Assessing the acid-base and conformational properties of histidine residues in human prion protein (125-228) by means of pK(a) calculations and molecular dynamics simulations

A thorough study of the acid-base behavior of the four histidines and the other titratable residues of the structured domain of human prion protein (125-228) is presented. By using multi-tautomer electrostatic calculations, average titration curves have been built for all titratable residues, using the whole bundles of NMR structures determined at pH 4.5 and 7.0. According to our results, (1) only histidine residues are likely to be involved in the first steps of the pH-driven conformational transition of prion protein; (2) the pK(a)'s of His140 and His177 are approximately 7.0, whereas those of His155 and His187 are < 5.5. 10-ns long molecular dynamics simulations have been performed on five different models, corresponding to the most significant combinations of histidine protonation states. A critical comparison between the available NMR structures and our computational results (1) confirms that His155 and His187 are the residues whose protonation is involved in the conformational rearrangement of huPrP in mildly acidic condition, and (2) shows how their protonation leads to the destructuration of the C-terminal part of HB and to the loss of the last turn of HA that represent the crucial microscopic steps of the rearrangement.     

Membrane environment alters the conformational structure of the recombinant human prion protein

The prion protein (PrP) in a living cell is associated with cellular membranes. However, all previous biophysical studies with the recombinant prion protein have been performed in an aqueous solution. To determine the effect of a membrane environment on the conformational structure of PrP, we studied the interaction of the recombinant human prion protein with model lipid membranes. The protein was found to bind to acidic lipid-containing membrane vesicles. This interaction is pH-dependent and becomes particularly strong under acidic conditions. Spectroscopic data show that membrane binding of PrP results in a significant ordering of the N-terminal part of the molecule. The folded C-terminal domain, on the other hand, becomes destabilized upon binding to the membrane surface, especially at low pH. Overall, these results show that the conformational structure and stability of the recombinant human PrP in a membrane environment are substantially different from those of the free protein in solution. These observations have important implications for understanding the mechanism of the conversion between the normal (PrP(C)) and pathogenic (PrP(Sc)) forms of prion protein.     

Molecular dynamics simulation of dimeric and monomeric forms of human prion protein: insight into dynamics and properties

A central theme in prion protein research is the detection of the process that underlies the conformational transition from the normal cellular prion form (PrP(C)) to its pathogenic isoform (PrP(Sc)). Although the three-dimensional structures of monomeric and dimeric human prion protein (HuPrP) have been revealed by NMR spectroscopy and x-ray crystallography, the process underlying the conformational change from PrP(C) to PrP(Sc) and the dynamics and functions of PrP(C) remain unknown. The dimeric form is thought to play an important role in the conformational transition. In this study, we performed molecular dynamics (MD) simulations on monomeric and dimeric HuPrP at 300 K and 500 K for 10 ns to investigate the differences in the properties of the monomer and the dimer from the perspective of dynamic and structural behaviors. Simulations were also undertaken with Asp178Asn and acidic pH, which is known as a disease-associated factor. Our results indicate that the dynamics of the dimer and monomer were similar (e.g., denaturation of helices and elongation of the beta-sheet). However, additional secondary structure elements formed in the dimer might result in showing the differences in dynamics and properties between the monomer and dimer (e.g., the greater retention of dimeric than monomeric tertiary structure).     

Exposure of hydrophobic core in human prion protein pathogenic mutant H187R

Pathogenesis studies have revealed that H187R mutation of human prion protein (huPrP) is related to GSS type of TSE diseases. Its pathogenic mechanism is still unclear. We here studied the globular domain of this mutant protein by molecular dynamics simulations. Compared to the wide-type protein, the mutant has similar dynamics and stability profiles in our simulation. Conformational rearrangements are concentrated around the mutation site, due to the introduction the positively charged side chain of Arg187. The strong electrostatic repulsion between Arg156 and Arg187 drives both side chains away from their original positions, leaving its hydrophobic core to be solvent accessible. Such a unfavorable conformational change may destabilize the mutant protein and make it more susceptible to unfolding.     

Prion protein-related proteins from zebrafish are complex glycosylated and contain a glycosylphosphatidylinositol anchor

A hallmark of prion diseases in mammals is a conformational transition of the cellular prion protein (PrP(C)) into a pathogenic isoform termed PrP(Sc). PrP(C) is highly conserved in mammals, moreover, genes of PrP-related proteins have been recently identified in fish. While there is only little sequence homology to mammalian PrP, PrP-related fish proteins were predicted to be modified with N-linked glycans and a C-terminal glycosylphosphatidylinositol (GPI) anchor. We biochemically characterized two PrP-related proteins from zebrafish in cultured cells and show that both zePrP1 and zeSho2 are imported into the endoplasmic reticulum and are post-translationally modified with complex glycans and a C-terminal GPI anchor.     

Sheep prion protein synthetic peptide spanning helix 1 and beta-strand 2 (residues 142-166) shows beta-hairpin structure in solution

According to the "protein only" hypothesis, a conformational conversion of the non-pathogenic "cellular" prion isoform into a pathogenic "scrapie" isoform is the fundamental event in the onset of prion diseases. During this pathogenic conversion, helix H1 and two adjacent surface loops L2 and L3 of the normal prion protein are thought to undergo a conformational transition into an extended beta-like structure, which is prompted by interactions with the pre-existing beta-sheet. To get more insight into the interaction between the helix and one of the beta-strands in the partially unfolded prion protein, the solution structure of a synthetic linear peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. We found that, in contrast to many prion fragments studied earlier, this peptide (i) is highly soluble and does not aggregate up to a millimolar concentration range in aqueous medium and (ii) exhibits an intrinsic propensity to a beta-hairpin like conformation at neutral pH. This beta-propensity can be one of the internal driving forces of the molecular rearrangement responsible for the pathogenic conversion of the prion protein.     

Bistability and the species barrier in prion diseases: stepping across the threshold or not

The infectious agent of transmissible spongiform encephalopathies is thought to be a cellular protein, the prion protein, which undergoes, under some circumstances, a dramatic conformational change leading to pathogenesis. The conversion between the normal and pathogenic isoforms corresponds to a autocatalytic mechanism and the metabolism of the prion protein exhibits switches between a normal, stable steady state and a pathogenic one. When the disease can be transmitted between two species, a primary infection from a heterologous donor has to be followed by two passages in the same host species so that the incubation period is stabilized. Sometimes, no pathogenic isoform of the prion protein is detected after the first passage, although corresponding brain extracts remain infectious. The observation that three and only three passages are needed in order to stabilize the strain strongly suggests that, during the course of the primary infection by the heterologous donor, an intermediary conformational species is formed. Within this assumption, a common mechanism involving only conformational changes of the prion protein can give a unifying interpretation of the problem of species barrier, lag characteristics and apparent lack of detection of the pathogenic isoform after the first passage in experiments dealing with interspecies transmission of prion diseases.     

Sequence-dependent dynamical instability of the human prion protein: a comparative simulation study

The present study aimed to explore the most probable regions of the human prion protein backbone for which the initial steps of conformational transitions as a result of intrinsic and extrinsic perturbing factors on the protein structure can be assigned. A total of 0.3-μs molecular dynamics simulations on several analog structures of the protein have been performed. To mimic the impact of the extrinsic and intrinsic destructive parameters on the dynamical characteristics of the protein, mild acidic conditions and R208H mutation have been simulated. The findings indicated that distribution of conformational flexibilities along the protein chain was almost independent of the induced perturbing factors, and was mostly centralized on certain distinct parts of the structure comprising residues 132–145 and 187–203. Analyses also revealed that the segment comprising residues 187–203 may be considered as a peptide sequence, possessing high potential to start the initial steps of conformational rearrangements due to the induced physicochemical alterations. Sequence alignment and molecular dynamics data also revealed that segment 178–203 prefers to accommodate in extended structures rather than α-helices. Region 178–203 may be considered as a peptide switch capable of initiating the conformational transitions due to the introduced modifications and perturbing parameters.     

The Unfolded State of the Murine Prion Protein and Properties of Single-point Mutants Related to Human Prion Diseases

The prion protein can exist both in a normal cellular isoform and in a pathogenic conformational isoform. The latter is responsible for the development of different neurodegenerative diseases, for example Creutzfeldt-Jakob disease or fatal familial insomnia. To convert the native benign state of the protein into a highly ordered fibrillar aggregate, large-scale rearrangements of the tertiary structure are necessary during the conversion process and intermediates that are at least partially unfolded are present during fibril formation. In addition to the sporadic conversion into the pathogenic isoform, more than 20 familial diseases are known that are caused by single point mutations increasing the probability of aggregation and neurodegeneration. Here, we demonstrate that the chemically denatured states of the mouse and human prion proteins have very similar structural and dynamic characteristics. Initial studies on the single point mutants E196K, F198S, V203I and R208H of the oxidized mouse construct, which are related to human prion diseases, reveal significant differences in the rate of aggregation. Aggregation for mutants V203I and R208H is slower than it is for the wild type, and the constructs E196K and F198S show accelerated aggregation. These differences in aggregation behaviour are not correlated with the thermal stability of the mutants, indicating different mechanisms promoting the conformational conversion process.     

The intrinsic helical propensities of the helical fragments in prion protein under neutral and low pH conditions: a replica exchange molecular dynamics study

Replica exchange molecular dynamics simulations in neutral and acidic aqueous solutions were employed to study the intrinsic helical propensities of three helices in both Syrian hamster (syPrP) and human (huPrP) prion proteins. The helical propensities of syPrP HA and huPrP HA are very high under both pH conditions, which implies that HA is barely involved in the helix-to-β transition. The SyPrP HB chain has a strong tendency to adopt an extended conformation, which is possibly involved in the mechanism of infectious prion diseases in Syrian hamster. HuPrP HC has more of a preference for the extended conformation than huPrP HA and huPrP HB do, which leads to the conjecture that it is more likely to be the source of β-rich structure for human prion protein. We also noticed that the presence of salt bridges is not correlated with helical propensity, indicating that salt bridges do not stabilize helices.     

Influence of pH on NMR structure and stability of the human prion protein globular domain

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 alpha-helices, comprising residues 144-156, 174-194, and 200-228, and a short anti-parallel beta-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 alpha1 is elongated at the C-terminal end with residues 153-156 forming a 310-helix, and the population of helical structure in the C-terminal two turns of helix alpha 2 is increased. The protonation of His155 and His187 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 C-terminal end of helix alpha 1 and residues 161-164 of beta-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 PrPC to PrPSc conformational transition resulting in transmissible spongiform encephalopathies.     

Possible role of region 152-156 in the structural duality of a peptide fragment from sheep prion protein

The conformational conversion of the nonpathogenic "cellular" prion isoform into a pathogenic "scrapie" protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a beta-like structure. A peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a beta-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152-156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein.     

Molecular dynamics studies on the buffalo prion protein

It was reported that buffalo is a low susceptibility species resisting to transmissible spongiform encephalopathies (TSEs) (same as rabbits, horses, and dogs). TSEs, also called prion diseases, are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of species (except for rabbits, dogs, horses, and buffalo), manifesting as scrapie in sheep and goats; bovine spongiform encephalopathy (BSE or “mad–cow” disease) in cattle; chronic wasting disease in deer and elk; and Creutzfeldt–Jakob diseases, Gerstmann–Sträussler–Scheinker syndrome, fatal familial insomnia, and Kulu in humans etc. In molecular structures, these neurodegenerative diseases are caused by the conversion from a soluble normal cellular prion protein (PrP^C), predominantly with α-helices, into insoluble abnormally folded infectious prions (PrP^Sc), rich in β-sheets. In this article, we studied the molecular structure and structural dynamics of buffalo PrP^C (BufPrP^C), in order to understand the reason why buffalo is resistant to prion diseases. We first did molecular modeling of a homology structure constructed by one mutation at residue 143 from the NMR structure of bovine and cattle PrP(124–227); immediately we found that for BufPrP^C(124–227), there are five hydrogen bonds (HBs) at Asn143, but at this position, bovine/cattle do not have such HBs. Same as that of rabbits, dogs, or horses, our molecular dynamics studies also revealed there is a strong salt bridge (SB) ASP178–ARG164 (O–N) keeping the β2–α2 loop linked in buffalo. We also found there is a very strong HB SER170–TYR218 linking this loop with the C-terminal end of α-helix H3. Other information, such as (i) there is a very strong SB HIS187–ARG156 (N–O) linking α-helices H2 and H1 (if mutation H187R is made at position 187, then the hydrophobic core of PrP^C will be exposed (L.H. Zhong (2010). Exposure of hydrophobic core in human prion protein pathogenic mutant H187R. Journal of Biomolecular Structure and Dynamics 28(3), 355–361)), (ii) at D178, there is a HB Y169–D178 and a polar contact R164–D178 for BufPrP^C instead of a polar contact Q168–D178 for bovine PrP^C (C.J. Cheng, & V. Daggett. (2014). Molecular dynamics simulations capture the misfolding of the bovine prion protein at acidic pH. Biomolecules 4(1), 181–201), (iii) BufPrP^C owns three 3₁₀ helices at 125–127, 152–156, and in the β2–α2 loop, respectively, and (iv) in the β2–α2 loop, there is a strong π–π stacking and a strong π–cation F175–Y169–R164.(N)NH2, has been discovered.     

Prion diseases: dynamics of the infection and properties of the bistable transition

Prion diseases are thought to result from a pathogenic, conformational change in a cellular protein, the prion protein. The pathogenic isoform seems to convert the normal isoform in an autocatalytic process. In contrast to the conditions used for in vitro studies of enzyme kinetics, the concentration of the catalyst is not much lower than that of the substrate in the course of infection. This feature may endow the system with a time-hierarchy allowing the pathogenic isoform to relax very slowly in the course of infection. This may contribute to the long incubation periods observed in prion diseases. The dynamic process of prion propagation, including turnover of the cellular prion protein, displays bistable properties. Sporadic prion diseases may result from a change in one of the parameters associated with metabolism of the prion protein. The bistable transition observed in sporadic disease is reversible, whereas that observed in cases of exogenous contamination is irreversible. This model is consistent with the occurrence of rare, sporadic forms of prion diseases. It may also explain why only some individuals of a cohort develop a prion disease following transient food contamination.     

Molecular mechanism for low pH triggered misfolding of the human prion protein

Conformational changes in the prion protein cause transmissible spongiform encephalopathies, also referred to as prion diseases. In its native state, the prion protein is innocuous (PrPC), but it can misfold into a neurotoxic and infectious isoform (PrPSc). The full-length cellular form of the prion protein consists of residues 23-230, with over half of the sequence belonging to the unstructured N-terminal domain and the remaining residues forming a small globular domain. During misfolding and aggregation, portions of both the structured and unstructured domains are incorporated into the aggregates. After limited proteolysis by proteinase K, the most abundant fragment from brain-derived prion fibrils is a 141-residue fragment composed of residues 90-230. Here we describe simulations of this fragment of the human prion protein at low pH, which triggers misfolding, and at neutral pH as a control. The simulations, in agreement with experiment, show that this biologically and pathologically relevant prion construct is stable and native-like at neutral pH. In contrast, at low pH the prion protein is destabilized via disruption of critical long-range salt bridges. In one of the low pH simulations this destabilization resulted in a conformational transition to a PrPSc-like isoform consistent with our previous simulations of a smaller construct.     

Prion disease: A loss of antioxidant function?

Prion disease, a neurodegenerative disorder, is widely believed to arise when a cellular prion protein (PrP(C)) undergoes conformational changes to a pathogenic isoform (PrP(Sc)). Recent data have shown PrP(C) to be copper binding and that it acquires antioxidant activity as a result. This enzymatic property is dependent mainly on copper binding to the octarepeats region. In normal human brain and human prion disease, there is a population of brain-derived PrP that has been truncated at the N-terminal which encompassed the octarepeats region. Increasing evidences have suggested imbalances of metal-catalyzed reactions to be the common denominator for several neurodegenerative diseases. Therefore, we propose that one of the causative factors for prion disease could be due to the imbalances in metal-catalyzed reactions resulting in an alteration of the antioxidant function. These result in an increase level of oxidative stress and, as such, trigger the neurodegenerative cascade.     

Kinetic intermediate in the folding of human prion protein

Transmissible spongiform encephalopathies are associated with the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). Here we have examined the kinetics of folding and unfolding reactions for the recombinant human prion protein C-terminal fragment 90-231 at pH 4.8 and 7.0. The stopped-flow data provide clear evidence for the population of an intermediate on the refolding pathway of the prion protein as indicated by a pronounced curvature in chevron plots and the presence of significant burst phase amplitude in the refolding kinetics. In addition to its role in the normal prion protein folding, this intermediate likely represents a crucial monomeric precursor of the pathogenic PrP(Sc) isoform.     

Structural and Dynamic Properties of the Human Prion Protein

Prion diseases involve the conformational conversion of the cellular prion protein (PrP^C) to its misfolded pathogenic form (PrP^Sc). To better understand the structural mechanism of this conversion, we performed extensive all-atom, explicit-solvent molecular-dynamics simulations for three structures of the wild-type human PrP (huPrP) at different pH values and temperatures. Residue 129 is polymorphic, being either Met or Val. Two of the three structures have Met in position 129 and the other has Val. Lowering the pH or raising the temperature induced large conformational changes of the C-terminal globular domain and increased exposure of its hydrophobic core. In some simulations, HA and its preceding S1-HA loop underwent large displacements. The C-terminus of HB was unstable and sometimes partially unfolded. Two hydrophobic residues, Phe-198 and Met-134, frequently became exposed to solvent. These conformational changes became more dramatic at lower pH or higher temperature. Furthermore, Tyr-169 and the S2-HB loop, or the X-loop, were different in the starting structures but converged to common conformations in the simulations for the Met-129, but not the Val-129, protein. α-Strands and β-strands formed in the initially unstructured N-terminus. α-Strand propensity in the N-terminus was different between the Met-129 and Val129 proteins, but β-strand propensity was similar. This study reveals detailed structural and dynamic properties of huPrP, providing insight into the mechanism of the conversion of PrP^C to PrP^Sc.     

Folding of prion protein to its native alpha-helical conformation is under kinetic control

The recombinant mouse prion protein (MoPrP) can be folded either to a monomeric alpha-helical or oligomeric beta-sheet-rich isoform. By using circular dichroism spectroscopy and size-exclusion chromatography, we show that the beta-rich isoform of MoPrP is thermodynamically more stable than the native alpha-helical isoform. The conformational transition from the alpha-helical to beta-rich isoform is separated by a large energetic barrier that is associated with unfolding and with a higher order kinetic process related to oligomerization. Under partially denaturing acidic conditions, MoPrP avoids the kinetic trap posed by the alpha-helical isoform and folds directly to the thermodynamically more stable beta-rich isoform. Our data demonstrate that the folding of the prion protein to its native alpha-helical monomeric conformation is under kinetic control.     

Mutation directional selection sheds light on prion pathogenesis

As mutations in the PRNP gene account for human hereditary prion diseases (PrDs), it is crucial to elucidating how these mutations affect the central pathogenic conformational transition of normal cellular prion protein (PrP^C) to abnormal scrapie isoform (PrP^Sc). Many studies proposed that these pathogenic mutations may make PrP more susceptible to conformational change through altering its structure stability. By evaluating the most recent observations regarding pathogenic mutations, it was found that the pathogenic mutations do not exert a uniform effect on the thermodynamic stability of the human PrP’s structure. Through analyzing the reported PrDs-related mutations, we found that 25 out of 27 mutations possess strong directional selection, i.e., enhancing hydrophobicity or decreasing negative and increasing positive charge. Based on the triggering role reported by previous studies of facilitating factors in PrP^C conversion, e.g., lipid and polyanion, we proposed that the mutation-induced changes may strengthen the interaction between PrP and facilitating factors, which will accelerate PrP conversion and cause PrDs.