Prion Protein mPrP[F175A](121–231): Structure and Stability in Solution

doi:10.1016/j.jmb.2012.08.011

Journal of Molecular Biology

Volume 423, Issue 4, 2 November 2012, Pages 496-502

https://doi.org/10.1016/j.jmb.2012.08.011 Get rights and content

Abstract

The three-dimensional structures of prion proteins (PrPs) in the cellular form (PrP^C) include a stacking interaction between the aromatic rings of the residues Y169 and F175, where F175 is conserved in all but two so far analyzed mammalian PrP sequences and where Y169 is strictly conserved. To investigate the structural role of F175, we characterized the variant mouse prion protein mPrP[F175A](121–231). The NMR solution structure represents a typical PrP^C-fold, and it contains a 3₁₀-helical β2–α2 loop conformation, which is well defined because all amide group signals in this loop are observed at 20 °C. With this “rigid‐loop PrP^C” behavior, mPrP[F175A](121–231) differs from the previously studied mPrP[Y169A](121–231), which contains a type I β-turn β2–α2 loop structure. When compared to other rigid‐loop variants of mPrP(121–231), mPrP[F175A](121–231) is unique in that the thermal unfolding temperature is lowered by 8 °C. These observations enable further refined dissection of the effects of different single-residue exchanges on the PrP^C conformation and their implications for the PrP^C physiological function.

Graphical Abstract

Highlights

► First rigid‐loop prion protein (RL-PrP^C) with reduced stability. ► Amino acid replacement of F175A induces extensive repacking of PrP^C core. ► Novel type of aromatic ring substitution in PrP^C core. ► β2–α2 loop polymorphism in PrP^C maintained after F175A exchange.

Introduction

NMR structure determination of prion proteins (PrPs) in the cellular form (PrP^C)[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11] has shown that the fold of a C-terminal globular domain in PrP^Cs is highly conserved (“PrP^C-fold”), with three α-helices and a short β-sheet. In this conserved scaffold, a surface area including the loop that connects the strand β2 with the helix α2 has attracted special interest due to its high sequence and three-dimensional (3D) structure variability among mammalian PrPs.[12], [13] For example, sequence variations in the β2–α2 loop by the substitution D167S¹⁰ and the two-residue substitution S170N/N174T⁶ have been shown to affect the conformation of the loop and relate to increased occurrence of spontaneous spongiform degeneration in transgenic mice,[14], [15], [16] indicating that a surface epitope that includes the β2–α2 loop plays an important role in prion pathology. With the use of different approaches, in vitro studies revealed correlations between the amino acid sequence of the β2–α2 loop and the propensity of PrP^C to undergo transitions to β-sheet-rich conformations.[17], [18], [19] Furthermore, steric zipper amyloid fibrils are formed by the hexapeptide SNQNNF, which corresponds to the loop-adjoining polypeptide segment S170–F175 in human and mouse PrP^Cs.²⁰ It is thus of keen interest to further investigate conformational properties of PrP^Cs in the region near the β2–α2 loop, in order to establish a structural basis that may help in rationalizing these observations.

This communication reports on the role of the residue F175 in mPrP(121–231). F175 is conserved in all but two mammalian PrPs, ferret PrP (Mustela putorius furo) and European polecat PrP (M. putorius), which contain L175.[21], [22] NMR structure determination of mPrP[F175A](121–231) and denaturation studies of this variant mouse prion protein (mPrP) are used to investigate the role of F175 for structure and stability of the protein and, in particular, its impact on the conformation of the β2–α2 loop.

Section snippets

Preparation of mPrP(121–231) variants with substitution of F175

We followed the strategy of producing the recombinant C-terminal globular domains of ferret PrP and three related variants of mPrP(121–231) and selecting the best-behaving protein for NMR structure determination. As no natural source DNA was available for ferret PrP, we replaced five amino acids in dog PrP(121–231) to obtain the gene encoding ferret PrP(121–231) (experimental details are given in Fig. 1a). Three new variants of mPrP(121–231) were cloned by introducing the single-residue

NMR structure of mPrP[F175A](121–231)

For the backbone resonance assignments, we recorded 3D HNCA, 3D HNCO, 3D HNCACO, 3D CBCACONH and 3D HNCACB triple‐resonance experiments. The side‐chain resonances were assigned with 3D ¹⁵N-resolved [¹H,¹H]-TOCSY (total correlated spectroscopy) and 3D HC(C)H–TOCSY data sets and with additional reference to the 3D ¹⁵N-resolved and ¹³C-resolved [¹H,¹H]-NOESY (nuclear Overhauser enhancement spectroscopy) spectra that were recorded for the collection of conformational constraints. Complete backbone

Stability of mPrP[F175A](121–231)

Figure 3 shows temperature-induced denaturation curves of mPrP(121–231) and mPrP[F175A](121–231) and lists the melting temperatures obtained with these experiments. Compared to mPrP(121–231), the mutation F175A reduces the melting temperature, T_m, by 8 °C at the solution conditions described in Fig. 3. Slow lowering of the temperature yielded renaturation of the protein in the extent of about 80%, but urea denaturation data confirmed that the reduced T_m value represents reduced stability of

Conclusions

Considering the close structural proximity of the aromatic rings of F175 and Y169 in mPrP(121–231) (Fig. 2b), it is remarkable that individual substitution of the two aromatic side chains with alanine generates two different β2–α2 loop conformations, with mPrP[Y169A](121–231) containing a temperature-insensitive type I β-turn structure²³ and mPrP[F175A](121–231) containing a 3₁₀-helix structure in conformational exchange with a lowly populated different structure.

The replacement of F175 with

Data bank accession codes

The atomic coordinates for mPrP[F175A](121–231) have been deposited in the Protein Data Bank (ID code 2L1E†), and chemical shifts have been deposited in the BioMagResBank (#17082‡).

Acknowledgements

Financial support by the Swiss National Science Foundation and the ETH Zurich through the National Center of Competence in Research “Structural Biology” and by the European Union (UPMAN) (project number 512052) is gratefully acknowledged.

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The inward movement of Met166 has facilitated the structural proximity of the β2-α2 loop and the C-terminal part of α3 by forming long-range hydrophobic interactions with Tyr218, Tyr225 and Tyr226. Consequently, the solvent-exposed surface epitope is formed by the β2-α2 loop and a part of α3 might act as a binding site to the effector molecules, thereby would cause the conversion of PrPC to the disease-associated PrPSc [12,15,75,76,78]. Apart from the aforementioned vital structural alterations, we further found another crucial change in the mutant associated with the transition of PrPC to the PrPSc isoform.
Misfolding of the cellular prion protein (PrP^C) into β-sheet-rich scrapie form (PrP^Sc) is associated with transmissible spongiform encephalopathies. A point mutation F198S is responsible for the development of a rare inherited Gerstmann-Straussler-Scheinker disease caused by the aggregation of PrP^C. Thus, identification and the structural characterization of aggregation-prone regions are essential to delineate the conversion of PrP^C to the disease-associated PrP^Sc upon F198S mutation. In the present study, molecular dynamics simulations on the wild-type PrP (WT-PrP) and its mutant were performed to explore the structural basis responsible for aggregation driven by the mutation. Secondary structure analysis revealed that the mutant exhibited a partial unfolding on α2 and the complete distortion in the 3₁₀-helix of the β2-α2 loop. Remarkably, the β2-α2 loop is in proximity to α3 attributed by the long-range hydrophobic interactions and such structural intimacy is not observed in the WT-PrP. Owing to this, the β1-α1-β2 regions have separated from α2-α3 domain resulting in the impairment on the hydrogen bond between α1 and α3. Thus, the present study provides a detailed structural description of the F198S mutant in line with previous experimental results and delivers insights into the structural basis responsible for the conversion of PrP^C to the disease-associated PrP^Sc.
A proposed mechanism for the promotion of prion conversion involving a strictly conserved tyrosine residue in the α<inf>2</inf>- β<inf>2</inf> loop of PrPC
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The transmission of infectious prions into different host species requires compatible prion protein (PrP) primary structures, and even one heterologous residue at a pivotal position can block prion infection. Mapping the key amino acid positions that govern cross-species prion conversion has not yet been possible, although certain residue positions have been identified as restrictive, including residues in the β₂-α₂ loop region of PrP. To further define how β₂-α₂ residues impact conversion, we investigated residue substitutions in PrP^C using an in vitro prion conversion assay. Within the β₂-α₂ loop, a tyrosine residue at position 169 is strictly conserved among mammals, and transgenic mice expressing mouse PrP having the Y169G, S170N, and N174T substitutions resist prion infection. To better understand the structural requirements of specific residues for conversion initiated by mouse prions, we substituted a diverse array of amino acids at position 169 of PrP. We found that the substitution of glycine, leucine, or glutamine at position 169 reduced conversion by ∼75%. In contrast, replacing tyrosine 169 with either of the bulky, aromatic residues, phenylalanine or tryptophan, supported efficient prion conversion. We propose a model based on a requirement for tightly interdigitating complementary amino acid side chains within specific domains of adjacent PrP molecules, known as “steric zippers,” to explain these results. Collectively, these studies suggest that an aromatic residue at position 169 supports efficient prion conversion.
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The salt bridge ASP177–ARG163 is just like a taut bow-string keeping the β2–α2 loop linked. This loop has been a focus on the studies of RaPrP molecular structure (Sweeting et al., 2009, 2013; Wen et al., 2010b; Christen et al., 2008, 2009, 2012, 2013; Damberger et al., 2011; Sigurdson et al., 2009, 2010, 2011; Pérez et al., 2010; Gossert et al., 2005; Lührs et al., 2003; Stanker et al., 2012; Cong et al., 2013; Bett et al., 2012; Meli et al., 2011; Rossetti et al., 2010; Kirby et al., 2010; Zhang, 2011, 2012). We also compared with NMR structures with X-ray structures of RaPrP.
Prion diseases, traditionally referred to as transmissible spongiform encephalopathies (TSEs), are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of mammalian species, 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. These neurodegenerative diseases are caused by the conversion from a soluble normal cellular prion protein $({PrP}^{C})$ into insoluble abnormally folded infectious prions $({PrP}^{Sc})$ , and the conversion of PrP^C to PrP^Sc is believed to involve conformational change from a predominantly α-helical protein to one rich in β-sheet structure. Such a conformational change may be amenable to study by molecular dynamics (MD) techniques. For rabbits, classical studies show that they have a low susceptibility to be infected by ${PrP}^{Sc}$ , but recently it was reported that rabbit prions can be generated through saPMCA (serial automated Protein Misfolding Cyclic Amplification) in vitro and the rabbit prion is infectious and transmissible. In this paper, we first do a detailed survey on the research advances of rabbit prion protein (RaPrP) and then we perform MD simulations on the NMR and X-ray molecular structures of rabbit prion protein wild-type and mutants. The survey shows to us that rabbits were not challenged directly in vivo with other known prion strains and the saPMCA result did not pass the test of the known BSE strain of cattle. Thus, we might still look rabbits as a prion resistant species. MD results indicate that the three α-helices of the wild-type are stable under the neutral pH environment (but under low pH environment the three α-helices have been unfolded into β-sheets), and the three α-helices of the mutants (I214V and S173N) are unfolded into rich β-sheet structures under the same pH environment. In addition, we found an interesting result that the salt bridges such as ASP201–ARG155, ASP177–ARG163 contribute greatly to the structural stability of RaPrP.
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¹: Present address: S. Hornemann, Institute of Neuropathology, University Hospital Zurich, Schmelzbergstrasse 12, CH‐8091 Zurich, Switzerland.

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Journal of Molecular Biology

CommunicationPrion Protein mPrP[F175A](121–231): Structure and Stability in Solution

Abstract

Graphical Abstract

Highlights

Introduction

Section snippets

Preparation of mPrP(121–231) variants with substitution of F175

NMR structure of mPrP[F175A](121–231)

Stability of mPrP[F175A](121–231)

Conclusions

Data bank accession codes

Acknowledgements

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

Biochem. Biophys. Res. Commun.

J. Mol. Biol.

J. Mol. Biol.

FEBS Lett.

J. Mol. Biol.

J. Mol. Biol.

Comput. Phys. Commun.

J. Mol. Graphics

NMR structure of the mouse prion protein domain PrP(121–231)

Nature

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

Proc. Natl Acad. Sci. USA

NMR solution structure of the human prion protein

Proc. Natl Acad. Sci. USA

NMR structure of the bovine prion protein

Proc. Natl Acad. Sci. USA

Prion protein NMR structures of cats, dogs, pigs, and sheep

Proc. Natl Acad. Sci. USA

Prion protein NMR structures of elk and of mouse/elk hybrids

Proc. Natl Acad. Sci. USA

Communication
Prion Protein mPrP[F175A](121–231): Structure and Stability in Solution