Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils

Abstract

A C-terminally truncated Y145Stop variant of the human prion protein (huPrP23-144) is associated with a hereditary amyloid disease known as PrP cerebral amyloid angiopathy. Previous studies have shown that recombinant huPrP23-144 can be efficiently converted in vitro to the fibrillar amyloid state, and that residues 138 and 139 play a critical role in the amyloidogenic properties of this protein. Here, we have used magic-angle spinning solid-state NMR spectroscopy to provide high-resolution insight into the protein backbone conformation and dynamics in fibrils formed by 13 C, 15 N-labeled huPrP23-144. Surprisingly, we find that signals from Ϸ100 residues (i.e., Ϸ80% of the sequence) are not detected above approximately ؊20°C in conventional solid-state NMR spectra. Sequential resonance assignments revealed that signals, which are observed, arise exclusively from residues in the region 112-141. These resonances are remarkably narrow, exhibiting average 13 C and 15 N linewidths of Ϸ0.6 and 1 ppm, respectively. Altogether, the present findings indicate the existence of a compact, highly ordered core of huPrP23-144 amyloid encompassing residues 112-141. Analysis of 13 C secondary chemical shifts identified likely ␤-strand segments within this core region, including ␤-strand 130 -139 containing critical residues 138 and 139. In contrast to this relatively rigid, ␤-sheet-rich amyloid core, the remaining residues in huPrP23-144 amyloid fibrils under physiologically relevant conditions are largely unordered, displaying significant conformational dynamics.protein structure ͉ solid-state NMR ͉ protein misfolding ͉ transmissible spongiform encephalopathies

Determination of amyloid core structure using chemical shifts

“…24,25 Instead, a variety of studies have investigated the structure of amyloid fibrils produced in vitro from recombinant PrP. [26][27][28][29][30][31][32][33][34][35] Hereditary prion diseases include C-terminally truncated variants of the PrP, Y145X, Q160X, Y226X, and Q227X. Previously, we showed that the b-sheet content is highly similar in amyloid fibrils of the Y145X and Q160X stop mutants of human PrP.…”

“…36 In addition, solid-state NMR spectroscopy demonstrated that residues 112-140 assume extended b-sheet conformation in a parallel, in register alignment in amyloid fibrils of the Y145X stop mutant. [29][30][31] We subjected the solid-state NMR chemical shifts reported by Helmus et al 29 for amyloid fibrils of the Y145X prion stop mutant to the adapted CSRosetta protocol. 17 Calculations were performed on five identical segments that were connected by 16-residue glycine-serine linkers.…”

Mutant Protein A30P α-Synuclein Adopts Wild-type Fibril Structure, Despite Slower Fibrillation Kinetics

“…Amyloid fibrils contain a rigid core, where dipolar-based experiments transfer polarization with high effi- ciency, and mobile regions, where polarization transfer is inefficient in dipolar-based experiments (34,54). Thus, the signal intensities report qualitatively on rigidity.…”

Progress towards structural understanding of infectious sheep PrP-amyloid

“…12 A lack of b-strands N-terminal of position 145 in full-length recPrP-fibrils as demonstrated by H/D exchange, 13 however, indicates that data on truncated PrP-constructs or PrP-fragments are difficult to be extrapolated to infer the full-length PrP-structure. For full-length recPrP(23-231)-fibrils, solid-state NMR studies on selectively 13 CO-labeled samples confirmed an in-register parallel arrangement of b-strands.…”