Cysteine variants behave like wild type NM
We created 37 individual cysteine-substitution mutations throughout NM (). Each was used to replace the wild-type (WT) SUP35 gene in vivo. All retained the capacity to support the [PSI+] and [psi−] states (Fig. S1a). None altered the stability of those states (data not shown).
Mapping the amyloid region of NM fibres.
Next, each protein was expressed in and purified from E.coli.
All spontaneously assembled into amyloid at the same rate as WT (Fig. S1b). Moreover, all fibres were indistinguishable from WT by electron microscopy and SDS (sodium dodecyl sulphate) solubility22
(data not shown). Finally, fibres made from each mutant seeded assembly as well as WT (Fig. S1c)17,20
. Having established that cysteine substitution proteins recapitulate prion behaviour in vivo
and in vitro
, we employed them to explore NM structure and assembly.
Boundaries of the cooperatively folded amyloid
We used two independent approaches to investigate the structure of NM fibres. First, in 37 sets of fibres assembled at 25°C from each variant, we probed the accessibility of cysteines to labelling with pyrene maleimide, and to a more hydrophilic reagent, Lucifer Yellow ( and data not shown). Proteins carrying cysteines between amino acid residues (aa) 25–58 were sparsely labelled. Proteins with cysteines between aa 2–21 and 68–112 showed partial accessibility. All cysteines in M were highly accessible.
Next, we used acrylodan to report on the conformational status of different segments of NM. Acrylodan exhibits an increase in fluorescence intensity and a blue shift in λmax when sequestered from solvent. When denatured, all cysteine-labelled proteins had a λmax around 530 nm. After assembly at 25°C, proteins labelled between aa 21–121 had strongly blue-shifted emissions (λmax486–488 nm) indicating sequestration from solvent (, zero guanidine hydrochloride, GdmCl, and data not shown). Proteins labelled in adjoining regions (residue 7 N-terminally and residues 137, 158 and 167 C-terminally) had partially blue-shifted emission maxima (λmax 493 to 525 nm). Proteins labelled at 2, 184, 225 and 234 had no significant blue shift, indicating that these residues remained exposed to solvent pre- and post-assembly ( and data not shown).
To determine which residues participate in the same cooperatively folded structure, we assessed their post-assembly GdmCl denaturation profiles, using 24 GdmCl concentrations for each of the fibres. All fibres labelled between aa 21–121 showed a similar drop in fluorescence intensity and a corresponding red shift in emission maxima with an inflection at 2.5M (± 0.15 M) GdmCl (). These profiles fitted a monophasic unfolding transition that corresponded to the major unfolding transition of WT NM protein (Cashikar and Lindquist, unpublished results). Adjacent cysteine variants with intermediate blue shifts on fibrillization also had distinct unfolding transitions in GdmCl.
These studies establish that: 1) a large portion of N is sequestered from solvent; 2) a sub-portion constitutes a distinct domain (formed by contiguous aa including 21–121) with an unusually stable structure and a single cooperative unfolding transition; 3) flanking sequences are structurally heterogeneous, with residues 137 and 158 having distinct, but cooperative, unfolding transitions and residues 2 and 7 biphasic transitions; and 4) beyond residue 158 M is flexible and solvent exposed. Residues in the N/M transition zone (aa 121, 137 and 158) were fully accessible to cysteine labelling but by acrylodan labelling and GdmCl denaturation were partially sequestered and structured. This likely reflects different sensitivities of the two techniques to structural instability. For example, if residue 121 adopts an open structure occasionally, it would exhibit a strong blue shift with acrylodan, but still be accessible to prolonged labelling.
Identifying intermolecular contacts
To determine which regions of NM make intermolecular contacts in fibres, we exploited the ability of pyrene-labelled proteins to form excimers (excited-state dimers). When two pyrenes lie within 4–10 Å of each other, the long fluorescence lifetime of pyrene allows an excited residue to interact with an unexcited residue before energy is emitted. This produces a strong red shift in fluorescence. Excimer fluorescence will occur between residues at β-strands that form the interface between two monomers () but not between residues that are distant from the interface (). (One caveat is that the pyrenes might alter the structure formed. Control experiments eliminated this concern. See Fig. S2c).
Intermolecular contacts in NM fibres.
In denaturant each of the pyrene-labelled proteins had multiple emission maxima between 384 and 405 nm (Fig. S2a and S2b). After assembly at 25°C, most proteins labelled in the N region exhibited a blue shift in fluorescence (Fig. S2b), indicating that they were sequestered from intermolecular contacts. Proteins labelled in two distinct regions (residues 25, 31, 38 and 91, 96, 106) produced strong red-shifted fluorescence (λmax ~465 nm; and Fig. S2a). These residues must lie at or near a contact between two NM molecules. We will refer to these intermolecular contact regions as the “Head” (aa 25–38) and “Tail” (aa 91–106). Residues between them, the “Central Core” (43–85), are also part of the cooperatively folded amyloid but are sequestered from intermolecular contacts.
Pyrene fluorescence patterns were virtually identical in repeat experiments (data not shown). They were also nearly identical in seeded and unseeded-rotated reactions (open and closed circles, ). The remarkable reproducibility of these excimer patterns establishes that the intermolecular contacts in seeded assembly quite precisely recapitulate those of spontaneous assembly.
Next, we mixed proteins labelled at two different cysteines in all pair-wise combinations ( and Fig. S3). Confirming that residues in the Central Core do not contribute to intermolecular contacts, all fibres in which either one or both of the proteins were labelled in this region produced low excimer signals (residues 51, 58 and 73; and Fig. S3). The strongest signals were again from the Head and Tail, but only when both proteins were labelled in the same region (, orange boxes). Fibres with one protein labelled in the Head and another in the Tail produced weak signals. We conclude that contacts between monomers in the fibre occur in a Head-to-Head and Tail-to-Tail fashion.
These data are not compatible with the parallel super-pleated sheet model for the Sup35 prion domain31
, in which individual NM molecules fold into long serpentine arrays, stacked in parallel along their entire length. They are compatible with a ß-helix model28
, and related ß-spiral and ß-sandwich models, wherein a contiguous stretch of amino acids forms the amyloid fold and a Central Core is sequestered from intermolecular contacts. Further, individual subunits must form contacts in a Head-to-Head and Tail-to-Tail fashion (see ).
An example of NM assembly that conforms to our data.
Constraints on inter-subunit relationships
To provide an independent assessment of inter-subunit interactions, we introduced two types of crosslinks into each of the individual cysteine-substituted proteins under denaturing conditions. Reaction with oxidized DTT produced disulfides with a bond length of ~2Å. Reaction with 1, 4-bis-maleimidobutane (BMB), a homobifunctional agent, produced crosslinks with a 10.9Å flexible linker.
Disulfide cross-links inhibited fibre formation at every position tested between aa 21–121 (, black bars; thioflavinT fluorescence levels equivalent to those of BSA aggregates). Disulfides in the extreme N terminus and in M had little effect on assembly. In contrast, with the flexible BMB linker, NM molecules cross-linked in the Head or Tail formed fibres very efficiently (, grey bars). BMB cross-links severely impeded fibre formation only in the Central Core.
The ~2Å bond length of a disulfide is closer than the inter-strand distances of ~4.7Å that characterize NM fibres20,28
. The distributions of residues for which disulfides inhibit amyloid formation support our earlier conclusion that a contiguous linear segment of aas, including aa 21–121, constitute a cooperatively folded unit. Improper intersubunit alignments of any two residues in this region prevent folding of the rest of the domain. The extreme N-terminus and the middle domain are outside this domain and have little influence on its capacity to fold.
With the longer linker, apposition of two NM proteins in the Head or Tail permits fibre formation. Apposition of Central Core residues prevents it. Thus, the separation of Central Core regions is not only a general characteristic of NM fibres but is essential for fibre formation.
Next we asked whether the structural information and the tools we had assembled could be employed to address two of the most enigmatic questions in prion biology. How is assembly nucleated? What is the structural basis of distinct prion strains?
Early events during nucleation and assembly
First, we first monitored kinetic changes in the fluorescence of proteins labelled with acrylodan. All tested proteins labelled in the cooperatively folded amyloid region (21–106) showed a very rapid increase in fluorescence, characteristic of a first-order reaction with no lag phase (). This fluorescence increase preceded conversion to amyloid: when amyloid formation was monitored by the acquisition of an SDS-insoluble state, each acrylodan-labelled protein had the same lag and assembly phase as WT protein (Fig. S4). In contrast, molecules labelled at residues 158 or 167 changed fluorescence simultaneously with amyloid formation ( and data not shown). Proteins labelled at residue 184, 203, and 225 showed no change in fluorescence ( and data not shown). We conclude that: 1) residues that form the cooperatively folded amyloid core rapidly enter a collapsed but non-amyloid state, 2) M residues proximal to N become structured only when N residues convert to amyloid, and 3) the distal region of M remains largely unstructured and exposed to solvent after amyloid assembly.
To determine which segments of the cooperatively folded amyloid region are the first to undergo conformational commitment, we took advantage of the fact that disulfide bonds anywhere in this region to prohibit assembly (). We reasoned that segments of NM that are the first to assume productive spatial relationships would also be the first to be protected from the spontaneous formation of disulfide bonds. Representative cysteine mutants were allowed to assemble in buffer without DTT, to facilitate the formation of disulfide bonds, and analysed on SDS gels without DTT. Cysteines in the Head (21, 25, and 31) formed fewer disulfides than cysteines at other positions (). Thus, during conformational conversion, strand spacings compatible with a productive fold (and incompatible with disulfide formation) are achieved in the Head region more rapidly than in other regions.
Next, we investigated the effects of adding a single charge at various positions in the amyloid region. In some ß-structures, such as the ß-helix, alternating residues point toward or away from solvent and the structures formed are very stable 32
. Although introduction of a single charge would be unlikely to perturb such structures once they form, charge repulsions in early nucleating segments of the molten, collapsed intermediate would reduce the frequency with which these segments come into proximity and, thereby, slow nucleation. Labelling individual NM cysteine residues with uncharged iodoacetamide had little effect on the quantity of protein converting to amyloid () or the kinetics of assembly (Fig. S5). In contrast iodoacetate labelling, which introduces a negatively charged moiety of similar size, inhibited assembly most strongly in the Head region ( and Fig. S5).
Finally, if Head-to-Head interactions are not only characteristic of early productive amyloid conformations, but actually cause a commitment to it, bringing Head regions in proximity with each other should promote nucleation. We compared assembly kinetics for several NM variants that were cross-linked with BMB under denaturing conditions and then transferred to assembly buffer. Crosslinks in the Central Core (aa 43,73) blocked assembly entirely, confirming that these regions must be separated from each other to form amyloid (). Cross-links in the Tail (96, 106) had little effect. Cross-links in the Head (aa 21,25,38) virtually eliminated the lag phase. Thus, the juxtaposition of residues in the Head region is an early event in amyloid formation and is, indeed, sufficient to nucleate it.
Structural distinctions between prion strain populations
To place the second critical question in prion biology – the basis of prion “strains” or variants – within the structural framework we have generated for NM fibres, we assembled the protein under conditions previously known to produce different strain populations (room temperature, RT, versus 4°C) 32
. We confirmed that fibres produced at 4°C, (1) assembled much more rapidly26
(data not shown), (2) were less stable to GdmCl denaturation (), and (3) produced mostly strong prion strains when used to transform cells from the [psi−
] non-prion state to the [PSI+
] prion state (, left) 32
Structural distinctions between prion strain populations
Do fibres enriched in different prion strains have distinct cooperatively folded amyloid domains? Denaturation profiles of fibres, independently assembled from 16 acrylodan-labelled proteins at either 4°C or 25°C, were determined as in . In 4°C fibres, residues 31–86 had strong blue shifts in fluorescence upon assembly and exhibited a single cooperative unfolding transition at D½ ~ 1.5 M GdmCl, ( and data not shown). Flanking residues 21, 25, 96, 112, and 121, had smaller blue shifts in fluorescence upon assembly and heterogeneous denaturation profiles after assembly, as had residues flanking the 25°C amyloid domain (2 and 7, 137 and 158; , , and data not shown). Thus, at both temperatures the cooperatively folded amyloid core has a similar character: it is formed by a contiguous stretch of amino acids and is flanked by residues that are structurally heterogeneous. However, the length of the region incorporated into the cooperative amyloid fold is much shorter in 4°C fibres than in 25°C fibres and, consequently, more easily denatured. Shorter less stable amyloid cores likely produce stronger more stably inherited prion strains because the fibres are more easily fragmented and transmitted to daughter cells.
Finally, we asked if intermolecular contacts differ in fibres assembled at 4°C and 25°C. The excimer fluorescence of residues in the Head region and in flanking residues (2, 7, and 16) changed modestly but in an extremely reproducible manner (seeded and unseeded reactions; ). Excimer fluorescence in the Tail shifted the most dramatically. Thus, as the number of residues that constitute the cooperatively folded amyloid domain change, the intersubunit interfaces change as well.
The Structural basis of prion strains
In fibres enriched for distinct prion strains we confirmed27
differences in the rate of assembly, and discovered differences in both the length of the amyloid core and intersubunit contacts. But are these features determinative? If so, proteins cross-linked in different places should strongly bias assembly reactions toward different strains.
Proteins cross-linked with BMB (as for ) were assembled at 25°C or 4°C and used to transform cells from the [psi−
] to the [PSI+
. Proteins cross-linked in the Central Core did not induce [PSI+
] above background levels (), consistent with their failure to undergo amyloid assembly (). Cross-links in the Head, which caused rapid assembly, biased fibres towards the production of strong strains. Conversely cross-links in the Tail, which should cause a longer segment to be incorporated into the amyloid fold, biased fibres towards weak strains (). Notably, the strain biases produced by different cross-link positions overcame those of different assembly temperatures. Whether the fibres used for transformation had been assembled at 4°C or at 25°C, proteins cross-linked in the Head produced primarily strong strains and proteins cross-linked in the Tail produced primarily weak strains (). Thus, strain distinctions are due to differences in the secondary and tertiary structures of individual NM molecules, as well as to the nature of NM::NM interactions.