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. 2022 Oct 20:16:960322.
doi: 10.3389/fnins.2022.960322. eCollection 2022.

Structural consequences of sequence variation in mammalian prion β2α2 loop segments

Calina Glynn  1 , Evelyn Hernandez  1 , Marcus Gallagher-Jones  1 , Jennifer Miao  1 , Christina J Sigurdson  2 , Jose A Rodriguez  1
Affiliations

Structural consequences of sequence variation in mammalian prion β2α2 loop segments

Calina Glynn et al. Front Neurosci. .

Abstract

Sequence variation in the β2α2 loop, residues 165-175 of the mammalian prion protein (PrP), influences its structure. To better understand the consequences of sequence variation in this region of the protein, we biochemically and biophysically interrogate natural and artificial sequence variants of the β2α2 loop of mammalian PrP. Using microcrystal electron diffraction (MicroED), we determine atomic resolution structures of segments encompassing residues 168-176 from the β2α2 loop of PrP with sequences corresponding to human, mouse/cow, bank vole/hamster, rabbit/pig/guinea pig, and naked mole rat (elk-T174S) β2α2 loops, as well as synthetic β2α2 loop sequences. This collection of structures presents two dominant amyloid packing polymorphisms. In the first polymorph, denoted "clasped", side chains within a sheet form polar clasps by facing each other on the same strand, exemplified by the mouse/cow, human, and bank vole/hamster sequences. Because its stability is derived from within a strand and through polar ladders within a sheet, the sequence requirements for the mating strand are less restrictive. A second polymorph, denoted "interdigitated," has sidechains interdigitate across mating sheets, exemplified by the elk, naked mole rat (elk T174S), and rabbit sequences. The two types of packing present distinct networks of stabilizing hydrogen bonds. The identity of residue 174 appears to strongly influence the packing adopted in these peptides, but consideration of the overall sequence of a given segment is needed to understand the stability of its assemblies. Incorporation of these β2α2 loop sequences into an 85 residue recombinant segment encoding wild-type bank vole PrP94-178 demonstrates that even single residue substitutions could impact fibril morphology as evaluated by negative stain electron microscopy. This is in line with recent findings supporting the accessibility of different structural geometries by varied mammalian prion sequences, and indicates that sequence-specific polymorphisms may be influenced by residues in the β2α2 loop.

Keywords: cryoEM; microED; prion; structure; transmission.

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Conflict of interest statement

JR is an equity stakeholder in Medstruc Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Analysis of sequence variability in polar residues of the β2α2 loop of the mammalian prion protein. The β2α2 loop of mammalian prions (left) takes on a loop structure in PrPC (center and top right) but misfolds into a beta sheet amyloid conformation (bottom right). Residues that vary between mammals (168, 170, and 174) are colored. Human PrPC structure with PDB ID 1QLX (Zahn et al., 2000) is shown.
FIGURE 2
FIGURE 2
Structures of crystalline mammalian prion β2α2 loop aggregates. A single pair of strands is shown for each 9-residue segment to illustrate the interface between protofilaments. Additionally, three published structures from peptides SNQNNF (Sawaya et al., 2007), NNQNTF (Wiltzius et al., 2009), and QYNNQNNFV (Gallagher-Jones et al., 2018) (starred) are included for reference. All structures except for the bottom row are class 2 (face-to-back) steric zippers that fall into packing category 1. The bottom row is composed of class 1 (face-to-face) steric zippers that fall into packing category 2.
FIGURE 3
FIGURE 3
β2α2 Loop Structures Fall Into Two Structural Categories. Packing arrangement for mouse, human, and bank vole (top) and mole rat, elk, and rabbit (bottom) β2α2 loops with black line highlighting the promiscuous (top) and selective (bottom) interfaces formed in each packing category. Insets show a conserved hydrogen bonding network across species between N171 and N173 (center column, blue boxes). For bank vole, mouse, and human loops (top) N172 and N174 form a clasp (center column, top blue box) while elk and naked mole rat structures (bottom) form a clasp between N170 and N172 (center column, center pink box), leaving Q168 free to form a polar ladder that can interdigitate with a complementary mated sheet. Residues that vary between species are colored in text and residue 174 is shown in color and underlined. Bound waters and ligands shown adjacent to their nearest peptide chain.
FIGURE 4
FIGURE 4
Hydrogen bonding in clasped structures of segments from the β2α2 Loop. Structures from the amyloid state of bank vole (top) mouse (center) and human (bottom) β2α2 loops show a conserved interface and sets of hydrogen bond networks (right). Species-specific differences in the hydrogen bonding networks between residues 168 and 170 give rise to differing stabilities for the three crystalline aggregates (left).
FIGURE 5
FIGURE 5
Comparison of naked mole rat and rabbit loop structures. (A) Naked mole rat (yellow) and rabbit (pink) loop structures share a common interface seen in packing category 2. (B) Interface 1 is composed of inward facing aromatic residues and a polar clasp between N171 and N173 on the same strand for all structures regardless of packing category with the exception of the rabbit loop structure (right). (C) A polar clasp is formed in both non-rabbit (left) and rabbit (right) structures, however, in the rabbit structure the clasp is formed by N171 and N173 on mated sheets rather than via interactions within a single sheet.
FIGURE 6
FIGURE 6
Denaturation of β2α2 loop crystals. (A) Wild type peptide crystals were subjected to 0.75M buffers at the specified pH (left column), increasing concentrations of guanidine HCl (center column), and increasing concentrations of urea (right column). (B) Human and (C) bank vole variants, including the wild type crystals shown in (A), plotted on the same axis as their wild type counterparts for direct comparison. All experiments were performed in triplicate; error bars correspond to standard deviation of the measurements.
FIGURE 7
FIGURE 7
Fiber Diffraction of β2α2 loop aggregates. Fiber diffraction patterns were collected for crystalline (bank vole, mouse, human, rabbit, and naked mole rat) and fibrillar (chimp, sheep R168 sequence polymorph, and elk) aggregates. All aggregates share ∼4.8 and ∼10Å reflections indicative of underlying amyloid structure.
FIGURE 8
FIGURE 8
Influence of the β2α2 loop on fibril morphology. Crystal structures for bank vole, mouse/cow, human, naked mole rat, and elk β2α2 loops are shown along with fibrils formed by rBvPrP94– 178 harboring the loop substitutions in residues 168-176 belonging to each species. Scalebars = 200nm.
FIGURE 9
FIGURE 9
Conformation of β2α2 loop segment in PrPSc fibril structures. (A) Sequence of residues associated with 263K, aRML, and ME7 prion fibrils with numbering according to hamster PrP. (B) Schematics of the recently reported structures of PrPSc determined by single particle cryo electron microscopy (cryoEM) (Hoyt et al., 2021; Kraus et al., 2021; Manka et al., 2021, 2022). Residues of interest are numbered, and hydrogen bonds between the β2α2 loop and N- and C-terminal cores are shown in as dashed lines.
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