Gregor Ilc

NMR structure of the human prion protein with the pathological Q212P mutation reveals unique structural features.

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrPC) conformer, denoted as infectious scrapie isoform or PrPSc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrPSc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β2–α2 loop region. This structure might provide new insights into the early events of conformational transition of PrPC into PrPSc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β2–α2 loop and α3 helix.

Toward the Molecular Basis of Inherited Prion Diseases: NMR Structure of the Human Prion Protein with V210I Mutation

The development of transmissible spongiform encephalopathies (TSEs) is associated with the conversion of the cellular prion protein (PrP(C)) into a misfolded, pathogenic isoform (PrP(Sc)). Spontaneous generation of PrP(Sc) in inherited forms of disease is caused by mutations in gene coding for PrP (PRNP). In this work, we describe the NMR solution-state structure of the truncated recombinant human PrP (HuPrP) carrying the pathological V210I mutation linked to genetic Creutzfeldt-Jakob disease. The three-dimensional structure of V210I mutant consists of an unstructured N-terminal part (residues 90-124) and a well-defined C-terminal domain (residues 125-228). The C-terminal domain contains three α-helices (residues 144-156, 170-194 and 200-228) and a short antiparallel β-sheet (residues 129-130 and 162-163). Comparison with the structure of the wild-type HuPrP revealed that although two structures share similar global architecture, mutation introduces some local structural differences. The observed variations are mostly clustered in the α(2)-α(3) inter-helical interface and in the β(2)-α(2) loop region. Introduction of bulkier Ile at position 210 induces reorientations of several residues that are part of hydrophobic core, thus influencing α(2)-α(3) inter-helical interactions. Another important structural feature involves the alteration of conformation of the β(2)-α(2) loop region and the subsequent exposure of hydrophobic cluster to solvent, which facilitates intermolecular interactions involved in spontaneous generation of PrP(Sc). The NMR structure of V210I mutant offers new clues about the earliest events of the pathogenic conversion process that could be used for the development of antiprion drugs.

Structural basis for the protective effect of the human prion protein carrying the dominant-negative E219K polymorphism.

The most common form of prion disease in humans is sCJD (sporadic Creutzfeldt-Jakob disease). The naturally occurring E219K polymorphism in the HuPrP (human prion protein) is considered to protect against sCJD. To gain insight into the structural basis of its protective influence we have determined the NMR structure of recombinant HuPrP (residues 90-231) carrying the E219K polymorphism. The structure of the HuPrP(E219K) protein consists of a disordered N-terminal tail (residues 90-124) and a well-structured C-terminal segment (residues 125-231) containing three α-helices and two short antiparallel β-strands. Comparison of NMR structures of the wild-type and HuPrPs with pathological mutations under identical experimental conditions revealed that, although the global architecture of the protein remains intact, replacement of Glu²¹⁹ with a lysine residue introduces significant local structural changes. The structural findings of the present study suggest that the protective influence of the E219K polymorphism is due to the alteration of surface charge distribution, in addition to subtle structural rearrangements localized within the epitopes critical for prion conversion.

Structural Rearrangements at Physiological pH: Nuclear Magnetic Resonance Insights from the V210I Human Prion Protein Mutant.

A major focus in prion structural biology studies is unraveling the molecular mechanism leading to the structural conversion of PrP(C) to its pathological form, PrP(Sc). In our recent studies, we attempted to understand the early events of the conformational changes leading to PrP(Sc) using as investigative tools point mutations clustered in the open reading frame of the human PrP gene and linked to genetic forms of human prion diseases. In the work presented here, we investigate the effect of pH on the nuclear magnetic resonance (NMR) structure of recombinant human PrP (HuPrP) carrying the pathological V210I mutation responsible for familial Creutzfeldt-Jakob disease. The NMR structure of HuPrP(V210I) determined at pH 7.2 shows the same overall fold as the previously determined structure of HuPrP(V210I) at pH 5.5. It consists of a disordered N-terminal tail (residues 90-124) and a globular C-terminal domain (residues 125-231) comprising three α-helices and a short antiparallel β-sheet. Detailed comparison of three-dimensional structures of HuPrP(V210I) at pH 7.2 and 5.5 revealed significant local structural differences, with the most prominent pH-related structural variations clustered in the α(2)-α(3) interhelical region, at the interface of the β(1)-α(1) loop, in helices α(1) and α(3), and in the β(2)-α(2) loop region. The detailed analysis of interactions among secondary structure elements suggests a higher degree of structural ordering of HuPrP(V210I) under neutral-pH conditions, thus implying that spontaneous misfolding of PrP(C) may occur under acidic-pH conditions in endosomal compartments.

Probing Early Misfolding Events in Prion Protein Mutants by NMR Spectroscopy

The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs) and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most α-helical motifs are replaced by β-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify “hot spots” in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research.

Biosimilar structural comparability assessment by NMR: from small proteins to monoclonal antibodies

Biosimilar drug products must have a demonstrated similarity with respect to the reference product’s molecules in order to ensure both the effectiveness of the drug and the patients’ safety. In this paper the fusion framework of a highly sensitive NMR fingerprinting approach for conformational changes and mathematically-based biosimilarity metrics is introduced. The final goal is to translate the complex spectral information into biosimilarity scores, which are then used to estimate the degree of similarity between the biosimilar and the reference product. The proposed method was successfully applied to a small protein, i.e., filgrastim (neutropenia treatment), which is the first biosimilar approved in the United States, and a relatively large protein, i.e., monoclonal antibody rituximab (lymphoma treatment). This innovative approach introduces a new level of sensitivity to structural changes that are induced by, e.g., a small pH shift or other changes in the protein formulation.

The N Terminus of the Prion Protein Mediates Functional Interactions with the Neuronal Cell Adhesion Molecule (NCAM) Fibronectin Domain.

The cellular form of the prion protein (PrPC) is a highly conserved glycoprotein mostly expressed in the central and peripheral nervous systems by different cell types in mammals. A misfolded, pathogenic isoform, denoted as prion, is related to a class of neurodegenerative diseases known as transmissible spongiform encephalopathy. PrPC function has not been unequivocally clarified, and it is rather defined as a pleiotropic protein likely acting as a dynamic cell surface scaffolding protein for the assembly of different signaling modules. Among the variety of PrPC protein interactors, the neuronal cell adhesion molecule (NCAM) has been studied in vivo, but the structural basis of this functional interaction is still a matter of debate. Here we focused on the structural determinants responsible for human PrPC (HuPrP) and NCAM interaction using stimulated emission depletion (STED) nanoscopy, SPR, and NMR spectroscopy approaches. PrPC co-localizes with NCAM in mouse hippocampal neurons, and this interaction is mainly mediated by the intrinsically disordered PrPC N-terminal tail, which binds with high affinity to the NCAM fibronectin type-3 domain. NMR structural investigations revealed surface-interacting epitopes governing the interaction between HuPrP N terminus and the second module of the NCAM fibronectin type-3 domain. Our data provided molecular details about the interaction between HuPrP and the NCAM fibronectin domain, and revealed a new role of PrPC N terminus as a dynamic and functional element responsible for protein-protein interaction.

NMR Structural Studies of Human Cellular Prion Proteins

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders associated with the conformational conversion of the cellular prion protein, PrP(C), into a pathological form known as prion or PrP(Sc). They can be classified into sporadic, inherited and infectious forms. Spontaneous generation of PrP(Sc) in inherited forms of prion diseases is caused by mutations in the human prion protein gene (PRNP). A major goal in prion biology is unraveling the molecular mechanism by which PrP(C) misfolds and leads to development of diseases. Structural characterization of various human PrP (HuPrP) variants may be helpful for better understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions. Here, we review the results of the recent high-resolution nuclear magnetic resonance (NMR) structural studies on HuPrPs with pathological Q212P and V210I mutations linked with Gerstmann-Sträussler-Scheinker (GSS) syndrome and familial Creutzfeldt-Jakob disease (fCJD), respectively, and HuPrP carrying naturally occurring E219K polymorphism considered to protect against sporadic CJD (sCJD). We describe subtle local differences between the three-dimensional (3D) structures of HuPrP mutants and the wild-type (WT) protein, providing new insights into the possible key structural determinants underlying conversion of PrP(C) into PrP(Sc). Also highlighted are the most recent findings from NMR studies about the effect of pH on the structural features of HuPrP with V210I mutation.

Structure and calcium-binding studies of calmodulin-like domain of human non-muscle alpha-actinin-1

The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.

19 F NMR studies provide insights into lipid membrane interactions of listeriolysin O, a pore forming toxin from Listeria monocytogenes

Listeria monocytogenes is a mammalian pathogen that causes gastroenteritis, miscarriages and infections of the central nervous system in immunocompromised individuals. Its main virulence factor is listeriolysin O (LLO), a pore-forming cholesterol-dependent cytolysin (CDC), which enables bacterial escape from the phagolysosome and contributes to bacterial pathogenicity. Details of cholesterol (Chol) recognition and membrane binding mechanisms by LLO are still not known. Here we used 19F-NMR spectroscopy in order to assess LLO-Chol interactions in solution and in a Chol-rich membrane environment. LLO has six tryptophan residues located in the region of the molecule that is first in contact with lipid membranes. 19F-LLO, which contained 5-fluoro-tryptophans, was prepared by using isotopic labelling in an E. coli expression system. Signals in the 19F-NMR spectrum of 19F-LLO were unambiguously assigned by using a series of single Trp → Phe point mutations. The results employing various cholesterol preparations in solution indicate that tryptophan residues are not directly involved in Chol binding in solution. However, significant chemical shift changes were observed upon LLO binding to Chol-rich membranes, highlighting the role of tryptophan residues in membrane interactions (W512) and oligomerisation (W189 and W489).