The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.
References
[1]
Maury CP (2009) Self-propagating beta-sheet polypeptide structures as prebiotic informational molecular entities: the amyloid world. Orig Life Evol Biosph 39: 141–150.
[2]
Greenwald J, Riek R (2010) Biology of amyloid: structure, function, and regulation. Structure 18: 1244–1260.
[3]
Greenwald J, Riek R (2012) On the possible amyloid origin of protein folds. J Mol Biol 421: 417–426.
[4]
Eichner T, Radford SE (2011) A diversity of assembly mechanisms of a generic amyloid fold. Mol Cell 43: 8–18.
[5]
Monsellier E, Chiti F (2007) Prevention of amyloid-like aggregation as a driving force of protein evolution. EMBO Rep 8: 737–742.
[6]
Blanco LP, Evans ML, Smith DR, Badtke MP, Chapman MR (2012) Diversity, biogenesis and function of microbial amyloids. Trends Microbiol 20: 66–73.
[7]
Shewmaker F, McGlinchey RP, Wickner RB (2011) Structural insights into functional and pathological amyloid. J Biol Chem 286: 16533–16540.
[8]
Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 75: 333–366.
[9]
Eisenberg D, Jucker M (2012) The amyloid state of proteins in human diseases. Cell 148: 1188–1203.
[10]
Lansbury PT (1994) Mechanism of scrapie replication. Science 265: 1510.
[11]
Walker LC, LeVine H 3rd (2012) Corruption and spread of pathogenic proteins in neurodegenerative diseases. J Biol Chem 287: 33109–33115.
[12]
Moreno-Gonzalez I, Soto C (2011) Misfolded protein aggregates: mechanisms, structures and potential for disease transmission. Semin Cell Dev Biol 22: 482–487.
[13]
Toyama BH, Weissman JS (2011) Amyloid structure: conformational diversity and consequences. Annu Rev Biochem 80: 557–585.
Liebman SW, Chernoff YO (2012) Prions in yeast. Genetics 191: 1014–1072.
[16]
Saupe SJ (2011) The [Het-s] prion of Podospora anserina and its role in heterokaryon incompatibility. Semin Cell Dev Biol 22: 460–468.
[17]
Coustou V, Deleu C, Saupe S, Begueret J (1997) The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog. Proc Natl Acad Sci U S A 94: 9773–9778.
[18]
Debets AJ, Dalstra HJ, Slakhorst M, Koopmanschap B, Hoekstra RF, et al. (2012) High natural prevalence of a fungal prion. Proc Natl Acad Sci U S A 109: 10432–10437.
[19]
Rizet G (1952) Les phénomènes de barrage chez Podospora anserina. I. Analyse de barrage entre les souches s et S. Rev Cytol Biol Veg 13: 51–92.
[20]
Bidard F, Clave C, Saupe SJ (2013) The Transcriptional Response to Nonself in the Fungus Podospora anserina. G3 (Bethesda) 3: 1015–1030.
[21]
Daskalov A, Paoletti M, Ness F, Saupe SJ (2012) Genomic Clustering and Homology between HET-S and the NWD2 STAND Protein in Various Fungal Genomes. Plos One 7: e34854.
[22]
Paoletti M, Saupe SJ (2009) Fungal incompatibility: evolutionary origin in pathogen defense? Bioessays 31: 1201–1210.
[23]
Beisson-Schecroun J (1962) Incompatibilité cellulaire et interactions nucléocytoplamsiques dans les phénomènes de barrage chez le Podospora anserina. Ann Genet 4: 3–50.
[24]
Dalstra HJ, Swart K, Debets AJ, Saupe SJ, Hoekstra RF (2003) Sexual transmission of the [Het-S] prion leads to meiotic drive in Podospora anserina. Proc Natl Acad Sci U S A 100: 6616–6621.
[25]
Balguerie A, Dos Reis S, Ritter C, Chaignepain S, Coulary-Salin B, et al. (2003) Domain organization and structure-function relationship of the HET-s prion protein of Podospora anserina. Embo J 22: 2071–2081.
[26]
Greenwald J, Buhtz C, Ritter C, Kwiatkowski W, Choe S, et al. (2010) The mechanism of prion inhibition by HET-S. Mol Cell 38: 889–899.
[27]
Ritter C, Maddelein ML, Siemer AB, Luhrs T, Ernst M, et al. (2005) Correlation of structural elements and infectivity of the HET-s prion. Nature 435: 844–848.
[28]
Van Melckebeke H, Wasmer C, Lange A, Ab E, Loquet A, et al. (2010) Atomic-resolution three-dimensional structure of HET-s(218–289) amyloid fibrils by solid-state NMR spectroscopy. J Am Chem Soc 132: 13765–13775.
[29]
Wasmer C, Lange A, Van Melckebeke H, Siemer AB, Riek R, et al. (2008) Amyloid fibrils of the HET-s(218–289) prion form a beta solenoid with a triangular hydrophobic core. Science 319: 1523–1526.
[30]
Seuring C, Greenwald J, Wasmer C, Wepf R, Saupe SJ, et al. (2012) The mechanism of toxicity in HET-S/HET-s prion incompatibility. PLoS Biol 10: e1001451.
[31]
Saupe SJ, Daskalov A (2012) The [Het-s] Prion, an Amyloid Fold as a Cell Death Activation Trigger. PLoS Pathog 8: e1002687.
[32]
Mathur V, Seuring C, Riek R, Saupe SJ, Liebman SW (2012) Localization of HET-S to the cell periphery, not to [Het-s] aggregates, is associated with [Het-s]-HET-S toxicity. Mol Cell Biol 32: 139–153.
[33]
Cai X, Chen J, Xu H, Liu S, Jiang QX, et al. (2014) Prion-like Polymerization Underlies Signal Transduction in Antiviral Immune Defense and Inflammasome Activation. Cell 156: 1207–1222.
[34]
Mizuno N, Baxa U, Steven AC (2011) Structural dependence of HET-s amyloid fibril infectivity assessed by cryoelectron microscopy. Proc Natl Acad Sci U S A 108: 3252–3257.
[35]
Van der Nest MA, Olson A, Lind M, Velez H, Dalman K, et al. (2014) Distribution and evolution of het gene homologs in the basidiomycota. Fungal Genet Biol 64: 45–57.
[36]
Gendoo DM, Harrison PM (2011) Origins and evolution of the HET-s prion-forming protein: searching for other amyloid-forming solenoids. Plos One 6: e27342.
[37]
Benkemoun L, Ness F, Sabate R, Ceschin J, Breton A, et al. (2011) Two structurally similar fungal prions efficiently cross-seed in vivo but form distinct polymers when coexpressed. Mol Microbiol 82: 1392–1405.
[38]
Wasmer C, Zimmer A, Sabate R, Soragni A, Saupe SJ, et al. (2010) Structural similarity between the prion domain of HET-s and a homologue can explain amyloid cross-seeding in spite of limited sequence identity. J Mol Biol 402: 311–325.
Wasmer C, Benkemoun L, Sabate R, Steinmetz MO, Coulary-Salin B, et al. (2009) Solid-state NMR spectroscopy reveals that E. coli inclusion bodies of HET-s(218–289) are amyloids. Angew Chem Int Ed Engl 48: 4858–4860.
[41]
Sabate R, Baxa U, Benkemoun L, Sanchez de Groot N, Coulary-Salin B, et al. (2007) Prion and non-prion amyloids of the HET-s prion forming domain. J Mol Biol 370: 768–783.
[42]
Wasmer C, Soragni A, Sabate R, Lange A, Riek R, et al. (2008) Infectious and noninfectious amyloids of the HET-s(218–289) prion have different NMR spectra. Angew Chem Int Ed Engl 47: 5839–5841.
[43]
Wan W, Bian W, McDonald M, Kijac A, Wemmer DE, et al. (2013) Heterogeneous seeding of a prion structure by a generic amyloid form of the fungal prion-forming domain HET-s(218–289). J Biol Chem 288: 29604–29612.
[44]
Wan W, Wille H, Stohr J, Baxa U, Prusiner SB, et al. (2012) Degradation of fungal prion HET-s(218–289) induces formation of a generic amyloid fold. Biophys J 102: 2339–2344.
[45]
Ferguson N, Becker J, Tidow H, Tremmel S, Sharpe TD, et al. (2006) General structural motifs of amyloid protofilaments. Proc Natl Acad Sci U S A 103: 16248–16253.
[46]
Williams AD, Shivaprasad S, Wetzel R (2006) Alanine scanning mutagenesis of Abeta(1–40) amyloid fibril stability. J Mol Biol 357: 1283–1294.
[47]
Ross ED, Edskes HK, Terry MJ, Wickner RB (2005) Primary sequence independence for prion formation. Proc Natl Acad Sci U S A 102: 12825–12830.
[48]
Derkatch IL, Chernoff YO, Kushnirov VV, Inge-Vechtomov SG, Liebman SW (1996) Genesis and variability of [PSI] prion factors in Saccharomyces cerevisiae. Genetics 144: 1375–1386.
[49]
Tanaka M, Chien P, Naber N, Cooke R, Weissman JS (2004) Conformational variations in an infectious protein determine prion strain differences. Nature 428: 323–328.
[50]
Marchante R, Rowe M, Zenthon J, Howard MJ, Tuite MF (2013) Structural Definition Is Important for the Propagation of the Yeast [PSI(+)] Prion. Mol Cell 50: 675–685.
[51]
Coustou V, Deleu C, Saupe SJ, Begueret J (1999) Mutational analysis of the [Het-s] prion analog of Podospora anserina. A short N-terminal peptide allows prion propagation. Genetics 153: 1629–1640.
[52]
Deleu C, Clave C, Begueret J (1993) A single amino acid difference is sufficient to elicit vegetative incompatibility in the fungus Podospora anserina. Genetics 135: 45–52.
[53]
Bailey TL, Boden M, Buske FA, Frith M, Grant CE, et al. (2009) MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37: W202–208.
[54]
Benkemoun L, Sabate R, Malato L, Dos Reis S, Dalstra H, et al. (2006) Methods for the in vivo and in vitro analysis of [Het-s] prion infectivity. Methods 39: 61–67.
[55]
Malato L, Dos Reis S, Benkemoun L, Sabate R, Saupe SJ (2007) Role of Hsp104 in the propagation and inheritance of the [Het-s] prion. Mol Biol Cell 18: 4803–4812.
[56]
Siemer AB, Ritter C, Ernst M, Riek R, Meier BH (2005) High-resolution solid-state NMR spectroscopy of the prion protein HET-s in its amyloid conformation. Angew Chem Int Ed Engl 44: 2441–2444.
[57]
Creighton TE (1993) Proteins: Structures and Molecular Properties, 2nd edition. New York: Freedman.
[58]
Luisi DL, Snow CD, Lin JJ, Hendsch ZS, Tidor B, et al. (2003) Surface salt bridges, double-mutant cycles, and protein stability: an experimental and computational analysis of the interaction of the Asp 23 side chain with the N-terminus of the N-terminal domain of the ribosomal protein l9. Biochemistry 42: 7050–7060.
[59]
Marqusee S, Sauer RT (1994) Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in lambda repressor. Protein Sci 3: 2217–2225.
[60]
Schreiber G, Fersht AR (1995) Energetics of protein-protein interactions: analysis of the barnase-barstar interface by single mutations and double mutant cycles. J Mol Biol 248: 478–486.
[61]
Santoro MM, Bolen DW (1988) Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27: 8063–8068.
[62]
Wang WaR, C.J., editor (2010) Aggregation of therapeutic proteins. Hoboken, New Jersey: Wiley.
[63]
Whitten ST, Wooll JO, Razeghifard R, Garcia-Moreno EB, Hilser VJ (2001) The origin of pH-dependent changes in m-values for the denaturant-induced unfolding of proteins. J Mol Biol 309: 1165–1175.
[64]
Batey S, Clarke J (2006) Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains. Proc Natl Acad Sci U S A 103: 18113–18118.
[65]
Myers JK, Pace CN, Scholtz JM (1995) Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci 4: 2138–2148.
[66]
Kajava AV, Baxa U, Steven AC (2010) Beta arcades: recurring motifs in naturally occurring and disease-related amyloid fibrils. FASEB J 24: 1311–1319.
[67]
Tanaka M, Collins SR, Toyama BH, Weissman JS (2006) The physical basis of how prion conformations determine strain phenotypes. Nature 442: 585–589.
[68]
Legname G, Nguyen HO, Peretz D, Cohen FE, DeArmond SJ, et al. (2006) Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes. Proc Natl Acad Sci U S A 103: 19105–19110.
[69]
Colby DW, Giles K, Legname G, Wille H, Baskakov IV, et al. (2009) Design and construction of diverse mammalian prion strains. Proc Natl Acad Sci U S A 106: 20417–20422.
[70]
Friedman R, Caflisch A (2013) Wild type and mutants of the HET-s(218–289) prion show different flexibility at fibrillar ends: A simulation study. Proteins 82: 399–404.
[71]
Chiti F, Webster P, Taddei N, Clark A, Stefani M, et al. (1999) Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc Natl Acad Sci U S A 96: 3590–3594.
[72]
Nelson R, Eisenberg D (2006) Recent atomic models of amyloid fibril structure. Curr Opin Struct Biol 16: 260–265.
[73]
Nelson R, Sawaya MR, Balbirnie M, Madsen AO, Riekel C, et al. (2005) Structure of the cross-beta spine of amyloid-like fibrils. Nature 435: 773–778.
[74]
Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, et al. (2007) Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature 447: 453–457.
[75]
Margittai M, Langen R (2006) Side chain-dependent stacking modulates tau filament structure. J Biol Chem 281: 37820–37827.
[76]
Tjernberg L, Hosia W, Bark N, Thyberg J, Johansson J (2002) Charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides. J Biol Chem 277: 43243–43246.
[77]
Zanuy D, Nussinov R (2003) The sequence dependence of fiber organization. A comparative molecular dynamics study of the islet amyloid polypeptide segments 22–27 and 22–29. J Mol Biol 329: 565–584.
[78]
Fernandez-Escamilla AM, Rousseau F, Schymkowitz J, Serrano L (2004) Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. Nat Biotechnol 22: 1302–1306.
[79]
Tartaglia GG, Pawar AP, Campioni S, Dobson CM, Chiti F, et al. (2008) Prediction of aggregation-prone regions in structured proteins. J Mol Biol 380: 425–436.
[80]
Trovato A, Chiti F, Maritan A, Seno F (2006) Insight into the structure of amyloid fibrils from the analysis of globular proteins. PLoS Comput Biol 2: e170.
[81]
El-Khoury R, Sellem CH, Coppin E, Boivin A, Maas MF, et al. (2008) Gene deletion and allelic replacement in the filamentous fungus Podospora anserina. Curr Genet 53: 249–258.
[82]
Takegoshi K, Nakamura S, Terao T (2001) 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Phys Lett 344: 631–637.
[83]
Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, et al. (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59: 687–696.
