, Glenner GG (1983) Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35(2):349–358 2. Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95(23):13363– 13383 3. Legname G, Baskakov IV, Nguyen H-OB, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB (2004) Synthetic mammalian prions. Science 305(5684):673–676 4. Griffith J (1967) Self-replication and scrapie. Nature 215(5105): 1043–1044 5. Stahl N, Baldwin MA, Teplow DB, Hood L, Gibson BW, Burlingame AL, Prusiner SB (1993) Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32(8):1991–2002 6. Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE (1993) Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA 90(23):10962–10966 7. Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS (1991) Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry 30(31): 7672–7680 8. Safar J, Roller PP, Gajdusek DC, Gibbs CJ (1993) Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity. Protein Sci 2(12):2206–2216 9. Stahl N, Prusiner SB (1991) Prions and prion proteins. FASEB J 5(13):2799–2807 10. Meyer RK, McKinley MP, Bowman KA, Braunfeld MB, Barry RA, Prusiner SB (1986) Separation and properties of cellular and scrapie prion proteins. Proc Natl Acad Sci USA 83(8):2310–2314 11. Oesch B, Westaway D, Wälchli M, McKinley MP, Kent S, Aebersold R, Barry RA, Tempst P, Teplow DB, Hood LE (1985) A cellular gene encodes scrapie PrP 27-30 protein. Cell 40(4):735–746 12. Donne DG, Viles JH, Groth D, Mehlhorn I, James TL, Cohen FE, Prusiner SB, Wright PE, Dyson HJ (1997) Structure of the recombinant full-length hamster prion protein PrP (29–231): the N terminus is highly flexible. Proc Natl Acad Sci USA 94(25):13452–13457 13. Riek R, Hornemann S, Wider G, Billeter M, Glockshuber R, Wüthrich K (1996) NMR structure of the mouse prion protein domain PrP (121–231). Nature 382(6587):180–182 14. Zahn R, Liu A, Lührs T, Riek R, Von Schroetter C, García FL, Billeter M, Calzolai L, Wider G, Wüthrich K (2000) NMR solution structure of the human prion protein. Proc Natl Acad Sci USA 97(1): 145–150 15. Knaus KJ, Morillas M, Swietnicki W, Malone M, Surewicz WK, Yee VC (2001) Crystal structure of the human prion protein reveals a mechanism for oligomerization. Nat Struct Mol Biol 8(9):770–774 16. Barducci A, Chelli R, Procacci P, Schettino V (2005) Misfolding pathways of the prion protein probed by molecular dynamics simulations. Biophys J 88(2):1334–1343 17. Chen J, Thirumalai D (2013) Helices 2 and 3 are the initiation sites in the PrP C→ PrP SC transition. Biochemistry 52(2):310–319 18. DeMarco ML, Daggett V (2007) Molecular mechanism for low pH triggered misfolding of the human prion protein. Biochemistry 46(11):3045–3054
J Mol Model (2014) 20:2106 19. DeMarco ML, Daggett V (2009) Characterization of cell‐surface prion protein relative to its recombinant analogue: insights from molecular dynamics simulations of diglycosylated, membrane‐bound human prion protein. J Neurochem 109(1):60–73 20. Garrec J, Tavernelli I, Rothlisberger U (2013) Two misfolding routes for the prion protein around pH 4.5. PLoS Comput Biol 9(5): e1003057 21. Issack BB, Berjanskii M, Wishart DS, Stepanova M (2012) Exploring the essential collective dynamics of interacting proteins: application to prion protein dimers. Proteins Struct Funct Genet 80(7):1847–1865 22. Christen B, Damberger FF, Pérez DR, Hornemann S, Wüthrich K (2013) Structural plasticity of the cellular prion protein and implications in health and disease. Proc Natl Acad Sci USA 110(21):8549– 8554 23. Yu H, Liu X, Neupane K, Gupta AN, Brigley AM, Solanki A, Sosova I, Woodside MT (2012) Direct observation of multiple misfolding pathways in a single prion protein molecule. Proc Natl Acad Sci USA 109(14):5283–5288 24. Lu X, Zeng J, Gao Y, Zhang JZ, Zhang D, Mei Y (2013) The intrinsic helical propensities of the helical fragments in prion protein under neutral and low pH conditions: a replica exchange molecular dynamics study. J Mol Model 19(11):4897–4908 25. Kong Q, Mills JL, Kundu B, Li X, Qing L, Surewicz K, Cali I, Huang S, Zheng M, Swietnicki W (2013) Thermodynamic stabilization of the folded domain of prion protein inhibits prion infection in vivo. Cell Rep 4(2):248–254 26. Schlepckow K, Schwalbe H (2013) Molecular mechanism of prion protein oligomerization at atomic resolution. Angew Chem 125(38): 10186–10189 27. Turk E, Teplow D, Hood LE, Prusiner SB (2005) Purification and properties of the cellular and scrapie hamster prion proteins. Eur J Biochem 176(1):21–30 28. Muramoto T, Scott M, Cohen FE, Prusiner SB (1996) Recombinant scrapie-like prion protein of 106 amino acids is soluble. Proc Natl Acad Sci USA 93(26):15457–15462 29. Herrmann LM, Caughey B (1998) The importance of the disulfide bond in prion protein conversion. Neuroreport 9(11):2457–2461 30. Maiti NR, Surewicz WK (2001) The role of disulfide bridge in the folding and stability of the recombinant human prion protein. J Biol Chem 276(4):2427–2431 31. Sang JC, Lee C-Y, Luh FY, Huang Y-W, Chiang Y-W, Chen RP-Y (2012) Slow spontaneous α-to-β structural conversion in a nondenaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein. Prion 6(5):489–497 32. Eghiaian F, Daubenfeld T, Quenet Y, van Audenhaege M, Bouin A-P, van der Rest G, Grosclaude J, Rezaei H (2007) Diversity in prion protein oligomerization pathways results from domain expansion as revealed by hydrogen/deuterium exchange and disulfide linkage. Proc Natl Acad Sci USA 104(18):7414–7419 33. Gu W, Wang T, Zhu J, Shi Y, Liu H (2003) Molecular dynamics simulation of the unfolding of the human prion protein domain under low pH and high temperature conditions. Biophys Chem 104(1):79– 94 34. van der Kamp MW, Daggett V (2010) Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding. J Mol Biol 404(4):732–748 35. Chen W, van der Kamp MW, Daggett V (2010) Diverse effects on the native β-sheet of the human prion protein due to disease-associated mutations. Biochemistry 49(45):9874–9881 36. Chen X, Duan D, Zhu S, Zhang J (2013) Molecular dynamics simulation of temperature induced unfolding of animal prion protein. J Mol Model 19(10):4433–4441 37. Langella E, Improta R, Barone V (2004) Checking the pH-induced conformational transition of prion protein by molecular dynamics
Page 7 of 8, 2106
38.
39.
40.
41.
42.
43. 44.
45.
46.
47.
48.
49.
50. 51.
52.
53. 54.
55.
56.
57.
58.
simulations: effect of protonation of histidine residues. Biophys J 87(6):3623–3632 Van der Kamp MW, Daggett V (2010) Influence of pH on the human prion protein: insights into the early steps of misfolding. Biophys J 99(7):2289–2298 Vila-Viçosa D, Campos SR, Baptista AM, Machuqueiro M (2012) Reversibility of prion misfolding: insights from constant-pH molecular dynamics simulations. J Phys Chem B 116(30):8812–8821 Day R, Bennion BJ, Ham S, Daggett V (2002) Increasing temperature accelerates protein unfolding without changing the pathway of unfolding. J Mol Biol 322(1):189–203 Daggett V, Levitt M (1993) Protein unfolding pathways explored through molecular dynamics simulations. J Mol Biol 232(2):600– 619 El-Bastawissy E, Knaggs MH, Gilbert IH (2001) Molecular dynamics simulations of wild-type and point mutation human prion protein at normal and elevated temperature. J Mol Graph Model 20(2):145– 154 Zhang J (2011) The structural stability of wild-type horse prion protein. J Biomol Struct Dyn 29(2):369–377 Bruccoleri RE, Karplus M (1990) Conformational sampling using high‐temperature molecular dynamics. Biopolymers 29(14):1847– 1862 Calzolai L, Zahn R (2003) Influence of pH on NMR structure and stability of the human prion protein globular domain. J Biol Chem 278(37):35592–35596 Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L, Schulten K (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26(16):1781–1802 MacKerell AD Jr, Bashford D, Bellott M, Dunbrack RL Jr, Evanseck J, Field M, Fischer S, Gao J, Guo H, Ha S (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102(18):3586–3616 Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79(2):926–935 Feller SE, Zhang Y, Pastor RW, Brooks BR (1995) Constant-pressure molecular-dynamics simulation-the Langevin piston method. J Chem Phys 103(11):4613–4621 Martyna GJ, Tobias DJ, Klein ML (1994) Constant pressure molecular dynamics algorithms. J Chem Phys 101:4177–4189 Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103: 8577–8593 Ryckaert JP, Ciccotti G, Berendsen HJC (1977) Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys 23(3):327–341 Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38 Frishman D, Argos P (2004) Knowledge-based protein secondary structure assignment. Proteins Struct Funct Bioinforma 23(4):566– 579 Jackson G, Hosszu L, Power A, Hill A, Kenney J, Saibil H, Craven C, Waltho J, Clarke A, Collinge J (1999) Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations. Science 283(5409):1935–1937 Guo J, Ning L, Ren H, Liu H, Yao X (2011) Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: insight from molecular dynamics simulations. Biochim Biophys Acta 1820(2):116–123 Santini S, Claude JB, Audic S, Derreumaux P (2003) Impact of the tail and mutations G131V and M129V on prion protein flexibility. Proteins Struct Funct Bioinforma 51(2):258–265 Campos SR, Machuqueiro M, Baptista AM (2010) Constant-pH molecular dynamics simulations reveal a β-rich form of the human prion protein. J Phys Chem B 114(39):12692–12700
2106, Page 8 of 8 59. Alonso DO, An C, Daggett V (2002) Simulations of biomolecules: characterization of the early steps in the pH-induced conformational conversion of the hamster, bovine and human forms of the prion protein. Phil Trans R Soc London A 360(1795):1165–1178 60. Riek R, Wider G, Billeter M, Hornemann S, Glockshuber R, Wüthrich K (1998) Prion protein NMR structure and familial human spongiform encephalopathies. Proc Natl Acad Sci USA 95(20): 11667–11672 61. Hosszu LL, Wells MA, Jackson GS, Jones S, Batchelor M, Clarke AR, Craven CJ, Waltho JP, Collinge J (2005) Definable equilibrium states in the folding of human prion protein. Biochemistry 44(50): 16649–16657
J Mol Model (2014) 20:2106 62. Kaneko K, Zulianello L, Scott M, Cooper CM, Wallace AC, James TL, Cohen FE, Prusiner SB (1997) Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proc Natl Acad Sci USA 94(19):10069– 10074 63. Meli M, Gasset M, Colombo G (2011) Dynamic diagnosis of familial prion diseases supports the β2-α2 loop as a universal interference target. PLoS ONE 6(4):e19093 64. Biljan I, Ilc G, Giachin G, Plavec J, Legname G (2012) Structural rearrangements at physiological pH: nuclear magnetic resonance insights from the V210I human prion protein mutant. Biochemistry 51(38):7465–7474
