Influence of pH on NMR Structure and Stability of the Human Prion Protein Globular Domain

Abstract

The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121-230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three ␣-helices, comprising residues 144 -156, 174 -194, and 200 -228, and a short anti-parallel ␤-sheet, comprising residues 128 -131 and 161-164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix ␣1 is elongated at the C-terminal end with residues 153-156 forming a 3 10 -helix, and the population of helical structure in the C-terminal two turns of helix ␣2 is increased. The protonation of His 155 and His 187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121-230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154 -157 at the Cterminal end of helix ␣1 and residues 161-164 of ␤-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a "starting point" for pH-induced unfolding and thus may be implicated in endosomic PrP C to PrP Sc conformational transition resulting in transmissible spongiform encephalopaties.The prion protein (PrP), 1 a predominantly synaptic protein present in all higher organisms (1-4), constitutes a major component of the infectious agent (prion) that causes transmissible spongiform encephalopathies (5, 6). The normal cellular isoform of the protein, PrP C , is soluble and protease-sensitive, whereas the disease-associated ␤-sheet-rich form, PrP Sc , is insoluble, partially resistant to protease digestion (7,8), and thought to propagate by converting PrP C molecules into an alternative conformation (9 -11). Recently, it has been shown that the accumulation of even small quantities of misfolded PrP in the cytosol is strongly neurotoxic in cultured cells and transgenic mice (12, 13). However, the subcellular localization of the conformational transition of PrP C into PrP Sc is controversial (14). There are indications that it takes place either at the cell surface, where the average interstitial milieu of the brain (15, 16) has a pH of 7.3, or after internalization of PrP Sc into endosomes (17)(18)(19), where pH values range between 4.7 and 5.8 (20).The in vitro conversion of human brain PrP C to a PrP Sc -like form is enhanced at acidic pH (21). Biophysical studies have shown that the free energy of unfolding of hPrP-(90 -231) is lower at acid pH than at neutral pH (22) and that in acidic guanidinium chloride hPrP-(90 -231) forms a folding intermediate that contains a large amount of ␤-sheet secondary structure. A ␤-sheet-rich folding intermediate has also been observed for mouse PrP-(121-231) at low pH in urea but is not seen at neutral pH (23). NMR structures are available for several recombinantly expressed mammalian prion prot...

Molecular Dynamics as an Approach to Study Prion Protein Misfolding and the Effect of Pathogenic Mutations

“…The first α-helix, HA, is the shortest. It spans residues 144-156, with the last 3 residues forming a 3 10 helix at neutral pH and a less regular structure at pH 4.5 [39,76]. The second and third α-helices, HB (res.…”

“…The C-terminal end of HB (res. 187-194) is significantly less stable than the rest; NMR studies reveal that it can exist in a disordered conformation [39] and hydrogen-exchange protection of the backbone amides is low [39, 76,79]. The many threonine residues in this sequence (HTVTTTTK, conserved in mammalian PrPs) cause this part of HB to have an inherently low helical propensity [80].…”

“…Human PrP extracted from brain cells forms detergent-insoluble aggregates at pH 3.5 and 1.5 M guanidinium hydrochloride [88]. Initial changes are accompanied by a decrease in thermodynamic stability of recPrP relative to neutral pH, as evidenced by continuous-flow fluorescence [89] and NMR [76]. Further, studies of human recPrP under acidic and mildly denaturing conditions suggest the presence of intermediate states during misfolding and oligomerization: a native like α-helical conformation was observed at pH 4.1 and a conformation with β-sheet characteristics at pH 3.6 [90].…”

“…The local protein environment can, however, change the pK a of individual residues significantly. Langella et al [99] calculated theoretical pK a values of the relevant sidechains based on the coordinates deposited for human recPrP obtained by protein NMR at pH 4.5 and pH 7.0 [39,76]. Although differences in the local conformation of residues between the various structures lead to a range of pK a values, their results suggest that two of the four histidine residues in the globular domain (His140 and His177) will be protonated at very mildly acidic pH (pH < 6.5).…”

Instability of familial spongiform encephalopathy-related prion mutants

“…were involved in the "starting point" of pH-induced unfolding and implicated in endosomic PrP C to PrP Sc conformational transition resulting in TSEs [15]. Therefore, elucidation of how the pH-induced local mobility change correlated with the mechanism of the extension of the S2 region should provide important clues regarding the molecular basis of prion diseases.…”