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• W2015444119 abstract "HET-S (97% identical to HET-s) has an N-terminal globular domain that exerts a prion-inhibitory effect in cis on its own prion-forming domain (PFD) and in trans on HET-s prion propagation. We show that HET-S fails to form fibrils in vitro and that it inhibits HET-s PFD fibrillization in trans. In vivo analyses indicate that β-structuring of the HET-S PFD is required for HET-S activity. The crystal structures of the globular domains of HET-s and HET-S are highly similar, comprising a helical fold, while NMR-based characterizations revealed no differences in the conformations of the PFDs. We conclude that prion inhibition is not encoded by structure but rather in stability and oligomerization properties: when HET-S forms a prion seed or is incorporated into a HET-s fibril via its PFD, the β-structuring in this domain induces a change in its globular domain, generating a molecular species that is incompetent for fibril growth. HET-S (97% identical to HET-s) has an N-terminal globular domain that exerts a prion-inhibitory effect in cis on its own prion-forming domain (PFD) and in trans on HET-s prion propagation. We show that HET-S fails to form fibrils in vitro and that it inhibits HET-s PFD fibrillization in trans. In vivo analyses indicate that β-structuring of the HET-S PFD is required for HET-S activity. The crystal structures of the globular domains of HET-s and HET-S are highly similar, comprising a helical fold, while NMR-based characterizations revealed no differences in the conformations of the PFDs. We conclude that prion inhibition is not encoded by structure but rather in stability and oligomerization properties: when HET-S forms a prion seed or is incorporated into a HET-s fibril via its PFD, the β-structuring in this domain induces a change in its globular domain, generating a molecular species that is incompetent for fibril growth. HET-S inhibits its own fibril formation as well as that of HET-s The structures of the N-terminal domains of HET-S and HET-s are very similar The prion inhibition mechanism relies on the stability of the N-terminal domain Prions are self-propagating, usually amyloid-like protein aggregates (Aguzzi et al., 2008Aguzzi A. Sigurdson C. Heikenwaelder M. Molecular mechanisms of prion pathogenesis.Annu. Rev. Pathol. 2008; 3: 11-40Crossref PubMed Scopus (272) Google Scholar). In mammals, prions cause fatal neurodegenerative disease, while in fungi they are detected as protein-based genetic elements (Wickner et al., 2008Wickner R.B. Shewmaker F. Kryndushkin D. Edskes H.K. Protein inheritance (prions) based on parallel in-register beta-sheet amyloid structures.Bioessays. 2008; 30: 955-964Crossref PubMed Scopus (71) Google Scholar). The fungal prion proteins have a modular organization comprising a so-called prion-forming domain (PFD) that is natively unfolded in the soluble form and that is appended to a globular domain. While the PFD of these proteins is both necessary and sufficient for prion propagation, the in vivo prion-forming abilities and aggregate morphology of a given protein can be strongly affected by mutations or polymorphism in the appended globular domain (Balguerie et al., 2003Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. Domain organization and structure-function relationship of the HET-s prion protein of Podospora anserina.EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (163) Google Scholar, Balguerie et al., 2004Balguerie A. Dos Reis S. Coulary-Salin B. Chaignepain S. Sabourin M. Schmitter J.M. Saupe S.J. The sequences appended to the amyloid core region of the HET-s prion protein determine higher-order aggregate organization in vivo.J. Cell Sci. 2004; 117: 2599-2610Crossref PubMed Scopus (27) Google Scholar, Fernandez-Bellot et al., 2000Fernandez-Bellot E. Guillemet E. Cullin C. The yeast prion [URE3] can be greatly induced by a functional mutated URE2 allele.EMBO J. 2000; 19: 3215-3222Crossref PubMed Scopus (52) Google Scholar, Liu et al., 2002Liu J.J. Sondheimer N. Lindquist S.L. Changes in the middle region of Sup35 profoundly alter the nature of epigenetic inheritance for the yeast prion.Proc. Natl. Acad. Sci. USA. 2002; 99: 16446-16453Crossref PubMed Scopus (121) Google Scholar, Maddelein and Wickner, 1999Maddelein M.L. Wickner R.B. Two prion-inducing regions of Ure2p are nonoverlapping.Mol. Cell. Biol. 1999; 19: 4516-4524Crossref PubMed Scopus (85) Google Scholar, Masison and Wickner, 1995Masison D.C. Wickner R.B. Prion-inducing domain of yeast Ure2p and protease resistance of Ure2p in prion-containing cells.Science. 1995; 270: 93-95Crossref PubMed Scopus (323) Google Scholar). This is apparently true for amyloidogenic proteins in general, as it has also been shown that amyloid toxicity of polyQ aggregates is strongly dependent on flanking domains (Dehay and Bertolotti, 2006Dehay B. Bertolotti A. Critical role of the proline-rich region in Huntingtin for aggregation and cytotoxicity in yeast.J. Biol. Chem. 2006; 281: 35608-35615Crossref PubMed Scopus (95) Google Scholar, Duennwald et al., 2006Duennwald M.L. Jagadish S. Muchowski P.J. Lindquist S. Flanking sequences profoundly alter polyglutamine toxicity in yeast.Proc. Natl. Acad. Sci. USA. 2006; 103: 11045-11050Crossref PubMed Scopus (221) Google Scholar). While considerable efforts have been devoted to the structural and functional characterization of the PFDs of fungal prions, the mechanistic basis of this cis-regulatory effect has been only scarcely studied despite its importance in the prion propagation mechanism. The het-s/het-S fungal prion may be the most blatant example of such a cis-acting prion-inhibitory domain (Balguerie et al., 2003Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. Domain organization and structure-function relationship of the HET-s prion protein of Podospora anserina.EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (163) Google Scholar, Balguerie et al., 2004Balguerie A. Dos Reis S. Coulary-Salin B. Chaignepain S. Sabourin M. Schmitter J.M. Saupe S.J. The sequences appended to the amyloid core region of the HET-s prion protein determine higher-order aggregate organization in vivo.J. Cell Sci. 2004; 117: 2599-2610Crossref PubMed Scopus (27) Google Scholar). HET-s and HET-S are natural polymorphic variants of the same protein that share a functional C-terminal PFD. Yet, in contrast to HET-s, full-length HET-S totally lacks prion-forming ability in vivo. In the filamentous fungus Podospora anserina, the prion state of the HET-s protein is involved in a programmed cell death (PCD) reaction termed heterokaryon incompatibility. Filamentous fungi have several incompatibility loci that regulate the fusion of mycelium between genetically distinct individuals (Glass et al., 2000Glass N.L. Jacobson D.J. Shiu P.K. The genetics of hyphal fusion and vegetative incompatibility in filamentous ascomycete fungi.Annu. Rev. Genet. 2000; 34: 165-186Crossref PubMed Scopus (281) Google Scholar, Saupe, 2000Saupe S.J. Molecular genetics of heterokaryon incompatibility in filamentous ascomycetes.Microbiol. Mol. Biol. Rev. 2000; 64: 489-502Crossref PubMed Scopus (241) Google Scholar). The het-s locus has two antagonistic alleles: het-s and het-S. Their encoded proteins, HET-s and HET-S, give rise to the compatibility phenotypes [Het-s] and [Het-S]. Although they differ by only 13 out of 289 residues (Turcq et al., 1991Turcq B. Deleu C. Denayrolles M. Begueret J. Two allelic genes responsible for vegetative incompatibility in the fungus Podospora anserina are not essential for cell viability.Mol. Gen. Genet. 1991; 228: 265-269Crossref PubMed Scopus (58) Google Scholar), only HET-s undergoes a transition to an infectious prion state that is correlated in vivo with the conversion from the [Het-s∗] to the [Het-s] phenotype (Coustou et al., 1997Coustou V. Deleu C. Saupe S. Begueret J. The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog.Proc. Natl. Acad. Sci. USA. 1997; 94: 9773-9778Crossref PubMed Scopus (388) Google Scholar). When a [Het-s] strain fuses with a [Het-S] strain, the fusion cell dies, whereas the fusion of [Het-s∗] with [Het-S] leads to a viable mixed cell (heterokaryon). Colonies with the [Het-s∗] phenotype convert spontaneously to [Het-s] at a very low frequency, but contact between [Het-s] and [Het-s∗] strains leads to infection of the latter and conversion of its phenotype to [Het-s]. Importantly, in vivo interactions between [Het-s] and [Het-S] strains can also lead to elimination of the [Het-s] prion state (Beisson-Schecroun, 1962Beisson-Schecroun J. Incompatibilité cellulaire et interactions nucléocytoplamsiques dans les phénomènes de barrage chez le Podospora anserina.Ann. Genet. 1962; 4: 3-50Google Scholar, Dalstra et al., 2003Dalstra H.J. Swart K. Debets A.J. Saupe S.J. Hoekstra R.F. Sexual transmission of the [Het-S] prion leads to meiotic drive in Podospora anserina.Proc. Natl. Acad. Sci. USA. 2003; 100: 6616-6621Crossref PubMed Scopus (67) Google Scholar, Rizet, 1952Rizet G. Les phénomènes de barrage chez Podospora anserina. I. Analyse de barrage entre les souches s et S.Rev. Cytol. Biol. Veg. 1952; 13: 51-92Google Scholar). This occurs, for instance, in a [Het-s] × [Het-S] sexual cross in which [Het-S] leads to complete curing of the [Het-s] prion in the meiotic progeny (daughter cells with the het-s allele are [Het-s∗]). Using microsurgical approaches, it was also shown that [Het-S] strongly inhibits [Het-s] propagation in vegetative hyphae (Beisson-Schecroun, 1962Beisson-Schecroun J. Incompatibilité cellulaire et interactions nucléocytoplamsiques dans les phénomènes de barrage chez le Podospora anserina.Ann. Genet. 1962; 4: 3-50Google Scholar). Therefore, [Het-S] not only triggers cell death upon interaction with [Het-s] but also exerts an inhibitory effect on its propagation. HET-s is a two-domain protein. It comprises a C-terminal PFD (residues 218–289) that is both necessary and sufficient for amyloid formation and prion propagation and an N-terminal globular domain (residues 1 to ∼227) that specifies the incompatibility type ([Het-s] or [Het-S]) (Balguerie et al., 2003Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. Domain organization and structure-function relationship of the HET-s prion protein of Podospora anserina.EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (163) Google Scholar). A chimeric protein construct with the HET-S globular domain appended to the HET-s PFD results in a protein of the [Het-S] type, and conversely a chimera associating the HET-s globular domain to the HET-S PFD displays the [Het-s] specificity. In other words, HET-S has a functional PFD, but the HET-S globular domain exerts a prion-inhibitory effect (in cis) on its own C-terminal PFD. A detailed analysis of the amino acid differences between HET-s and HET-S revealed that a single amino acid substitution in HET-S (H33P) converted its specificity to [Het-s] in vivo, whereas the conversion of [Het-s] to [Het-S] requires minimally two amino acid substitutions (D23A/P33H) (Deleu et al., 1993Deleu C. Clave C. Begueret J. A single amino acid difference is sufficient to elicit vegetative incompatibility in the fungus Podospora anserina.Genetics. 1993; 135: 45-52PubMed Google Scholar). The molecular mechanism of HET-s-mediated heterokaryon incompatibility is not known, nor has the toxic entity that leads to cell death been identified. The reaction that occurs when incompatible strains fuse is a form of PCD that is spatially restricted to the fusion cell or one or two cells to each side. In all fungal incompatibility systems except het-s, one of the het genes involved encodes for a protein with a HET domain (no relation to the HET-s protein). It has been shown that overexpression of the HET domain alone is sufficient to induce PCD, and that it depends on the presence of two other genes (Paoletti and Clave, 2007Paoletti M. Clave C. The fungus-specific HET domain mediates programmed cell death in Podospora anserina.Eukaryot. Cell. 2007; 6: 2001-2008Crossref PubMed Scopus (49) Google Scholar). In contrast, no other genes have been found that are essential for het-s-mediated PCD, so it is possible that HET-S and HET-s form a toxic species that directly initiates PCD. Deletions in the globular domain of HET-S not only alleviate the prion-inhibitory effect of the domain but also abolish HET-S activity in incompatibility. Thus, the HET-S globular domain (but not the HET-s globular domain) is essential for PCD (Balguerie et al., 2004Balguerie A. Dos Reis S. Coulary-Salin B. Chaignepain S. Sabourin M. Schmitter J.M. Saupe S.J. The sequences appended to the amyloid core region of the HET-s prion protein determine higher-order aggregate organization in vivo.J. Cell Sci. 2004; 117: 2599-2610Crossref PubMed Scopus (27) Google Scholar). The recently reported structures of the C-terminal PFD of HET-s in its fibril form have shed light on the mechanism of prion formation and propagation (Wasmer et al., 2008Wasmer C. Lange A. Van Melckebeke H. Siemer A.B. Riek R. Meier B.H. Amyloid fibrils of the HET-s(218-289) prion form a beta solenoid with a triangular hydrophobic core.Science. 2008; 319: 1523-1526Crossref PubMed Scopus (782) Google Scholar, Wasmer et al., 2009Wasmer C. Schutz A. Loquet A. Buhtz C. Greenwald J. Riek R. Bockmann A. Meier B.H. The molecular organization of the fungal prion HET-s in its amyloid form.J. Mol. Biol. 2009; 394: 119-127Crossref PubMed Scopus (62) Google Scholar). These reports revealed that the PFD aggregates into a highly ordered β-solenoid fold. However, the question remains as to how the N-terminal domain of HET-S can exert a prion-inhibitory effect on the PFD both in cis and in trans. To probe this question, we studied the functional properties of the HET-S protein in vitro and in vivo and solved the structure of the HET-s and HET-S N-terminal domains by X-ray crystallography. We expressed and purified a soluble full-length HET-S protein (see the Experimental Procedures) allowing the comparison of the in vitro fibrillization of HET-s and HET-S. Both proteins tend to aggregate in vitro, and this can be partially suppressed by storage at 4°C. However, as revealed by negatively stained electron micrographs (Figure 1A ), the aggregation of HET-s leads to long (>1 μm), well-ordered, single fibrils, while HET-S aggregates are amorphous and range in size from 10 to 100 nm. The HET-s fibril growth can be seeded by fibrils of the isolated PFD, greatly shortening the nucleation time, while the addition of PFD seeds did not lead to HET-S fibrils (data not shown). The finding that HET-S does not readily form fibrils in vitro explains the fact that [Het-S] strains do not have a prion-associated phenotype and directly illustrates the cis-acting prion inhibition of the HET-S globular domain. Since the above experiments suggest that the prion inhibition mechanism of HET-S in cis can be recapitulated in vitro, we wondered whether the same was true for the trans-acting prion-inhibitory effect of HET-S on [Het-s] propagation. Thus, we looked at the ability of HET-S to inhibit fibril formation in trans. The aggregation kinetics of the PFD from HET-s were monitored by solution NMR in the presence of unlabeled HET-S. In this experiment, only the monomeric PFD is detected, so that the decay of the signal is proportional to the amount of PFD that has been recruited into large, presumably fibrillar aggregates. In order to minimize the stochastic nature of self-seeded aggregation kinetics, the experiments were started with the addition of 30 nM PFD fibril seeds (see the Experimental Procedures). We found that HET-S is able to delay the onset of fibrillization in substoichiometric ratios as low as 1:700 (HET-S: PFD). The results plotted in Figure 1B show that at a ratio of 1:70, the onset of aggregation is delayed by several hours yet still proceeds relatively quickly once it begins. At the 1:7 ratio, the aggregation is delayed by more than 65 hr but eventually occurs sometime before 110 hr (data collection was not continuous after 24 hr). As expected, the addition of HET-s at substoichiometric ratios (up to 1:7) does not have an inhibitory affect. Also, HET-S(1–227) does not significantly inhibit at a 1:7 ratio, thus demonstrating that the PFD of HET-S is required for the in vitro inhibition of PFD aggregation in trans (data summarized in Table S1, available online). Having demonstrated that the HET-S prion-inhibitory effect is an intrinsic property of the protein, we set out to determine the structural basis of this functional difference between the HET-s and HET-S proteins. We have previously shown that the HET-s in its soluble form is composed of an N-terminal folded domain comprising residues ∼1–227 followed by a flexible and highly dynamic C-terminal tail (Balguerie et al., 2003Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. Domain organization and structure-function relationship of the HET-s prion protein of Podospora anserina.EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (163) Google Scholar). Since HET-S differs from HET-s in only 13 out of its 289 residues, it is not surprising that we find the same domain composition for HET-S. As in the case of HET-s, the [15N,1H]-TROSY spectrum of HET-S has two qualitatively different kinds of peaks: a first group of about 220 broad cross peaks with full line widths at half height along 1H of ∼25 Hz and a second group of about 70 sharp, more intense cross peaks with line widths of ∼14 Hz indicative of amino acid residues in a flexible conformation. Comparison with the [15N,1H]-TROSY spectrum of HET-S(1–227) (Figure S1) shows that the first group of peaks has their counterpart in HET-S(1–227). We established the sequential assignment of HET-S, and the 13Cα chemical shifts are consistent with these domain boundaries: the per-residue chemical shift deviation of HET-S (Figure S2) shows that it is composed of an N-terminal mostly helical structured domain comprising at least residues 13–222 followed by an unstructured and flexible C-terminal tail. In addition to having near-random coil 13Cα chemical shifts, the C-terminal tails of both HET-S and HET-s are highly dynamic as evidenced by low 15N{1H}-NOE values (Figure S3). The structure of HET-s(1–227) was solved by heavy atom phasing, and the refined coordinates were used as a starting model for the structure determination by molecular replacement of HET-S(1–227) and of HET-s[D23A,P33H](13–221), the globular domain of a mutant that exhibits the [Het-S] phenotype in vivo (structure factors and coordinates were deposited in the Protein Data Bank, see Table 1). All three proteins contain the same α-helical fold of 8–9 helices with a short two-stranded β sheet (Figure 2). The first helix, α1 (residues 2–8) is only visible in the HET-S crystals. Its absence in the HET-s structure may be a crystallographic artifact, and the HET-s[D23A,P33H](13–221) construct lacks the residues that make up this helix. This shorter construct was designed based on the defined regions in the initial structure determination of HET-s(1-227), in an effort to improve the diffraction by crystallizing a protein without the flexible ends. Indeed, it did yield the best diffracting crystals (Table 1), although the construct itself is significantly less stable (see below). The first five helices of HET-S pack in a regular antiparallel bundle followed by a loop and short β strand that connects α5 and α6. This loop (L5-6) comprising residues 137–147 and strand β2 thread back through the molecule (between α2 and α4) so that α5 and α6 are parallel but on opposite sides of the molecule. The last three helices (α7–α9) form a three-helix bundle substructure that packs approximately perpendicular to the first six helices. The overall shape of the fold is roughly an equilateral triangle of ∼55 Å on a side and a thickness of ∼25 Å.Table 1Crystallographic Data and Refinement StatisticsModel (PDB ID)HET-s(1–227) (2wvn)HET-S(1–227) (2wvo)HET-s[D23A,P33H](13–221) (2wvq)Space groupp432p3221p21Unit cell dimensions a, b, c (Å)122.695.1, 95.1, 170.449.3, 83.2, 67.4 α, β, γ (°)9090, 90, 12090.0, 104.5, 90.0Resolution range (outer shell) (Å)40–2.62 (2.77–2.62)40–2.3 (2.44–2.3)65–2.0 (2.12–2)Number of unique reflections10,04440,55634,078Redundancy10.1 (10.2)7.0 (6.2)3.2 (3.0)I/sigma24.1 (3.9)23.8 (3.6)10.5 (3.7)RmeasaRmeas = ∑h (nh/(nh−1))1/2 ∑i |Ii(h) − < I(h) > | / ∑h∑i Ii(h), where Ii(h) and < I(h) > are the ith and mean intensity, and nh is the multiplicity over all symmetry-equivalent reflections h (Diederichs and Karplus, 1997).0.060 (0.463)0.048 (0.569)0.088 (0.459)Completeness %99.7 (99.0)99.3 (99.1)95.2 (93.3)Number of protein monomers in A.U.122Number of nonhydrogen atoms in refinement Protein169635333363 DTT––16 Chloride ion–4– Water–135198R factor/free R factorbR = ∑‖FC |− |FO‖ / ∑|FO|, where |FC|is the calculated structure factor amplitude of the model, and |FO|is the observed structure factor amplitude; the free R factor was calculated against a random 5% test set of reflections that was not used during refinement. The same test set was used for all seven data sets.0.216/0.2700.225/0.2570.225/0.269Rmsd bond lengths/anglescRmsd, root-mean-square deviation from the parameter set for ideal stereochemistry (Engh and Huber, 1991).0.018/1.8430.010/1.4110.016/1.739a Rmeas = ∑h (nh/(nh−1))1/2 ∑i |Ii(h) − < I(h) > | / ∑h∑i Ii(h), where Ii(h) and < I(h) > are the ith and mean intensity, and nh is the multiplicity over all symmetry-equivalent reflections h (Diederichs and Karplus, 1997Diederichs K. Karplus P.A. Improved R-factors for diffraction data analysis in macromolecular crystallography.Nat. Struct. Biol. 1997; 4: 269-275Crossref PubMed Scopus (758) Google Scholar).b R = ∑‖FC |− |FO‖ / ∑|FO|, where |FC|is the calculated structure factor amplitude of the model, and |FO|is the observed structure factor amplitude; the free R factor was calculated against a random 5% test set of reflections that was not used during refinement. The same test set was used for all seven data sets.c Rmsd, root-mean-square deviation from the parameter set for ideal stereochemistry (Engh and Huber, 1991Engh R.A. Huber R. Accurate bond and angle parameters for X-ray protein structure refinement.Acta Crystallogr. A. 1991; 47: 392-400Crossref Scopus (2501) Google Scholar). Open table in a new tab There are to date more than 30 homologs of the HET-s N-terminal globular domain that can be identified with a PSI-BLAST search, all of which come from filamentous fungi and none of which have a known function (Figure S4). One of the homologs was identified in a screen for mutants that affect the pathogenicity of Leptosphaeria maculans, the fungus that causes blackleg disease of Brassica napus (rapeseed). This loss-of-pathogenicity (LOP-B) protein has 529 amino acids, and its N terminus is 31% similar to the HET-s globular domain. Henceforth, we refer to this particular fungal domain with its helical fold as the HeLo domain (HET-s/LOP-B). The conserved residues of the HeLo domain correspond primarily to buried residues in the HET-S(1–227) structure. The lack of surface-exposed residue conservation suggests that the fold is well conserved but that the function or target of the domain may differ among the HeLo domain-containing proteins. The degree of conservation is highest in the first ∼60 residues, with little or no conservation in the loops between secondary structures. The sequence of the last ∼50 residues, comprising the three terminal helices, is less well conserved (Figure S4). Despite the fact that all three constructs were crystallized in diverse conditions with different numbers of molecules in their asymmetric units (Table 1), they exhibit only minor differences. An overlay of HET-s and HET-S gives a root-mean-square deviation (rmsd) of 0.82 Å (0.81 Å for chain B) for the main-chain atoms in residues 11–223. For comparison, the overlay of chain A and B of HET-S within the same crystal gives an rmsd of 0.31 Å. Overlays of HET-s[D23A,P33H] with HET-s and HET-S give similarly low numbers. Hence, the structures are so similar that local differences need to be interpreted carefully, taking into account possible crystal packing effects. The first major difference between HET-s and HET-S is the N-terminal helix α1 that is present only in HET-S. However, the solution NMR spectra of neither HET-S nor HET-s indicate the presence of helix α1 in solution: the 13Cα and 13Cβ chemical shifts for the residues in this region do not exhibit helical deviations from their random coil values (Figure S2). Furthermore, we could not detect a difference in the helical content of the two proteins by CD spectroscopy (data not shown). There are two factors that may explain the discrepancy between the crystal structures and solution structures: (1) Since helix α1 resides at the dimer interface, it may not be stable in the monomeric form. If so, the 0.5–1 mM protein used for the NMR measurements may have led to an insufficient dimer population to support the helix (note that the crystals contain 15–20 mM protein). (2) The crystallization conditions of HET-S (4.0 M NaCl) may have stabilized the hydrophobic interaction that the helix α1 makes with itself at the interface, while the low salt conditions for HET-s (30% PEG 4000, ∼100 mM NaCl) may have had less of a stabilizing influence. The helix is necessarily absent from the mutant structure because it is not in the construct that was crystallized (residues 13–221). Therefore, we cannot conclude from the crystal structures that helix α1 is unique to HET-S, but it appears to be a quasistable structure that is absent from both HET-S and HET-s in solution. The second major structural difference occurs in the loop L5-6, the residues of which have higher B factors and are generally less well ordered than the other loops (Figure 2A and Figure S5). In the case of chain B from the mutant, its electron density is too weak to be confidently modeled. In HET-s there is a salt bridge between K145 of the loop L5-6 and D23 of helix α2 that stabilizes the loop conformation, while for HET-S and the mutant this interaction is absent (Figure S5B). HET-S and the mutant have an alanine at position 23, and L5-6 in their structures adopts a slightly more open conformation, moving 2–4 Å away from α2. In the HET-S(1–227) and HET-s[D23A,P33H](13–221) crystal structures, the residual B factors for α7-α9 are higher than those for α1-α6 (Figure S5C). In the HET-s structure these three helices do not have heightened B factors, but their lower sequence conservation is indicative of a somewhat independent structural unit. Although none of the structures was solved at atomic resolution, precluding an anisotropic refinement of the B factors, there was evidence of anisotropy in the molecule. We refined the models with the inclusion of four TLS (translation, libration, screw) domains, which led to a significant lowering of the crystallographic R factor Rfree (0.7%, 1.8%, and 2.4% for HET-s[1–227], HET-s[D23A,P33H][13–221] and HET-S[1–227], respectively). In the three structures reported here as well as every other crystal form of the different HET-s constructs that we obtained (total of 12 asymmetric molecules in 5 crystal forms), the crystal packing incorporated a common 2-fold symmetric dimer that was related by either crystallographic symmetry or by proper noncrystallographic symmetry (Figure 2B). Due to their poorer data quality, the other crystal forms were not pursued once they were solved by molecular replacement and are not discussed further here. The HET-S dimer interface consists of an equal amount of charged and nonpolar residues (nine residues or 39%) and has a moderate surface area of 944 Å2 (863 Å2 for HET-s; smaller due to lack of α1). During purification of these molecules on a Superdex 75 gel filtration column, their elution volumes were more consistent with their being monomeric based on calibration with globular proteins. However, the ubiquitousness of the dimer in the crystals led us to investigate whether it also existed in solution. To test the oligomerization state of the different HET-s constructs, we measured the masses of the solution species by multiangle light scattering (MALS) coupled to the flow from a G2000SWXL gel filtration column. By injecting protein at concentrations ranging from 25 to 2300 μM, we observed varying degrees of partitioning between a monomer and dimer (Figure 3A ). The association constant of the dimer is low enough that there is significant dissociation during the dilution and separation that occurs on the column, resulting in a single peak. However, at higher protein concentrations there is a clear decrease in mass from the start to the end of the peak with a concomitant broadening and decrease in retention time. Although such a low-affinity interaction does not at first appear to be biologically relevant, the fact that HET-s and possibly HET-S can oligomerize via their C-terminal PFDs implies that the N-terminal domain might also oligomerize at the high local concentration that exists in a fibrillar state (see below). An interesting mutant of HET-S (E86K) that was found in a genetic screen (Coustou et al., 1999Coustou V. Deleu C. Saupe S.J. Begueret J. Mutational analysis of the [Het-s] prion analog of Podospora anserina. A short N-terminal peptide allows prion propagation.Genetics. 1999; 153: 1629-1640PubMed Google Scholar) unexpectedly relates to the dimer. When expressed in P. anserina, HET-S[E86K] giv" @default.
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• W2015444119 title "The Mechanism of Prion Inhibition by HET-S" @default.
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