scholarly journals PrPSc-induced conformational changes and strain-specific structures of PrPSc revealed by Disulfide-crosslink scanning

2017 ◽  
Author(s):  
Yuzuru Taguchi ◽  
Noriyuki Nishida ◽  
Hermann S. Schatzl
Keyword(s):  
Conformational Changes ◽  
Genetic Material ◽  
Distal Region ◽  
Prion Strain ◽  
Local Structures ◽  
Prion Strains ◽  
Previous Hypothesis ◽  
Structural Details ◽  
Cysteine Scanning ◽  
Α Helix

ABSTRACTThere exist many phenotypically-varied prion strains, like viruses, despite the absence of conventional genetic material which codes the phenotypic information. As prion is composed solely of the pathological isoform (PrPSc) of prion protein (PrP), the strain-specific traits are hypothesized to be enciphered in the structural details of PrPSc. Identification of the structures of PrPSc is therefore vital for the understanding of prion biology, though they remain unidentified due to the incompatibility of PrPSc with conventional high-resolution structural analyses. Based on our previous hypothesis that the region between the first and the second α-helix (H1∼H2) and the distal region of the third helix (Ctrm) of the cellular isoform of PrP (PrPC) have important roles for efficient interactions with PrPSc, we created series of mutant PrPs with two cysteine substitutions (C;C-PrP) which were systematically designed to form an intramolecular disulfide crosslink between H1∼H2 and Ctrm and assessed their conformational changes by prions: Specifically, a cysteine substitution in H1∼H2 from 165 to 169 was combined with cysteine-scanning along Ctrm from 220 to 229. C;C-PrPs with the crosslinks were expressed normally with the similar glycosylation patterns and subcellular localization as the wild-type PrP albeit with varied expression levels. Interestingly, some of the C;C-PrPs converted to the protease-resistant isoforms in the N2a cells persistently infected with 22L prion strain, whereas the same mutants did not convert in the cells infected with another prion strain Fukuoka1, indicating that local structures of PrPSc in these regions vary among prion strains and contribute to prion-strain diversity. Moreover, patterns of the crosslinks of the convertible C;C-PrPs implied drastic changes in positional relations of H1∼H2 and Ctrm in the PrPSc-induced conformational changes by 22L prion. Thus, disulfide-crosslink scanning is a useful approach for investigation of strain-specific structures of PrPSc, and would be applicable to other types of amyloids as well.

scholarly journals In silico evidence of unique behaviors of methionine in an in-register parallel beta-sheet amyloid, suggestive of its possible contribution to strain diversity of amyloids

2019 ◽  
Author(s):  
Hiroki Otaki ◽  
Yuzuru Taguchi ◽  
Noriyuki Nishida
Keyword(s):  
Md Simulation ◽  
Genetic Material ◽  
Side Chain ◽  
Critical Position ◽  
Local Structures ◽  
Strain Diversity ◽  
Hydrophobic Amino Acids ◽  
Α Helix ◽  
Species Specific ◽  
Β Sheet

AbstractMechanism of strain diversity of prions is a long-standing conundrum, because prions consist solely of abnormal isoform of prion protein (PrPSc) devoid of genetic material. Pathogenic properties of prions are determined by conformations of the constituent PrPScaccording to the protein-only hypothesis, and alterations to even a single residue can drastically change the properties when the residue is located at a critical position for the structure of PrPSc. Interestingly, methionine (Met) is often recognized as the polymorphic or species-specific residues responsible for species/strain barriers of prions, implying its unique influences on the structures of PrPSc. However, how it is unique is difficult to demonstrate due to lack of the detailed structures of PrPSc. Here we analyzed influences of Met substitutions on structures of an in-register parallel β-sheet amyloid of α-synuclein (αSyn) by molecular dynamics (MD) simulation, to extrapolate the results to PrPSc. The MD simulation revealed that Met uniquely stabilized a U-shaped β-arch of the Greek-key αSyn amyloid, whereas other hydrophobic amino acids tended to destabilize it. The stabilizing effect of Met was attributable to the long side chain without Cβ branching. Our findings exemplify specifically how and in what structure of an in-register parallel β-sheet amyloid Met can uniquely behave, and are suggestive of its influences on structures of PrPScand strain diversity of prions. We also discuss about relations between α-helix propensity and local structures of in-register parallel amyloids.

scholarly journals Mechanisms of Strain Diversity of Disease-Associated in-Register Parallel β-Sheet Amyloids and Implications About Prion Strains

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 110 ◽  
Author(s):  
Yuzuru Taguchi ◽  
Hiroki Otaki ◽  
Noriyuki Nishida
Keyword(s):  
Electron Microscopy ◽  
Resonance Spectroscopy ◽  
Post Translational Modifications ◽  
Prion Strain ◽  
Local Structures ◽  
Strain Diversity ◽  
Cryo Electron Microscopy ◽  
Prion Strains ◽  
Dynamics Simulations ◽  
Β Sheet

The mechanism of prion strain diversity remains unsolved. Investigation of inheritance and diversification of protein-based pathogenic information demands the identification of the detailed structures of abnormal isoforms of the prion protein (PrPSc); however, achieving purification is difficult without affecting infectivity. Similar prion-like properties are recognized also in other disease-associated in-register parallel β-sheet amyloids including Tau and α-synuclein (αSyn) amyloids. Investigations into structures of those amyloids via solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy recently made remarkable advances due to their relatively small sizes and lack of post-translational modifications. Herein, we review advances regarding pathogenic amyloids, particularly Tau and αSyn, and discuss implications about strain diversity mechanisms of prion/PrPSc from the perspective that PrPSc is an in-register parallel β-sheet amyloid. Additionally, we present our recent data of molecular dynamics simulations of αSyn amyloid, which suggest significance of compatibility between β-sheet propensities of the substrate and local structures of the template for stability of amyloid structures. Detailed structures of αSyn and Tau amyloids are excellent models of pathogenic amyloids, including PrPSc, to elucidate strain diversity and pathogenic mechanisms.

scholarly journals The key amino acids of E protein involved in early flavivirus infection: viral entry

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Tao Hu ◽  
Zhen Wu ◽  
Shaoxiong Wu ◽  
Shun Chen ◽  
Anchun Cheng
Keyword(s):  
Amino Acids ◽  
Conformational Changes ◽  
Viral Entry ◽  
Envelope Protein ◽  
Cell Attachment ◽  
Envelope Proteins ◽  
Infection Process ◽  
Early Infection ◽  
Protein Amino Acids ◽  
Α Helix

AbstractFlaviviruses are enveloped viruses that infect multiple hosts. Envelope proteins are the outermost proteins in the structure of flaviviruses and mediate viral infection. Studies indicate that flaviviruses mainly use envelope proteins to bind to cell attachment receptors and endocytic receptors for the entry step. Here, we present current findings regarding key envelope protein amino acids that participate in the flavivirus early infection process. Among these sites, most are located in special positions of the protein structure, such as the α-helix in the stem region and the hinge region between domains I and II, motifs that potentially affect the interaction between different domains. Some of these sites are located in positions involved in conformational changes in envelope proteins. In summary, we summarize and discuss the key envelope protein residues that affect the entry process of flaviviruses, including the process of their discovery and the mechanisms that affect early infection.

scholarly journals Reovirus core proteins λ1 and σ2 promote stability of disassembly intermediates and influence early replication events

2020 ◽  
Author(s):  
Stephanie Gummersheimer ◽  
Pranav Danthi
Keyword(s):  
Conformational Changes ◽  
Genetic Material ◽  
Point Mutations ◽  
Host Cells ◽  
Capsid Proteins ◽  
Endosomal Membrane ◽  
The Core ◽  
Core Proteins ◽  
Early Replication ◽  
Outer Capsid

ABSTRACTThe capsids of mammalian reovirus contain two concentric protein shells, the core and the outer capsid. The outer capsid is comprised of µ1-σ3 heterohexamers which surround the core. The core is comprised of λ1 decamers held in place by σ2. After entry into the endosome, σ3 is proteolytically degraded and µ1 is cleaved and exposed to form ISVPs. ISVPs undergo further conformational changes to form ISVP*s, resulting in the release of µ1 peptides which facilitate the penetration of the endosomal membrane to release transcriptionally active core particles into the cytoplasm. Previous work has identified regions or specific residues within reovirus outer capsid that impact the efficiency of cell entry. We examined the functions of the core proteins λ1 and σ2. We generated a reovirus T3D reassortant that carries strain T1L derived σ2 and λ1 proteins (T3D/T1L L3S2). This virus displays a lower ISVP stability and therefore converts to ISVP*s more readily. To identify the basis for lability of T3D/T1L L3S2, we screened for hyper-stable mutants of T3D/T1L L3S2 and identified three point mutations in µ1 that stabilize ISVPs. Two of these mutations are located in the C-terminal ϕ region of µ1, which has not previously been implicated in controlling ISVP stability. Independent from compromised ISVP stability, we also found that T3D/T1L L3S2 launches replication more efficiently and produces higher yields in infected cells. In addition to identifying a new role for the core proteins in disassembly events, these data highlight that core proteins may influence multiple stages of infection.IMPORTANCEProtein shells of viruses (capsids) have evolved to undergo specific changes to ensure the timely delivery of genetic material to host cells. The 2-layer capsid of reovirus provides a model system to study the interactions between capsid proteins and the changes they undergo during entry. We tested a virus in which the core proteins were derived from a different strain than the outer capsid. We found that this mismatched virus was less stable and completed conformational changes required for entry prematurely. Capsid stability was restored by introduction of specific changes to the outer capsid, indicating that an optimal fit between inner and outer shells maintains capsid function. Separate from this property, mismatch between these protein layers also impacted the capacity of virus to initiate infection and produce progeny. This study reveals new insights into the roles of capsid proteins and their multiple functions during viral replication.

scholarly journals Coinfecting Prion Strains Compete for a Limiting Cellular Resource

2010 ◽  
Vol 84 (11) ◽  
pp. 5706-5714 ◽  
Author(s):  
Ronald A. Shikiya ◽  
Jacob I. Ayers ◽  
Charles R. Schutt ◽  
Anthony E. Kincaid ◽  
Jason C. Bartz
Keyword(s):  
Protein Misfolding ◽  
Neuronal Loss ◽  
Infectious Agent ◽  
Prion Strain ◽  
Prion Strains ◽  
The Central Nervous System ◽  
Transmissible Mink Encephalopathy ◽  
Prion Conversion ◽  
Cellular Components

ABSTRACT Prion strain interference can influence the emergence of a dominant strain from a mixture; however, the mechanisms underlying prion strain interference are poorly understood. In our model of strain interference, inoculation of the sciatic nerve with the drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent prior to superinfection with the hyper (HY) strain of TME can completely block HY TME from causing disease. We show here that the deposition of PrPSc, in the absence of neuronal loss or spongiform change, in the central nervous system corresponds with the ability of DY TME to block HY TME infection. This suggests that DY TME agent-induced damage is not responsible for strain interference but rather prions compete for a cellular resource. We show that protein misfolding cyclic amplification (PMCA) of DY and HY TME maintains the strain-specific properties of PrPSc and replicates infectious agent and that DY TME can interfere, or completely block, the emergence of HY TME. DY PrPSc does not convert all of the available PrPC to PrPSc in PMCA, suggesting the mechanism of prion strain interference is due to the sequestering of PrPC and/or other cellular components required for prion conversion. The emergence of HY TME in PMCA was controlled by the initial ratio of the TME agents. A higher ratio of DY to HY TME agent is required for complete blockage of HY TME in PMCA compared to several previous in vivo studies, suggesting that HY TME persists in animals coinfected with the two strains. This was confirmed by PMCA detection of HY PrPSc in animals where DY TME had completely blocked HY TME from causing disease.

Orthorhombic lysozyme crystallization at acidic pH values driven by phosphate binding

2018 ◽  
Vol 74 (5) ◽  
pp. 480-489 ◽  
Author(s):  
Marina Plaza-Garrido ◽  
M. Carmen Salinas-Garcia ◽  
Ana Camara-Artigas
Keyword(s):  
Conformational Changes ◽  
Protein Function ◽  
Protein Crystallization ◽  
Amyloid Formation ◽  
Acidic Ph ◽  
Phosphate Binding ◽  
Cell Parameters ◽  
Ph Values ◽  
Phosphate Ion ◽  
Α Helix

The structure of orthorhombic lysozyme has been obtained at 298 K and pH 4.5 using sodium chloride as the precipitant and in the presence of sodium phosphate at a concentration as low as 5 mM. Crystals belonging to space groupP212121(unit-cell parametersa= 30,b= 56,c= 73 Å, α = β = γ = 90.00°) diffracted to a resolution higher than 1 Å, and the high quality of these crystals permitted the identification of a phosphate ion bound to Arg14 and His15. The binding of this ion produces long-range conformational changes affecting the loop containing Ser60–Asn74. The negatively charged phosphate ion shields the electrostatic repulsion of the positively charged arginine and histidine residues, resulting in higher stability of the phosphate-bound lysozyme. Additionally, a low-humidity orthorhombic variant was obtained at pH 4.5, and comparison with those previously obtained at pH 6.5 and 9.5 shows a 1.5 Å displacement of the fifth α-helix towards the active-site cavity, which might be relevant to protein function. Since lysozyme is broadly used as a model protein in studies related to protein crystallization and amyloid formation, these results indicate that the interaction of some anions must be considered when analysing experiments performed at acidic pH values.

scholarly journals Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Poutoum Samire ◽  
Bastien Serrano ◽  
Denis Duché ◽  
Emeline Lemarié ◽  
Roland Lloubès ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cell Envelope ◽  
Genetic Material ◽  
Phage Infection ◽  
Filamentous Phage ◽  
Cell Physiology ◽  
Inner Membrane ◽  
Existing Structures ◽  
Energy Consuming ◽  
Filamentous Phages

ABSTRACT Filamentous phages are nonlytic viruses that specifically infect bacteria, establishing a persistent association with their host. The phage particle has no machinery for generating energy and parasitizes its host’s existing structures in order to cross the bacterial envelope and deliver its genetic material. The import of filamentous phages across the bacterial periplasmic space requires some of the components of a macrocomplex of the envelope known as the Tol system. This complex uses the energy provided by the proton motive force (pmf) of the inner membrane to perform essential and highly energy-consuming functions of the cell, such as envelope integrity maintenance and cell division. It has been suggested that phages take advantage of pmf-driven conformational changes in the Tol system to transit across the periplasm. However, this hypothesis has not been formally tested. In order to decouple the role of the Tol system in cell physiology and during phage parasitism, we used mutations on conserved essential residues known for inactivating pmf-dependent functions of the Tol system. We identified impaired Tol complexes that remain fully efficient for filamentous phage uptake. We further demonstrate that the TolQ-TolR homologous motor ExbB-ExbD, normally operating with the TonB protein, is able to promote phage infection along with full-length TolA. IMPORTANCE Filamentous phages are widely distributed symbionts of Gram-negative bacteria, with some of them being linked to genome evolution and virulence of their host. However, the precise mechanism that permits their uptake across the cell envelope is poorly understood. The canonical phage model Fd requires the TolQRA protein complex in the host envelope, which is suspected to translocate protons across the inner membrane. In this study, we show that phage uptake proceeds in the presence of the assembled but nonfunctional TolQRA complex. Moreover, our results unravel an alternative route for phage import that relies on the ExbB-ExbD proteins. This work provides new insights into the fundamental mechanisms of phage infection and might be generalized to other filamentous phages responsible for pathogen emergence.

Conformational changes in the third PDZ domain of the neuronal postsynaptic density protein 95

2019 ◽  
Vol 75 (4) ◽  
pp. 381-391 ◽  
Author(s):  
Ana Camara-Artigas ◽  
Javier Murciano-Calles ◽  
Jose C. Martínez
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pdz Domain ◽  
Postsynaptic Density ◽  
Crystal Form ◽  
Space Groups ◽  
The Third ◽  
Conformational Plasticity ◽  
Α Helix ◽  
Postsynaptic Density Protein

PDZ domains are protein–protein recognition modules that interact with other proteins through short sequences at the carboxyl terminus. These domains are structurally characterized by a conserved fold composed of six β-strands and two α-helices. The third PDZ domain of the neuronal postsynaptic density protein 95 has an additional α-helix (α3), the role of which is not well known. In previous structures, a succinimide was identified in the β2–β3 loop instead of Asp332. The presence of this modified residue results in conformational changes in α3. In this work, crystallographic structures of the following have been solved: a truncated form of the third PDZ domain of the neuronal postsynaptic density protein 95 from which α3 has been removed, D332P and D332G variants of the protein, and a new crystal form of this domain showing the binding of Asp332 to the carboxylate-binding site of a symmetry-related molecule. Crystals of the wild type and variants were obtained in different space groups, which reflects the conformational plasticity of the domain. Indeed, the overall analysis of these structures suggests that the conformation of the β2–β3 loop is correlated with the fold acquired by α3. The alternate conformation of the β2–β3 loop affects the electrostatics of the carboxylate-binding site and might modulate the binding of different PDZ-binding motifs.

A Mass Spectrometry Investigation of the Conformational Changes in Adrenocorticotropic Hormones

2002 ◽  
Vol 8 (5) ◽  
pp. 381-387 ◽  
Author(s):  
Hui Lin ◽  
Chhabil Dass
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Aqueous Media ◽  
Charge State Distribution ◽  
Random Coil ◽  
Helical Conformation ◽  
Ionization Mass ◽  
Time Resolved ◽  
Esi Ms ◽  
Α Helix

Electrospray ionization-mass spectrometry (ESI-MS) was employed to study methanol-induced conformational changes in adrenocorticotrophic hormone (ACTH). ACTH, a 39–residue peptide, is a member of the proopiomelanocortin family of peptides. Charge-state distribution (CSD) and hydrogen–deuterium (H/D) exchange were used to monitor the conformational changes as a function of methanol concentration. The latter experiments were conducted via time-resolved ESI-MS in a continuous-flow apparatus. The CSD and the H/D exchange experimental data both reveal that ACTH exists, presumably in a random coil open structure in aqueous media, but assumes a more compact helical conformation with increased concentration of methanol. The H/D exchange experiments also reveal that 79% of ACTH is present as α-helix in mixed water-methanol solvent media.

Activity and conformational changes of horseradish peroxidase in trifluoroethanol

2002 ◽  
Vol 80 (2) ◽  
pp. 205-213 ◽  
Author(s):  
Hong-Wei Zhou ◽  
Yan Xu ◽  
Hai-Meng Zhou
Keyword(s):  
Enzyme Activity ◽  
Horseradish Peroxidase ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Intrinsic Fluorescence ◽  
Two Phase ◽  
Main Effect ◽  
Α Helix ◽  
Kinetics Of

The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5–25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25–40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The α-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.Key words: horseradish peroxidase, trifluoroethanol, unfolding, Soret.

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