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. 2010 Jun 25;38(6):889-99.
doi: 10.1016/j.molcel.2010.05.019.

The mechanism of prion inhibition by HET-S

Jason Greenwald  1 , Carolin Buhtz, Christiane Ritter, Witek Kwiatkowski, Senyon Choe, Marie-Lise Maddelein, Frederique Ness, Sandra Cescau, Alice Soragni, Dominik Leitz, Sven J Saupe, Roland Riek
Affiliations

The mechanism of prion inhibition by HET-S

Jason Greenwald et al. Mol Cell. .

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 beta-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 beta-structuring in this domain induces a change in its globular domain, generating a molecular species that is incompetent for fibril growth.

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Figures

Figure 1
Figure 1. HET-S Does Not Form Fibrils and Can Inhibit Aggregation of HET-s PFD
(A) Electron micrographs of negatively stained aggregates of HET-s (left) and HET-S (right), both at the same magnification (scale bar in the lower right of image is 500 nm). Proteins at 15 μM were incubated 24 hr at 37°C with light agitation in 100 mM Tris-HCl (pH 8), 150 mM NaCl. (B) Coaggregation kinetics of HET-s PFD in the presence of HET-S. The 15N-filtered 1D NMR signal of the 15N-labeled PFD was integrated between 6.5 and 9.0 ppm for each time point, the data normalized to the first time point, and represented as the fraction soluble. All measurements included 52 μM PFD and the plot symbols are as follows: open circles, control PFD alone; gray diamonds, 75 nM HET-S; gray squares, 750 nM HET-S; gray triangles, 7.5 μM HET-S; black diamonds, 750 nM HET-S[E86K]. The arrows represent the 95% confidence intervals for the 50% aggregation time for the measurements of 0, 75, and 750 nM HET-S (three or four measurements each with the closest to the average shown in plot). The 7.5 μM HET-S sample retained more than 70% of the PFD in solution after 65 hr, and complete aggregation occurred sometime between 65 and 110 hr.
Figure 2
Figure 2. The Structure of the HET-S N-Terminal Domain
(A) Ribbon diagram of HET-S(1–227). The ribbon color is a rainbow gradient from N (red) to C terminus (purple). Regions of the molecule are highlighted as follows: flexible loop L5–6 in black, residues A23 and H33 as black spheres, location of point mutants that can convert HET-S to [HET-s] phenotype as red spheres, and residue E86 as yellow sphere. The helices are numbered 1–9 and β1–β2 for the strands. (B) Perpendicular views of the crystallographically observed HET-S(1–227) dimer with consensus residues from the β-aggregation prediction algorithms (see the Experimental Procedures) as black tubes.
Figure 3
Figure 3. The HET-S Solution Dimer Shares the Same Interface as the Crystallographic Dimer
(A) Gel filtration and MALS profiles reveal the presence of a dimer of HET-S in solution. The proteins were injected onto the sizing column at the three concentrations indicated, and the eluted proteins were simultaneously analyzed for concentration (solid lines) and molecular weight (dotted lines). The vertical dashed lines indicate the elution volume. The concentration-dependent peak broadening, elution time, and calculated mass of HET-S compared to HET-S[E86K] all indicate that the former self-associates in solution. (B) The weighted chemical shift differences (ΔδH,N) between the highest and lowest concentrations (see Figure S6) were mapped to the 3D structure of HET-S(1–227) as a gradient of white (ΔδH,N < 0.02 ppm) to red (ΔδH,N > 0.08 ppm) with unassigned residues in dark gray. The circle inlay highlights the charged residues at the interface near E86. (C) Per residue plot of the concentration-dependent ΔδH,N (2.5 mM–25 μM) for HET-S[E86K](1–227) and (500 μM–25 μM) for HET-S(1–227) and the isoconcentration ΔδH,N for HET-S(1–227) wild-type versus the mutant (500 μM). The isoconcentration ΔδH,N is plotted on a separate y scale (~40%) to make a better comparison of the per residue shift pattern. The missing assignments appear as gaps in the plot. The dashed line represents the distance of each Cα to the interface with shortest distances nearest the top, showing the inverse correlation between distance from interface and ΔδH,N.
Figure 4
Figure 4. HET-s Protein Constructs Are More Stable than HET-S
Overlay of the CD thermal denaturation profiles (gray lines) and their fits (black lines) for the N-terminal domain constructs. The arrows indicate the effects of the mutations or changes in the domain boundaries: (1) HET-s→HET-s [D23A,P33H]; (2) HET-s[D23A,P33H] (1–227)→(13–221); (3) HET-S→HET-S [E86K]. From low to high thermal denaturation temperature, the traces correspond to HET-s[D23A,P33H](13–221), HET-S(1–227), HET-S[E86K](1–227), HET-s[D23A,P33H](1–227), and HET-s(1–227).
Figure 5
Figure 5. HET-S Can Associate with HET-s Aggregates In Vivo
A P. anserina strain coexpressing HET-S-GFP and HET-s-RFP was analyzed by fluorescence microscopy. The hyphae of the transformants displayed various degrees of growth alteration and in the three fields shown, strong vacuolisation and formation of lipid droplets, the hallmarks of incompatibility. Where HET-s-RFP aggregates are visible, HET-S-GFP signal is also detected. Scale bar is 4 μm.
Figure 6
Figure 6. Proposed Model for HET-S Fibril Inhibition and Induction of Toxicity
(1) The HET-s fibril (formed from alternating light/dark gray HET-s monomers) is approached by a soluble HET-S (rainbow HeLo domain with random coil PFD in black). (2) Upon addition of HET-S to the HET-s fibril, the terminal helices of its HeLo domain unwind to accommodate the prion fold. (3) The second addition of a HET-S monomer to the fibril leads to a dimerization of the HeLo domains which facilitates a re/unfolding of the HeLo domains with the potential for further aggregation of HeLo domains that (in 4) blocks further growth of the fibril and activates a toxic form of HET-S. The model for the HET-s fibril was created from the PFD fibril structure (Wasmer et al., 2008) and the HeLo domain with an unwinding the last three helices of the HeLo domain (residues 177–222) to make space for the HeLo domains around the fibril. The HET-s HeLo domains are depicted as dimers between adjacent monomers in the fibril, but these are speculative and it should be emphasized that the structures of the HeLo domains of HET-s and HET-S, in the context of a fibril, are not known except that HET-s suffers a loss of tertiary structure (more molten globule-like), with a local loss of secondary structure around residues 190–220 (Wasmer et al., 2009).
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