scholarly journals Novel quaternary structures of the human prion protein globular domain

2020 ◽  
Author(s):  
Leandro Oliveira Bortot ◽  
Victor Lopes Rangel ◽  
Francesca A. Pavlovici ◽  
Kamel El Omari ◽  
Armin Wagner ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Prion Protein ◽  
3D Structure ◽  
Disulfide Bridge ◽  
Cellular Prion Protein ◽  
Crystallographic Structure ◽  
Globular Domain ◽  
X Ray Crystallography ◽  
Quaternary Structures ◽  
Dynamics Simulations

AbstractPrion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process.

New structural insights into anomeric carbohydrate recognition by frutalin: an α-d-galactose-binding lectin from breadfruit seeds

2019 ◽  
Vol 476 (1) ◽  
pp. 101-113 ◽  
Author(s):  
Antonio Eufrásio Vieira Neto ◽  
Felipe Domingos de Sousa ◽  
Humberto D'Muniz Pereira ◽  
Frederico Bruno Mendes Batista Moreno ◽  
Marcos Roberto Lourenzoni ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activities ◽  
3D Structure ◽  
Carbohydrate Binding ◽  
X Ray Crystallography ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
New Perspective ◽  
Dynamics Simulations ◽  
Binding Lectin

Abstract Frutalin (FTL) is a multiple-binding lectin belonging to the jacalin-related lectin (JRL) family and derived from Artocarpus incisa (breadfruit) seeds. This lectin specifically recognizes and binds α-d-galactose. FTL has been successfully used in immunobiological research for the recognition of cancer-associated oligosaccharides. However, the molecular bases by which FTL promotes these specific activities remain poorly understood. Here, we report the whole 3D structure of FTL for the first time, as determined by X-ray crystallography. The obtained crystals diffracted to 1.81 Å (Apo-frutalin) and 1.65 Å (frutalin–d-Gal complex) of resolution. The lectin exhibits post-translational cleavage yielding an α- (133 amino acids) and β-chain (20 amino acids), presenting a homotetramer when in solution, with a typical JRL β-prism. The β-prism was composed of three 4-stranded β-sheets forming three antiparallel Greek key motifs. The carbohydrate-binding site (CBS) involved the N-terminus of the α-chain and was formed by four key residues: Gly25, Tyr146, Trp147 and Asp149. Together, these results were used in molecular dynamics simulations in aqueous solutions to shed light on the molecular basis of FTL-ligand binding. The simulations suggest that Thr-Ser-Ser-Asn (TSSN) peptide excision reduces the rigidity of the FTL CBS, increasing the number of interactions with ligands and resulting in multiple-binding sites and anomeric recognition of α-d-galactose sugar moieties. Our findings provide a new perspective to further elucidate the versatility of FTL in many biological activities.

scholarly journals Zn(II) binding causes interdomain changes in the structure and flexibility of the human prion protein

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maciej Gielnik ◽  
Michał Taube ◽  
Lilia Zhukova ◽  
Igor Zhukov ◽  
Sebastian K. T. S. Wärmländer ◽  
...  
Keyword(s):  
Prion Protein ◽  
Metal Ion ◽  
Structural Transition ◽  
Cellular Prion Protein ◽  
Zinc Homeostasis ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathies ◽  
Structural Conversion ◽  
Protein Size ◽  
Dynamics Simulations

AbstractThe cellular prion protein (PrPC) is a mainly α-helical 208-residue protein located in the pre- and postsynaptic membranes. For unknown reasons, PrPC can undergo a structural transition into a toxic, β-sheet rich scrapie isoform (PrPSc) that is responsible for transmissible spongiform encephalopathies (TSEs). Metal ions seem to play an important role in the structural conversion. PrPC binds Zn(II) ions and may be involved in metal ion transport and zinc homeostasis. Here, we use multiple biophysical techniques including optical and NMR spectroscopy, molecular dynamics simulations, and small angle X-ray scattering to characterize interactions between human PrPC and Zn(II) ions. Binding of a single Zn(II) ion to the PrPC N-terminal domain via four His residues from the octarepeat region induces a structural transition in the C-terminal α-helices 2 and 3, promotes interaction between the N-terminal and C-terminal domains, reduces the folded protein size, and modifies the internal structural dynamics. As our results suggest that PrPC can bind Zn(II) under physiological conditions, these effects could be important for the physiological function of PrPC.

scholarly journals A Promising Antiprion Trimethoxychalcone Binds to the Globular Domain of the Cellular Prion Protein and Changes Its Cellular Location

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
N. C. Ferreira ◽  
L. M. Ascari ◽  
A. G. Hughson ◽  
G. R. Cavalheiro ◽  
C. F. Góes ◽  
...  
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Prion Protein ◽  
Molecular Mechanisms ◽  
Cellular Prion Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Globular Domain ◽  
Mrna Levels ◽  
Spongiform Encephalopathies

ABSTRACTThe search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identifiedin vitroto be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPCmRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23–231) as the substrate and PrPScseeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90–231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPCglobular domain. J8 also reduced the fibrillation of mouse rPrP23–231seeded within vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPCon the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPCcontent on the cell surface.

scholarly journals The structural transformation comparison of the human Prion protein mutants V176G, E196A, and I215V by using molecular dynamics simulation

2021 ◽  
Author(s):  
Ashraf Fadhil Jomah ◽  
Sepideh Parvizpour ◽  
Jafar Razmara ◽  
Mohd Shahir Shamsir
Keyword(s):  
Prion Protein ◽  
Aqueous Media ◽  
Point Mutations ◽  
Cellular Prion Protein ◽  
Dynamics Simulation ◽  
Globular Domain ◽  
Salt Bridges ◽  
Wild Type ◽  
Structural Determinants ◽  
C Terminus

Abstract The point mutations in the gene coding of prion protein (PrP) originate human familial prion protein (HuPrP) diseases. Such diseases are caused by several amino acid mutations of HuPrP including V176G, I215V, and E196A located at the second, third native helix and in their loop, respectively. Determining the transition from cellular prion protein (PrPc) to pathogenic conformer (PrPSc) in the globular domain of HuPrP that results in pathogenic mutations is the key issue. The effects of mutation on monomeric PrP are detected in the absence of an unstructured N-terminal domain only. A MD simulation for each of these wild type mutants is performed to examine their structure in the aqueous media. The structural determinants are discerned to be different for wild-type HuPrP (125–228) variants compare to that of HuPrP mutations. These three mutations exhibiting diverse effects on the dynamical properties of PrP are attributed to the variations in the secondary structure, solvent accessible surface areas (SASAs), and salt bridges in the globular domain of HuPrP. High fluctuations that are evidenced around residues of the C-terminus of the helix 1 for V176G cause Gerstmann-Straussler-Scheinker (GSS) syndrome. Conversely, the occurrence of fluctuations around residues of helix 2, helix 3, and the loss of salt bridges in these regions for E196A and I215V mutants is responsible for Creutzfeldt-Jakob disease. Furthermore, small changes in the overall SASAs mutations strongly influence the intermolecular interactions during the aggregation process. The comparative results in this study demonstrate that the three mutants undergo different pathogenic transformations.

scholarly journals Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

2021 ◽  
Vol 77 (5) ◽  
pp. 645-662
Author(s):  
Risako Tamura-Sakaguchi ◽  
Rie Aruga ◽  
Mika Hirose ◽  
Toru Ekimoto ◽  
Takuya Miyake ◽  
...  
Keyword(s):  
Complex Formation ◽  
Structure Determination ◽  
Structural Changes ◽  
Insertion Site ◽  
3D Structure ◽  
Binding Pocket ◽  
Crystallographic Analysis ◽  
Stable Complex ◽  
X Ray Crystallography ◽  
Dynamics Simulations

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

scholarly journals Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope tag insertion

2020 ◽  
Author(s):  
Risako Tamura-Sakaguchi ◽  
Rie Aruga ◽  
Mika Hirose ◽  
Toru Ekimoto ◽  
Takuya Miyake ◽  
...  
Keyword(s):  
Structural Change ◽  
Complex Formation ◽  
Structure Determination ◽  
Insertion Site ◽  
3D Structure ◽  
Binding Pocket ◽  
Stable Complex ◽  
X Ray Crystallography ◽  
Antibody Complex ◽  
Dynamics Simulations

AbstractAntibody labeling has been extensively conducted for structure determination in both x-ray crystallography and EM analysis. However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, we have developed an alternative method to prepare an antibody-complex by inserting an exogenous epitope into the target. We have already demonstrated that the Fab of monoclonal antibody NZ-1 could form a stable complex with the target containing a PA12 tag as an inserted epitope. Nevertheless, we also found that the complex formation through the inserted PA12 tag inevitably caused structural change around the insertion site of the target. Hence, we here attempted to improve the insertion method and consequently discovered that utilization of a PA14 tag significantly reduced the structural change in the target. By adopting a closed ring-like conformation inside the antigen-binding pocket, the inserted PA14 tag had less impact on the folding of the target. Due to this structural property, the PA14 tag could also be inserted into the sterically hindered loop for labeling. Molecular dynamics simulations also indicated that the folding of the target was rigid regardless of the PA14 insertion and the complex formation with the NZ-1 Fab. Using the improved labeling technique, we performed negative-stain EM on a bacterial site-2 protease, which enabled us to approximate the domain arrangement based on the docking mode of the NZ-1 Fab.

scholarly journals Liquid-liquid phase separation and aggregation of the prion protein globular domain modulated by a high-affinity DNA aptamer

2019 ◽  
Author(s):  
Carolina O. Matos ◽  
Yulli M. Passos ◽  
Mariana J. do Amaral ◽  
Bruno Macedo ◽  
Matheus Tempone ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Prion Protein ◽  
Liquid Phase Separation ◽  
Cellular Prion Protein ◽  
Globular Domain ◽  
Structural Conversion ◽  
High Affinity ◽  
Liquid Liquid Phase Separation

ABSTRACTStructural conversion of cellular prion protein (PrPC) into scrapie PrP (PrPSc) and subsequent aggregation are key events for the onset of Transmissible Spongiform Encephalopathies (TSEs). Experimental evidences support the role of nucleic acids (NAs) in assisting the protein conversion process. Here, we used the SELEX methodology to identify two 25-mer DNA aptamers against the globular domain of recombinant murine PrP (rPrP90-231), namely A1 and A2. High-affinity binding of A1 and A2 to rPrP was verified by ITC. Aptamers structure was characterized by theoretical predictions, CD, NMR and SAXS, revealing that A1 adopts a hairpin conformation. Aptamer binding caused dynamic aggregation of rPrP90-231, resulting from the ability of rPrP90-231to undergo liquid-liquid phase separation (LLPS). While free rPrP90-231phase separated into large droplets, aptamer binding increased the amount but reduced the size of the condensates. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced transition to an ordered state, suggestive of amyloid formation on the surface of the droplets. Our results describe for the first time PrP:NA interaction leading to LLPS and modulation of this effect depending on NA structure and binding stoichiometry, shedding light on the role of NAs in PrP misfolding and TSEs.

scholarly journals 3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Xing Zhang ◽  
Lei Zhang ◽  
Huimin Tong ◽  
Bo Peng ◽  
Matthew J. Rames ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Individual Particle ◽  
Single Particle Reconstruction ◽  
X Ray Crystallography ◽  
Igg1 Antibody ◽  
Structural Fluctuation ◽  
Dynamics Simulations

Abstract Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

scholarly journals The channel activities of the full-length prion and truncated proteins

2022 ◽  
Author(s):  
Jinming Wu ◽  
Asvin KK Lakkaraju ◽  
Adriano KK Aguzzi ◽  
Jinghui Luo
Keyword(s):  
Prion Protein ◽  
Prion Disease ◽  
Molecular Mechanisms ◽  
Membrane Permeabilization ◽  
Cellular Prion Protein ◽  
Full Length ◽  
Functional Domain ◽  
Globular Domain ◽  
Channel Activity ◽  
Cellular Membranes

Prion disease is a fatal neurodegenerative disorder, in which the cellular prion protein PrPC is converted to a misfolded prion which in turn is hypothesized to permeabilize cellular membranes. The pathways leading to toxicity in prion disease are not yet completely elucidated and whether it also includes formation of membrane pores remains to be answered. Prion protein consists of two domains: a globular domain (GD) and a flexible N-terminus (FT) domain. Although a proximal nine polybasic amino acid (FT(23-31)) sequence of FT is a prerequisite for cellular membrane permeabilization, other functional domain regions may influence FT(23-31) and its permeabilization. By using single-channel electrical recordings, we reveal that FT(23-50) dominates the membrane permeabilization within the full-length mouse PrP (mPrP(23-230)). The other domain of FT(51-110) or C-terminal domain down-regulates the channel activity of FT(23-50) and the full-length mouse PrP (mPrP(23-230)). The addition of prion mimetic antibody, POM1 significantly enhances mPrP(23-230) membrane permeabilization, whereas POM1-Y104A, a POM1 mutant that binds to PrP but cannot elicit toxicity has negligible effect on membrane permeabilization. Additionally, anti-N-terminal antibody POM2 or Cu2+ stabilizes FT domain, thus provoking FT(23-110) channel activity. Furthermore, our setup provides a more direct method without an external fused protein to study the channel activity of truncated PrP in the lipid membranes. We therefore hypothesize that the primary N-terminal residues are essential for membranes permeabilization and other functional segments play a vital role to modulate the pathological effects of PrP-medicated neurotoxicity. This may yield essential insights into molecular mechanisms of prion neurotoxicity to cellular membranes in prion disease.

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