Prion and nonprion forms of proteins are believed to differ solely in their three-dimensional structure, which is therefore of paramount importance for the prion function. However, no atomic-resolution structure of the fibrillar state that is likely infectious has been reported to date. We present a structural model based on solid-state nuclear magnetic resonance restraints for amyloid fibrils from the prion-forming domain (residues 218 to 289) of the HET-s protein from the filamentous fungus Podospora anserina. On the basis of 134 intra- and intermolecular experimental distance restraints, we find that HET-s(218-289) forms a left-handed beta solenoid, with each molecule forming two helical windings, a compact hydrophobic core, at least 23 hydrogen bonds, three salt bridges, and two asparagine ladders. The structure is likely to have broad implications for understanding the infectious amyloid state.

SUMMARY RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

Prions are believed to be infectious self-propagating polymers of otherwise soluble host-encoded proteins 1,2 . This concept is now strongly supported by the recent findings that amyloid fibrils of recombinant prion proteins from yeast 3-5 , Podospora anserina 6 , and mammals 7 can induce prion phenotypes in the corresponding hosts. However, the structural basis of prion infectivity remains largely elusive because acquisition of atomic resolution structural properties of amyloid fibrils represents a largely unsolved technical challenge. HET-s, the prion protein of P. anserina, contains a C-terminal prion domain comprising residues 218-289. Amyloid fibrils of are necessary and sufficient for the induction and propagation of prion infectivity 6 . Here, we have used fluorescence studies, quenched hydrogen exchange NMR and solid state NMR to determine the sequence specific positions of secondary structure elements of amyloid fibrils of . This revealed four β-strands constituted by two pseudo repeat sequences, each forming a β-strandturn-β-strand motif. We show that this conformation is the functional and infectious entity of the HET-s prion by using a structure-based mutagenesis approach. These results correlate for the first time distinct structural elements with prion infectivity.The prion form of the protein HET-s is involved in a programmed cell death phenomenon termed heterokaryon incompatibility 8,9 . This reaction occurs in filamentous fungi when cells of incompatible genotype fuse and form a mixed cell. In P. anserina, two incompatible genotypes, called het-s and het-S, encode for the proteins HET-s and HET-S. They are both 289 amino acids long and differ in only 13 residues 10 . However, only HET-s can form a prion 11 : P. anserina cells expressing the HET-s protein exist either in a prion state called [Hets] Correspondence and requests for materials should be addressed to R.R. (e-mail: riek@salk.edu).. $ these authors contributed equally to this work.Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). (Fig. 1a) contained one assigned cross-peak for each backbone amide of with the exception of 289, enabling a residuespecific determination of the hydrogen exchange rates. Upon exchange in D 2 O buffer for 6 weeks the intensity of about 45% of the resonances was significantly reduced or absent from the spectrum (Fig. 1b). This suggested that the corresponding amides have undergone exchange with solvent deuterons, which are not visible in this experiment. NIH Public AccessThe hydrogen exchange was followed over a total period of 3 months. All residues displayed a mono-exponential decay (Fig. S1) indicating that the structure of the fibrils was well defined and homogeneous. The summarized hydrogen exchange data (Fig. 1e) show that due to exchange rates faster than 5·h -1 , the 8 N-terminal residues, the 5 C-terminal residues and residues 247-261 are only weakly or not protected and may therefore be conformationally disordered. Four segments were observed that d...

The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic β sheets. Two such β sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.

We report the observation of undetected (until now) residues of the prion protein fragment HET-s(218-289) which give rise to well-resolved (13)C, (15)N, and (1)H NMR resonances under high-resolution magic-angle spinning (HRMAS) conditions. The observed signals belong to large polymeric units as shown by measuring the lateral diffusion constants. The amino acids identified in the spectra are compatible with their localization in the segments of the protein which could not be detected in earlier solid-state NMR experiments. The observed chemical shifts indicate that these residues are in a random-coil conformation. Complementary experiments which detect only dynamic or static residues, respectively, strongly suggest that they belong to different parts of the same molecule.