The structural basis of yeast prion strain variants

Abstract

Among the many surprises to arise from studies of prion biology, perhaps the most unexpected is the strain phenomenon whereby a single protein can misfold into structurally distinct, infectious states that cause distinguishable phenotypes. Similarly, proteins can adopt a spectrum of conformations in non-infectious diseases of protein folding; some are toxic and others are well tolerated. However, our understanding of the structural differences underlying prion strains and how these differences alter their physiological impact remains limited. Here we use a combination of solution NMR, amide hydrogen/deuterium (H/D) exchange and mutagenesis to study the structural differences between two strain conformations, termed Sc4 and Sc37 (ref. 5), of the yeast Sup35 prion. We find that these two strains have an overlapping amyloid core spanning most of the Gln/Asn-rich first 40 amino acids that is highly protected from H/D exchange and very sensitive to mutation. These features indicate that the cores are composed of tightly packed beta-sheets possibly resembling 'steric zipper' structures revealed by X-ray crystallography of Sup35-derived peptides. The stable structure is greatly expanded in the Sc37 conformation to encompass the first 70 amino acids, revealing why this strain shows increased fibre stability and decreased ability to undergo chaperone-mediated replication. Our findings establish that prion strains involve large-scale conformational differences and provide a structural basis for understanding a broad range of functional studies, including how conformational changes alter the physiological impact of prion strains.

Distinct conformations of in vitro and in vivo amyloids of huntingtin-exon1 show different cytotoxicity

“…This apparent inconsistency of previous results regarding amyloid toxicity may result from the structural diversity of amyloid, as amyloid-forming protein often misfolds into multiple conformations and each conformation could exert distinct physiological effects (19,23). Previous reports indicate that the thermodynamic parameter of temperature can modulate protein folding and dynamics of amyloid-forming proteins, leading to different amyloid conformations (19,(32)(33)(34). Here we took advantages of this fact by making distinct thtt amyloids simply by polymerizing thtt protein at 4°C or 37°C.…”

Ultrasonication-dependent production and breakdown lead to minimum-sized amyloid fibrils

“…Once the nucleus is formed, the growth phase proceeds via the incorporation of the monomers into the ends of seed fibrils in a template-dependent manner (9). Under normal extension conditions without the fragmenting effect of ultrasonication, the energy landscape is broad, and might be modulated by additional mechanisms including breaking down and rejoining reactions especially for long fibrils (22)(23)(24)(25), resulting in a wide range of fibril lengths (black line in Fig. 1).…”

“…The position of the energy minimum and the steepness of the landscape of fragmentation would be determined by a balance between the elongation kinetics and fragmentation efficiency, the latter of which is presumably determined by the frequency of ultrasonication pulses. Additionally, the intrinsic physical properties such as fragility of individual fibrils (23,(25)(26)(27) and intensity of ultrasonication would affect the equilibrated fibril size.…”

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

“…In all cases, the [PSI ϩ ] phenotype was restored upon complementation. At the colony level, the diploid phenotype of wild-type and heterozygous mutants was indistinguishable, with both forming white colonies on rich media and in the absence of adenine ( Figure 3B and data not shown), but the effects of loss of even one copy of NAT1 could still be detected at the single-cell level using a more sensitive fluorescence-based read-through reporter, with the wild-type strains exhibiting a higher level of stop codon read-through than heterozygous mutants (Supplemental Figure S3) (Derkatch et al, 1996;King and Diaz-Avalos, 2004;Tanaka et al, 2004;Toyama et al, 2007), is crossed to a wild-type [psi Ϫ ] strain, the resulting diploid retains the weak phenotype, forming pink colonies on rich media that fail to grow in the absence of adenine ( Figure 3B). Thus, the reemergence of the [PSI ϩ ] phenotype upon complementation of the NatA defect indicates that self-replication of the parental Sup35 [PSI ϩ ] conformation persists but is somehow phenotypically masked in NatA-deficient strains.…”