Destabilization of polar interactions in the prion protein triggers misfolding and oligomerization

Abstract

The prion protein (PrP) misfolds and oligomerizes at pH 4 in the presence of physiological salt concentrations. Low pH and salt cause structural perturbations in the monomeric prion protein that lead to misfolding and oligomerization. However, the changes in stability within different regions of the PrP prior to oligomerization are poorly understood. In this study, we have characterized the local stability in PrP at high resolution using amide temperature coefficients (TC) measured by nuclear magnetic resonance (NMR) spectroscopy. The local stability of PrP was investigated under native as well as oligomerizing conditions. We have also studied the rapidly oligomerizing PrP variant (Q216R) and the protective PrP variant (A6). We report that at low pH, salt destabilizes PrP at several polar residues, and the hydrogen bonds in helices α2 and α3 are weakened. In addition, salt changes the curvature of the α3 helix, which likely disrupts α2–α3 contacts and leads to oligomerization. These results are corroborated by the TC values of rapidly oligomerizing Q216R‐PrP. The poly‐alanine substitution in A6‐PrP stabilizes α2, which prevents oligomerization. Altogether, these results highlight the importance of native polar interactions in determining the stability of PrP and reveal the structural disruptions in PrP that lead to misfolding and oligomerization.

A fine balance of hydrophobic-electrostatic communication pathways in a pH-switching protein

“…The view from MD simulations is strongly consistent and synergistic with experiments. Exploiting underexplored chemical shift differences and cycle analyses may provide high-resolution insights for many systems, including a wide variety of proteins and peptide assemblies ( 16 , 21 – 26 ), and serve as a valuable resource to complement, test, and refine molecular simulations.…”

“…However, as the many intra- and intermolecular contributions to chemical shift vary in complex ways with temperature, a quantitative structural interpretation of TCs has remained elusive. Recent studies in our group and others have shown how the complexities of amide temperature dependencies may be simplified by comparing related protein variants, yielding nuanced information on changes in structural stability and sampling of alternative states significant for natural function, disease, drug discovery, and materials ( 6 , 16 – 26 ).…”

Mutations of evolutionarily conserved aromatic residues suggest that misfolding of the mouse prion protein may commence in multiple ways

“…In the case of PUF1, the sequence segments covering β1 (96–144), the α1‐β2 loop, the β2‐α2 loop (168–173), the C‐terminal end of α2 (182–196), the second half of α3 (210–216), and the sequence segment 225–231 are found to have become disordered. An earlier NMR study of the temperature dependence of chemical shifts seen for wt moPrP (Bhate et al., 2021) had indicated that in the absence of added salt, many residues in β1 and the β1‐α1 loop (in sequence segment 96–144), as well as Gln211 in the second half of α3 (in sequence segment 210–216), which is adjacent in structure, sample at least one alternative conformation. Both these sequence segments are disordered in PUF1 suggesting that it is the alternative conformation that is sampled.…”

“…segment 225-231 are found to have become disordered. An earlier NMR study of the temperature dependence of chemical shifts seen for wt moPrP(Bhate et al, 2021) had indicated that in the absence of added salt, many residues in β1 and the β1-α1 loop (in sequence segment 96-144), as well as Gln211 in the second half of α3 (in sequence segment 210-216), which is adjacent in structure, sample at least one alternative conformation. Both these sequence segments are disordered in PUF1 suggesting that it is the alternative conformation that is sampled.It is not yet known how the addition of the salt (150 mM NaCl) affects the extents to which the PUFs are populated.…”

“…Both these sequence segments are disordered in PUF1 suggesting that it is the alternative conformation that is sampled.It is not yet known how the addition of the salt (150 mM NaCl) affects the extents to which the PUFs are populated. The NMR study of the temperature dependence of chemical shifts(Bhate et al, 2021), indicated that, upon the addition of the salt, the alternative conformation in which the residues in β1 and the β1-α1 loop as well as Gln211 in the second half of α3 are perturbed (see above), becomes populated to a greater extent. This could mean that PUF1 becomes populated to a greater extent.…”