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Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.

The structures of prion protein (PrP)-based mammalian prions have long been elusive. However, cryo-EM has begun to reveal the near-atomic resolution structures of fully infectious ex vivo mammalian prion fibrils as well as relatively innocuous synthetic PrP amyloids. Comparisons of these various types of PrP fibrils are now providing initial clues to structural features that correlate with pathogenicity. As first indicated by electron paramagnetic resonance and solid-state NMR studies of synthetic amyloids, all sufficiently resolved PrP fibrils of any sort (n 10) have parallel in-register intermolecular β-stack architectures. Cryo-EM has shown that infectious brain-derived prion fibrils of the rodent-adapted 263K and RML scrapie strains have much larger ordered cores than the synthetic fibrils. These bona fide prion strains share major structural motifs, but the conformational details and the overall shape of the fibril cross sections differ markedly. Such motif variations, as well as differences in sequence within the ordered polypeptide cores, likely contribute to strain-dependent templating. When present, N-linked glycans and glycophosphatidylinositol (GPI) anchors project outward from the fibril surface. For the mouse RML strain, these posttranslational modifications have little effect on the core structure. In the GPI-anchored prion structures, a linear array of GPI anchors along the twisting fibril axis appears likely to bind membranes in vivo, and as such, may account for pathognomonic membrane distortions seen in prion diseases. In this review, we focus on these infectious prion structures and their implications regarding prion replication mechanisms, strains, transmission barriers, and molecular pathogenesis.

Little is known about the structural basis of prion strains. Here we provide a high (3.0 Å) resolution cryo-electron microscopy-based structure of brain-derived fibrils of the mouse anchorless RML scrapie strain which, like the recently determined hamster 263K strain, has a parallel in-register β-sheet-based core. However, detailed comparisons reveal that variations in shared structural motifs provide a basis for prion strain determination.One-sentence summaryCryo-electron microscopy reveals a near-atomic structure of an infectious, brain-derived murine prion fibril and strain differences.

Classical mammalian prions are assemblies of prion protein molecules that are extraordinarily transmissible, with a microgram of protein containing up to 108 lethal doses of infectivity1,2. Unlike most other pathologic and amyloidogenic proteins, prions typically contain glycolipid anchors 3 and abundant asparagine‐linked glycans4‐6. The infectious nature, complexity, and biophysical properties of prions have complicated structural analyses and stymied any prior elucidation of 3D conformation at the polypeptide backbone level7. Here we have determined the structure of the core of a fully infectious, brain‐derived prion by cryo‐electron microscopy with ∼3.1 Å resolution. The purified prions are amyloid fibrils comprised of monomers assembled with parallel in‐register intermolecular beta sheets and connecting chains. Residues ∼95‐227 of each monomer provide one rung of the ordered fibril core, with the glycans and glycolipid anchor projecting from the lateral surfaces of the fibril. The fibril ends, where prion growth occurs, are formed by single monomers in an extended serpentine combination of β‐ arches, a Greek key, and loops that presumably template the refolding of incoming monomers. Our results describe an atomic model to underpin detailed molecular hypotheses of how pathologic prion proteins can propagate as infectious agents, and how such propagation and associated pathogenesis might be impeded.

Although prion protein fibrils can have either parallel-in-register intermolecular β-sheet (PIRIBS) or, probably, β-solenoid architectures, the plausibility of PIRIBS architectures for the usually glycosylated natural prion strains has been questioned based the expectation that such glycans would not fit if stacked in-register on each monomer within a fibril. To directly assess this issue, we have added N-linked glycans to a recently reported cryo-electron microscopy-based human prion protein amyloid model with a PIRIBS architecture and performed in silico molecular dynamics studies to determine if the glycans can fit. Our results show that triantennary glycans can be sterically accommodated in-register on both N-linked glycosylation sites of each monomer. Additional simulations with an artificially mutated β-solenoid model confirmed that glycans can be accommodated when aligned with ∼4.8 A spacing on every rung of a fibril. Altogether, we conclude that steric intermolecular clashes between glycans do not, in themselves, preclude PIRIBS architectures for prions.

Within the extensive range of self-propagating pathologic protein aggregates of mammals, prions are the most clearly infectious (e.g., ∼109 lethal doses per milligram). The structures of such lethal assemblies of PrP molecules have been poorly understood. Here we report a near-atomic core structure of a brain-derived, fully infectious prion (263K strain). Cryo-electron microscopy showed amyloid fibrils assembled with parallel in-register intermolecular β sheets. Each monomer provides one rung of the ordered fibril core, with N-linked glycans and glycolipid anchors projecting outward. Thus, single monomers form the templating surface for incoming monomers at fibril ends, where prion growth occurs. Comparison to another prion strain (aRML) revealed major differences in fibril morphology but, like 263K, an asymmetric fibril cross-section without paired protofilaments. These findings provide structural insights into prion propagation, strains, species barriers, and membrane pathogenesis. This structure also helps frame considerations of factors influencing the relative transmissibility of other pathologic amyloids.