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Protein Folding
Molecular genetics: Chaperone protein gets personal
Apr 20, 2017   Nature Add nature.com free-link Cancel
Siegal ML
Nanodiscs for INPHARMA NMR Characterization of GPCRs: Ligand Binding to the Human A2A Adenosine Receptor
Apr 21, 2017   Angewandte Chemie (International Ed. In English)
Fredriksson K, Lottmann P, Hinz S, Onila I, Shymanets A, Harteneck C, Müller CE, Griesinger C, Exner TE
Nanodiscs for INPHARMA NMR Characterization of GPCRs: Ligand Binding to the Human A2A Adenosine Receptor
Apr 21, 2017
Angewandte Chemie (International Ed. In English)
G-protein-coupled-receptors (GPCRs) are of fundamental importance for signal transduction through cell membranes. This makes them important drug targets, but structure-based drug design (SBDD) is still hampered by the limitations for structure determination of unmodified GPCRs. We show that the interligand NOEs for pharmacophore mapping (INPHARMA) method can provide valuable information on ligand poses inside the binding site of the unmodified human A2A adenosine receptor reconstituted in nanodiscs. By comparing experimental INPHARMA spectra with back-calculated spectra based on ligand poses obtained from molecular dynamics simulations, a complex structure for A2A R with the low-affinity ligand 3-pyrrolidin-1-ylquinoxalin-2-amine was determined based on the X-ray structure of ligand ZM-241,358 in complex with a modified A2A R. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Role of the Ion Channel Extracellular Collar in AMPA Receptor Gating
Apr 22, 2017   Scientific Reports
Yelshanskaya MV, Mesbahi-Vasey S, Kurnikova MG, Sobolevsky AI
Role of the Ion Channel Extracellular Collar in AMPA Receptor Gating
Apr 22, 2017
Scientific Reports
AMPA subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission and are implicated in numerous neurological diseases. Ionic currents through AMPA receptor channels can be allosterically regulated via different sites on the receptor protein. We used site-directed mutagenesis and patch-clamp recordings to probe the ion channel extracellular collar, the binding region for noncompetitive allosteric inhibitors. We found position and substitution-dependent effects for introduced mutations at this region on AMPA receptor gating. The results of mutagenesis suggested that the transmembrane domains M1, M3 and M4, which contribute to the ion channel extracellular collar, undergo significant relative displacement during gating. We used molecular dynamics simulations to predict an AMPA receptor open state structure and rationalize the results of mutagenesis. We conclude that the ion channel extracellular collar plays a distinct role in gating and represents a hub for powerful allosteric modulation of AMPA receptor function that can be used for developing novel therapeutics.
Toward a direct and scalable identification of reduced models for categorical processes
Apr 22, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Gerber S, Horenko I
Toward a direct and scalable identification of reduced models for categorical processes
Apr 22, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
The applicability of many computational approaches is dwelling on the identification of reduced models defined on a small set of collective variables (colvars). A methodology for scalable probability-preserving identification of reduced models and colvars directly from the data is derived-not relying on the availability of the full relation matrices at any stage of the resulting algorithm, allowing for a robust quantification of reduced model uncertainty and allowing us to impose a priori available physical information. We show two applications of the methodology: (i) to obtain a reduced dynamical model for a polypeptide dynamics in water and (ii) to identify diagnostic rules from a standard breast cancer dataset. For the first example, we show that the obtained reduced dynamical model can reproduce the full statistics of spatial molecular configurations-opening possibilities for a robust dimension and model reduction in molecular dynamics. For the breast cancer data, this methodology identifies a very simple diagnostics rule-free of any tuning parameters and exhibiting the same performance quality as the state of the art machine-learning applications with multiple tuning parameters reported for this problem.
The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor Turnover
Apr 21, 2017   Cell
Tawo R, Pokrzywa W, Kevei É, Akyuz ME, Balaji V, Adrian S, Höhfeld J, Hoppe T
The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor Turnover
Apr 21, 2017
Cell
Aging is attended by a progressive decline in protein homeostasis (proteostasis), aggravating the risk for protein aggregation diseases. To understand the coordination between proteome imbalance and longevity, we addressed the mechanistic role of the quality-control ubiquitin ligase CHIP, which is a key regulator of proteostasis. We observed that CHIP deficiency leads to increased levels of the insulin receptor (INSR) and reduced lifespan of worms and flies. The membrane-bound INSR regulates the insulin and IGF1 signaling (IIS) pathway and thereby defines metabolism and aging. INSR is a direct target of CHIP, which triggers receptor monoubiquitylation and endocytic-lysosomal turnover to promote longevity. However, upon proteotoxic stress conditions and during aging, CHIP is recruited toward disposal of misfolded proteins, reducing its capacity to degrade the INSR. Our study indicates a competitive relationship between proteostasis and longevity regulation through CHIP-assisted proteolysis, providing a mechanistic concept for understanding the impact of proteome imbalance on aging. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Structural and Functional Analysis of a β2-Adrenergic Receptor Complex with GRK5
Apr 21, 2017   Cell
Komolov KE, Du Y, Duc NM, Betz RM, Rodrigues JPGLM, Leib RD, Patra D, Skiniotis G, Adams CM, Dror RO, Chung KY, Kobilka BK, Benovic JL
Structural and Functional Analysis of a β2-Adrenergic Receptor Complex with GRK5
Apr 21, 2017
Cell
The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β2-adrenergic receptor (β2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the β2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP. Copyright © 2017 Elsevier Inc. All rights reserved.
The burden of trisomy 21 disrupts the proteostasis network in Down syndrome
Apr 21, 2017   PloS One
Aivazidis S, Coughlan CM, Rauniyar AK, Jiang H, Liggett LA, Maclean KN, Roede JR
The burden of trisomy 21 disrupts the proteostasis network in Down syndrome
Apr 21, 2017
PloS One
Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21. Abnormalities in chromosome number have the potential to lead to disruption of the proteostasis network (PN) and accumulation of misfolded proteins. DS individuals suffer from several comorbidities, and we hypothesized that disruption of proteostasis could contribute to the observed pathology and decreased cell viability in DS. Our results confirm the presence of a disrupted PN in DS, as several of its elements, including the unfolded protein response, chaperone system, and proteasomal degradation exhibited significant alterations compared to euploid controls in both cell and mouse models. Additionally, when cell models were treated with compounds that promote disrupted proteostasis, we observed diminished levels of cell viability in DS compared to controls. Collectively our findings provide a cellular-level characterization of PN dysfunction in DS and an improved understanding of the potential pathogenic mechanisms contributing to disrupted cellular physiology in DS. Lastly, this study highlights the future potential of designing therapeutic strategies that mitigate protein quality control dysfunction.
Piecing Together the Allosteric Patterns of Chaperonin GroEL
Apr 21, 2017   The Journal Of Physical Chemistry. B
Chen J, Zhang Q, Ren W, Li W
Piecing Together the Allosteric Patterns of Chaperonin GroEL
Apr 21, 2017
The Journal Of Physical Chemistry. B
Despite considerable effort, elucidating allostery of large macromolecular assemblies at a molecular level in solution remains technically challenging due to its structural complexity. Here we have employed an approach combining amide backbone hydrogen/deuterium exchange coupled with mass spectrometry, fluorescence spectroscopy and molecular simulations to characterize allosteric patterns of chaperonin GroEL, a ~800 kDa tetradecamer from E. coli. Using available crystal structures of GroEL, we quantitatively map out GroEL allosteric changes in solution by resolving exchange behaviors of 133 overlapping proteolytic peptides with more than 95% sequence coverage. This comprehensive analysis gives a refined resolution down to 5 residues to pilot the GroEL allosteric determinants, of which the localized dynamics is monitored by tryptophan-mutated GroEL. Furthermore, the GroEL conformational transition is evaluated by molecular dynamics simulations with an atomic-interaction-based coarse-grained model. Collectively, we provide a practical methodology to analyze GroEL allostery in solution.
Atomistic non-adiabatic dynamics of the LH2 complex with a GPU-accelerated ab initio exciton model
Apr 21, 2017   Physical Chemistry Chemical Physics : PCCP
Sisto A, Stross C, van der Kamp MW, O'Connor M, McIntosh-Smith S, Johnson GT, Hohenstein EG, Manby FR, Glowacki DR, Martinez TJ
Atomistic non-adiabatic dynamics of the LH2 complex with a GPU-accelerated ab initio exciton model
Apr 21, 2017
Physical Chemistry Chemical Physics : PCCP
We recently outlined an efficient multi-tiered parallel ab initio excitonic framework that utilizes time dependent density functional theory (TDDFT) to calculate ground and excited state energies and gradients of large supramolecular complexes in atomistic detail - enabling us to undertake non-adiabatic simulations which explicitly account for the coupled anharmonic vibrational motion of all the constituent atoms in a supramolecular system. Here we apply that framework to the 27 coupled bacterio-chlorophyll-a chromophores which make up the LH2 complex, using it to compute an on-the-fly nonadiabatic surface-hopping (SH) trajectory of electronically excited LH2. Part one of this article is focussed on calibrating our ab initio exciton Hamiltonian using two key parameters: a shift δ, which corrects for the error in TDDFT vertical excitation energies; and an effective dielectric constant ε, which describes the average screening of the transition-dipole coupling between chromophores. Using snapshots obtained from equilibrium molecular dynamics simulations (MD) of LH2, we tune the values of both δ and ε through fitting to the thermally broadened experimental absorption spectrum, giving a linear absorption spectrum that agrees reasonably well with experiment. In part two of this article, we construct a time-resolved picture of the coupled vibrational and excitation energy transfer (EET) dynamics in the sub-picosecond regime following photo-excitation. Assuming Franck-Condon excitation of a narrow eigenstate band centred at 800 nm, we use surface hopping to follow a single nonadiabatic dynamics trajectory within the full eigenstate manifold. Consistent with experimental data, this trajectory gives timescales for B800→B850 population transfer (τB800→B850) between 650-1050 fs, and B800 population decay (τ800→) between 10-50 fs. The dynamical picture that emerges is one of rapidly fluctuating LH2 eigenstates that are delocalized over multiple chromophores and undergo frequent crossing on a femtosecond timescale as a result of the atomic vibrations of the constituent chromophores. The eigenstate fluctuations arise from disorder that is driven by vibrational dynamics with multiple characteristic timescales. The scalability of our ab initio excitonic computational framework across massively parallel architectures opens up the possibility of addressing a wide range of questions, including how specific dynamical motions impact both the pathways and efficiency of electronic energy-transfer within large supramolecular systems.
The HSP90 chaperone machinery
Apr 21, 2017   Nature Reviews. Molecular Cell Biology
Schopf FH, Biebl MM, Buchner J
The HSP90 chaperone machinery
Apr 21, 2017
Nature Reviews. Molecular Cell Biology
The heat shock protein 90 (HSP90) chaperone machinery is a key regulator of proteostasis under both physiological and stress conditions in eukaryotic cells. As HSP90 has several hundred protein substrates (or 'clients'), it is involved in many cellular processes beyond protein folding, which include DNA repair, development, the immune response and neurodegenerative disease. A large number of co-chaperones interact with HSP90 and regulate the ATPase-associated conformational changes of the HSP90 dimer that occur during the processing of clients. Recent progress has allowed the interactions of clients with HSP90 and its co-chaperones to be defined. Owing to the importance of HSP90 in the regulation of many cellular proteins, it has become a promising drug target for the treatment of several diseases, which include cancer and diseases associated with protein misfolding.
Thermal Conductivity Enhancement of Coaxial Carbon@Boron Nitride Nanotube Arrays
Apr 21, 2017   ACS Applied Materials & Interfaces
Jing L, Samani MK, Liu B, Li H, Tay RY, Tsang SH, Cometto O, Nylander A, Liu J, Teo EHT, Tok AIY
Thermal Conductivity Enhancement of Coaxial Carbon@Boron Nitride Nanotube Arrays
Apr 21, 2017
ACS Applied Materials & Interfaces
We demonstrate the thermal conductivity enhancement of the vertically aligned carbon nanotube (CNT) arrays (from ~15.5 to 29.5 W/mK, ~90% increase) by encapsulating outer boron nitride nanotube (BNNT, 0.97 nm-thick with ~3-4 walls). The heat transfer enhancement mechanism of the coaxial C@BNNT was further revealed by molecular dynamics simulations. Owing to their highly coherent lattice structures, the outer BNNT serves as additional heat conducting path without impairing the thermal conductance of inner CNT. This work provides deep insights into tailoring the heat transfer of arbitrary CNT arrays and will enable their broader applications as thermal interface material.
The Dynamics of the Reaction of FeO+ and H2 : A Model for Inorganic Oxidation
Apr 21, 2017   Angewandte Chemie (International Ed. In English)
Essafi S, Tew DP, Harvey JN
The Dynamics of the Reaction of FeO+ and H2 : A Model for Inorganic Oxidation
Apr 21, 2017
Angewandte Chemie (International Ed. In English)
Extensive density functional theory (DFT) calculations using the B3LYP functional were used to explore the sextet and quartet energy potential energy surfaces (PESs) of the title reaction, and as a basis to fit global analytical reactive PESs. Surface-hopping dynamics on these PESs reproduce the experimentally observed reactivity and confirm that hydrogen activation rather than spin-state change is rate-limiting at low reaction energy, where the main products are Fe+ and H2 O. A change in spin state is inefficient in the product region so that excited-state 4 Fe+ is the dominant product. At higher energies, spin-allowed hydrogen atom abstraction to form FeOH+ predominates. At intermediate energy, a previously unexpected rebound mechanism contributes significantly to the reactivity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Aggregation of cyclic polypeptoids bearing zwitterionic end-groups with attractive dipole-dipole and solvophobic interactions: a study by small-angle neutron scattering and molecular dynamics simulation
Apr 21, 2017   Physical Chemistry Chemical Physics : PCCP
Du P, Li A, Li X, Zhang Y, Do C, He L, Rick SW, John VT, Kumar R, Zhang D
Aggregation of cyclic polypeptoids bearing zwitterionic end-groups with attractive dipole-dipole and solvophobic interactions: a study by small-angle neutron scattering and molecular dynamics simulation
Apr 21, 2017
Physical Chemistry Chemical Physics : PCCP
Aggregation behavior of cyclic polypeptoids bearing zwitterionic end-groups in methanol has been studied using a combination of experimental and simulation techniques. The data from SANS and cryo-TEM indicate that the solution contains small clusters of these cyclic polypeptoids, ranging from a single polypeptoid chain to small oligomers, while the linear counterpart shows no cluster formation. Atomistic molecular dynamics simulations reveal that the driving force for this clustering behavior is due to the interplay between the effective repulsion due to the solvation of the dipoles formed by the charged end-groups in each polypeptoid chain and the attractive forces due to dipole-dipole interactions and the solvophobic effect.
Chaperones as Suppressors of Protein Misfolded Oligomer Toxicity
Apr 20, 2017   Frontiers In Molecular Neuroscience
Mannini B, Chiti F
Chaperones as Suppressors of Protein Misfolded Oligomer Toxicity
Apr 20, 2017
Frontiers In Molecular Neuroscience
Chaperones have long been recognized to play well defined functions such as to: (i) assist protein folding and promote formation and maintenance of multisubunit complexes; (ii) mediate protein degradation; (iii) inhibit protein aggregation; and (iv) promote disassembly of undesired aberrant protein aggregates. In addition to these well-established functions, it is increasingly clear that chaperones can also interact with aberrant protein aggregates, such as pre-fibrillar oligomers and fibrils, and inhibit their toxicity commonly associated with neurodegenerative diseases without promoting their disassembly. In particular, the evidence collected so far in different labs, exploiting different experimental approaches and using different chaperones and client aggregated proteins, indicates the existence of two distinct mechanisms of action mediated by the chaperones to neutralize the toxicity of aberrant proteins oligomers: (i) direct binding of the chaperones to the hydrophobic patches exposed on the oligomer/fibril surface, with resulting shielding or masking of the moieties responsible for the aberrant interactions with cellular targets; (ii) chaperone-mediated conversion of aberrant protein aggregates into large and more innocuous species, resulting in a decrease of their surface-to-volume ratio and diffusibility and in deposits more easily manageable by clearance mechanisms, such as autophagy. In this review article we will describe the in vitro and in vivo evidence supporting both mechanisms and how this results in a suppression of the detrimental effects caused by protein misfolded aggregates.
Decreased Levels of Foldase and Chaperone Proteins Are Associated with an Early-Onset Amyotrophic Lateral Sclerosis
Apr 21, 2017   Frontiers In Molecular Neuroscience
Filareti M, Luotti S, Pasetto L, Pignataro M, Paolella K,   . . . . . .   , Chiò A, Corbo M, Bendotti C, Beghi E, Bonetto V
Decreased Levels of Foldase and Chaperone Proteins Are Associated with an Early-Onset Amyotrophic Lateral Sclerosis
Apr 21, 2017
Frontiers In Molecular Neuroscience
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive upper and lower motor neuron degeneration. One of the peculiar clinical characteristics of ALS is the wide distribution in age of onset, which is probably caused by different combinations of intrinsic and exogenous factors. We investigated whether these modifying factors are converging into common pathogenic pathways leading either to an early or a late disease onset. This would imply the identification of phenotypic biomarkers, that can distinguish the two populations of ALS patients, and of relevant pathways to consider in a therapeutic intervention. Toward this aim a differential proteomic analysis was performed in peripheral blood mononuclear cells (PBMC) from a group of 16 ALS patients with an age of onset ≤55 years and a group of 16 ALS patients with an age of onset ≥75 years, and matched healthy controls. We identified 43 differentially expressed proteins in the two groups of patients. Gene ontology analysis revealed that there was a significant enrichment in annotations associated with protein folding and response to stress. We next validated a selected number of proteins belonging to this functional group in 85 patients and 83 age- and sex-matched healthy controls using immunoassays. The results of the validation study confirmed that there was a decreased level of peptidyl-prolyl cis-trans isomerase A (also known as cyclophilin A), heat shock protein HSP 90-alpha, 78 kDa glucose-regulated protein (also known as BiP) and protein deglycase DJ-1 in PBMC of ALS patients with an early onset. Similar results were obtained in PBMC and spinal cord from two SOD1G93A mouse models with an early and late disease onset. This study suggests that a different ability to upregulate proteins involved in proteostasis, such as foldase and chaperone proteins, may be at the basis of a different susceptibility to ALS, putting forward the development of therapeutic approaches aiming at boosting the protein quality control system.
Protein Quality Control by Molecular Chaperones in Neurodegeneration
Apr 21, 2017   Frontiers In Neuroscience
Ciechanover A, Kwon YT
Protein Quality Control by Molecular Chaperones in Neurodegeneration
Apr 21, 2017
Frontiers In Neuroscience
Protein homeostasis (proteostasis) requires the timely degradation of misfolded proteins and their aggregates by protein quality control (PQC), of which molecular chaperones are an essential component. Compared with other cell types, PQC in neurons is particularly challenging because they have a unique cellular structure with long extensions. Making it worse, neurons are postmitotic, i.e., cannot dilute toxic substances by division, and, thus, are highly sensitive to misfolded proteins, especially as they age. Failure in PQC is often associated with neurodegenerative diseases, such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), and prion disease. In fact, many neurodegenerative diseases are considered to be protein misfolding disorders. To prevent the accumulation of disease-causing aggregates, neurons utilize a repertoire of chaperones that recognize misfolded proteins through exposed hydrophobic surfaces and assist their refolding. If such an effort fails, chaperones can facilitate the degradation of terminally misfolded proteins through either the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (hereafter autophagy). If soluble, the substrates associated with chaperones, such as Hsp70, are ubiquitinated by Ub ligases and degraded through the proteasome complex. Some misfolded proteins carrying the KFERQ motif are recognized by the chaperone Hsc70 and delivered to the lysosomal lumen through a process called, chaperone-mediated autophagy (CMA). Aggregation-prone misfolded proteins that remain unprocessed are directed to macroautophagy in which cargoes are collected by adaptors, such as p62/SQSTM-1/Sequestosome-1, and delivered to the autophagosome for lysosomal degradation. The aggregates that have survived all these refolding/degradative processes can still be directly dissolved, i.e., disaggregated by chaperones. Studies have shown that molecular chaperones alleviate the pathogenic symptoms by neurodegeneration-causing protein aggregates. Chaperone-inducing drugs and anti-aggregation drugs are actively exploited for beneficial effects on symptoms of disease. Here, we discuss how chaperones protect misfolded proteins from aggregation and mediate the degradation of terminally misfolded proteins in collaboration with cellular degradative machinery. The topics also include therapeutic approaches to improve the expression and turnover of molecular chaperones and to develop anti-aggregation drugs.
CMA down-regulates p53 expression through degradation of HMGB1 protein to inhibit irradiation-triggered apoptosis in hepatocellular carcinoma
Apr 21, 2017   World Journal Of Gastroenterology
Wu JH, Guo JP, Shi J, Wang H, Li LL, Guo B, Liu DX, Cao Q, Yuan ZY
CMA down-regulates p53 expression through degradation of HMGB1 protein to inhibit irradiation-triggered apoptosis in hepatocellular carcinoma
Apr 21, 2017
World Journal Of Gastroenterology
To investigate the mechanism of chaperone-mediated autophagy (CMA)-induced resistance to irradiation-triggered apoptosis through regulation of the p53 protein in hepatocellular carcinoma (HCC). Firstly, we detected expression of lysosome-associated membrane protein 2a (Lamp-2a), which is the key protein of CMA, by western blot in HepG2 and SMMC7721 cells after irradiation. We further used shRNA Lamp-2a HCC cells to verify the radioresistance induced by CMA. Next, we detected the HMGB1 and p53 expression after irradiation by western blot, and we further used RNA interference and ethyl pyruvate (EP), as a HMGB1 inhibitor, to observe changes of p53 expression. Finally, an immunoprecipitation assay was conducted to explore the interaction between Lamp-2a and HMGB1, and the data were analyzed. We found the expression of Lamp-2a was increased on irradiation while apoptosis decreased in HepG2 and SMMC7721 cells. The apoptosis was increased markedly in the shRNA Lamp-2a HepG2 and SMMC7721 cells as detected by western blot and colony formation assay. Next, we found p53 expression was gradually reduced on irradiation but obviously increased in shRNA Lamp-2a cells. Furthermore, p53 increased the cell apoptosis on irradiation in Hep3B (p53-/-) cells. Finally, p53 levels were regulated by HMGB1 as measured through RNA interference and the EP treatment. HMGB1 was able to combine with Lamp-2a as seen by immunoprecipitation assay and was degraded via the CMA pathway. The decreased HMGB1 inhibited p53 expression induced by irradiation and further reduced the apoptosis in HCC cells. CMA pathway activation appears to down-regulate the susceptibility of HCC to irradiation by degrading HMGB1 with further impact on p53 expression. These findings have clinical relevance for radiotherapy of HCC.
Quantitative 3D evolution of colloidal nanoparticle oxidation in solution
Apr 21, 2017   Science (New York, N.Y.)
Sun Y, Zuo X, Sankaranarayanan SKRS, Peng S, Narayanan B, Kamath G
Quantitative 3D evolution of colloidal nanoparticle oxidation in solution
Apr 21, 2017
Science (New York, N.Y.)
Real-time tracking of the three-dimensional (3D) evolution of colloidal nanoparticles in solution is essential for understanding complex mechanisms involved in nanoparticle growth and transformation. We used time-resolved small-angle and wide-angle x-ray scattering simultaneously to monitor oxidation of highly uniform colloidal iron nanoparticles, enabling the reconstruction of intermediate 3D morphologies of the nanoparticles with a spatial resolution of ~5 angstroms. The in situ observations, combined with large-scale reactive molecular dynamics simulations, reveal the details of the transformation from solid metal nanoparticles to hollow metal oxide nanoshells via a nanoscale Kirkendall process-for example, coalescence of voids as they grow and reversal of mass diffusion direction depending on crystallinity. Our results highlight the complex interplay between defect chemistry and defect dynamics in determining nanoparticle transformation and formation. Copyright © 2017, American Association for the Advancement of Science.
Key features of an Hsp70 chaperone allosteric landscape revealed by ion mobility native mass spectrometry and double electron-electron resonance
Apr 21, 2017   The Journal Of Biological Chemistry
Lai AL, Clerico EM, Blackburn ME, Patel NA, Robinson CV, Borbat PP, Freed JH, Gierasch LM
Key features of an Hsp70 chaperone allosteric landscape revealed by ion mobility native mass spectrometry and double electron-electron resonance
Apr 21, 2017
The Journal Of Biological Chemistry
Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains (viz, the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates. However, it remains unclear how the Hsp70 conformational ensemble is populated at each point of the allosteric cycle and how ligands control these populations. We have mapped the conformational species present under different ligand-binding conditions throughout the allosteric cycle of the E. coli Hsp70 DnaK by two complementary methods, ion mobility mass spectrometry and double electron-electron resonance. Our results obtained under biologically relevant ligand-bound conditions confirm the current picture derived from NMR and crystallographic data of domain docking upon ATP binding and undocking in response to ADP and substrate. Additionally, we find that the helical lid of DnaK is a highly dynamic unit of the structure in all ligand-bound states. Importantly, we demonstrate that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnaK allosteric landscape. Because Hsp70s are emerging as potential drug targets for many diseases, fully mapping an allosteric landscape of a molecular chaperone like DnaK will facilitate the development of small molecules that modulate Hsp70 function via allosteric mechanisms. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.
Polar N-terminal residues conserved in type 2 secretion pseudopilins determine subunit targeting and membrane extraction steps during fibre assembly
Apr 21, 2017   Journal Of Molecular Biology
Santos-Moreno J, East A, Guilvout I, Nadeau N, Bond PJ, Van Nhieu GT, Francetic O
Polar N-terminal residues conserved in type 2 secretion pseudopilins determine subunit targeting and membrane extraction steps during fibre assembly
Apr 21, 2017
Journal Of Molecular Biology
Bacterial type 2 secretion systems (T2SS), type 4 pili (T4P) and archaeal flagella assemble fibres from initially membrane-embedded pseudopilin and pilin subunits. Fibre subunits are made as precursors with positively charged N-terminal anchors, whose cleavage via the prepilin peptidase, essential for pilin membrane extraction and assembly, is followed by N-methylation of the mature (pseudo)pilin N-terminus. The conserved Glu residue at position 5 (E5) of mature (pseudo)pilins is essential for assembly. Unlike T4 pilins, where E5 residue substitutions also abolish N-methylation, the E5A variant of T2SS pseudopilin PulG remains N-methylated, but is affected in interaction with the T2SS component PulM. Here, biochemical and functional analyses showed that the PulM interaction defect only partly accounts for the PulGE5A assembly defect. First, PulGT2A variant, equally defective in PulM interaction, remained partially functional. Furthermore, pseudopilus assembly defect of pulG(E5A) mutant was stronger than that of the pulM deletion mutant. To understand the dominant effect of E5A mutation, we used molecular dynamics simulations of PulGE5A, methylated PulGWT (MePulGWT) and MePulGE5A variant in a model membrane. These simulations pointed to a key role for an intra-molecular interaction between the pseudopilin N-terminal amine and E5 to limit polar interactions with membrane phospholipids. N-methylation of the N-terminal amine further limited it interactions with phospholipid head-groups to facilitate pseudopilin membrane escape. By binding to polar residues in the conserved N-terminal region of PulG, we propose that PulM acts as chaperone to promote pseudopilin recruitment and coordinate its membrane extraction with subsequent steps of the fibre assembly process. Copyright © 2017. Published by Elsevier Ltd.
Design and synthesis of neolamellarin a derivatives targeting heat shock protein 90
Apr 20, 2017   European Journal Of Medicinal Chemistry
Jiang L, Yin R, Wang X, Dai J, Li J, Jiang T, Yu R
Design and synthesis of neolamellarin a derivatives targeting heat shock protein 90
Apr 20, 2017
European Journal Of Medicinal Chemistry
In this study, we designed and synthesized a novel family of neolamellarin A derivatives that showed high inhibitory activity toward heat shock protein 90 (Hsp90), a kinase associated with cell proliferation. The 3,4-bis(catechol)pyrrole scaffold and the benzyl group with methoxy modification at N position of pyrrole are essential to the Hsp90 inhibitory activity and cytotoxicity of these compounds. Western blot analysis demonstrated that these compounds induced dramatic depletion of the examined client proteins of Hsp90, and accelerated cancer cell apoptosis. Docking simulations suggested that the binding mode of 9p was similar to that of the VER49009, a potent inhibitor of Hsp90. Further molecular dynamics simulation indicated that the hydrophobic interactions as well as the hydrogen bonds contributed to the high affinity of 9p to Hsp90. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
The influence of cell membrane and SNAP25 linker loop on the dynamics and unzipping of SNARE complex
Apr 20, 2017   PloS One
Shi Y, Zhang Y, Lou J
The influence of cell membrane and SNAP25 linker loop on the dynamics and unzipping of SNARE complex
Apr 20, 2017
PloS One
The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is composed of three neuronal proteins VAMP2, Syntaxin and SNAP25, which plays a core role during the process of membrane fusion. The zipping assembly of the SNARE complex releases energies and drives the vesicle and cell membrane into close proximity. In this study, we use all-atom molecular dynamics simulations to probe the dynamics of SNARE and its unzipping process in the context of membrane at the atomistic details. Our results indicated that the NTD of SNARE core domain is relatively more stable than CTD, which is in agreement with previous experiments. More importantly, possible interactions between the linker loop (LL) region of SNAP25 and VAMP2 are observed, suggests that the LL region may facilitate VAMP2 binding and SNARE initiation. The forced unzipping of SNARE in the presence of membrane and LL of SNAP25 reveals the possible pathway for energy generation of SNARE zipping, provides information to understand how force may regulate the cooperativity between the membrane and the SNARE complex.
Cheminformatics-aided discovery of small-molecule Protein-Protein Interaction (PPI) dual inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL)
Apr 20, 2017   PLoS Computational Biology
Melagraki G, Ntougkos E, Rinotas V, Papaneophytou C, Leonis G, Mavromoustakos T, Kontopidis G, Douni E, Afantitis A, Kollias G
Cheminformatics-aided discovery of small-molecule Protein-Protein Interaction (PPI) dual inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL)
Apr 20, 2017
PLoS Computational Biology
We present an in silico drug discovery pipeline developed and applied for the identification and virtual screening of small-molecule Protein-Protein Interaction (PPI) compounds that act as dual inhibitors of TNF and RANKL through the trimerization interface. The cheminformatics part of the pipeline was developed by combining structure-based with ligand-based modeling using the largest available set of known TNF inhibitors in the literature (2481 small molecules). To facilitate virtual screening, the consensus predictive model was made freely available at: http://enalos.insilicotox.com/TNFPubChem/. We thus generated a priority list of nine small molecules as candidates for direct TNF function inhibition. In vitro evaluation of these compounds led to the selection of two small molecules that act as potent direct inhibitors of TNF function, with IC50 values comparable to those of a previously-described direct inhibitor (SPD304), but with significantly reduced toxicity. These molecules were also identified as RANKL inhibitors and validated in vitro with respect to this second functionality. Direct binding of the two compounds was confirmed both for TNF and RANKL, as well as their ability to inhibit the biologically-active trimer forms. Molecular dynamics calculations were also carried out for the two small molecules in each protein to offer additional insight into the interactions that govern TNF and RANKL complex formation. To our knowledge, these compounds, namely T8 and T23, constitute the second and third published examples of dual small-molecule direct function inhibitors of TNF and RANKL, and could serve as lead compounds for the development of novel treatments for inflammatory and autoimmune diseases.
Tyrosine Kinase Activation and Conformational Flexibility: Lessons from Src-Family Tyrosine Kinases
Apr 20, 2017   Accounts Of Chemical Research
Meng Y, Pond MP, Roux B
Tyrosine Kinase Activation and Conformational Flexibility: Lessons from Src-Family Tyrosine Kinases
Apr 20, 2017
Accounts Of Chemical Research
Protein kinases are enzymes that catalyze the covalent transfer of the γ-phosphate of an adenosine triphosphate (ATP) molecule onto a tyrosine, serine, threonine, or histidine residue in the substrate and thus send a chemical signal to networks of downstream proteins. They are important cellular signaling enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Unregulated protein kinase activity is often associated with a wide range of diseases, therefore making protein kinases major therapeutic targets. A prototypical system of central interest to understand the regulation of kinase activity is provided by tyrosine kinase c-Src, which belongs to the family of Src-related non-receptor tyrosine kinases (SFKs). Although the broad picture of autoinhibition via the regulatory domains and via the phosphorylation of the C-terminal tail is well characterized from a structural point of view, a detailed mechanistic understanding at the atomic-level is lacking. Advanced computational methods based on all-atom molecular dynamics (MD) simulations are employed to advance our understanding of tyrosine kinase activation. The computational studies suggest that the isolated kinase domain (KD) is energetically most favorable in the inactive conformation when the activation loop (A-loop) of the KD is not phosphorylated. The KD makes transient visits to a catalytically competent active-like conformation. The process of bimolecular trans-autophosphorylation of the A-loop eventually locks the KD in the active state. Activating point mutations may act by slightly increasing the population of the active-like conformation, enhancing the availability of the A-loop to be phosphorylated. The Src-homology 2 (SH2) and Src-homology 3 (SH3) regulatory domains, depending upon their configuration, either promote the inactive or the active state of the kinase domain. In addition to the roles played by the SH3, SH2, and KD, the Src-homology 4-Unique domain (SH4-U) region also serves as a key moderator of substrate specificity and kinase function. Thus, a fundamental understanding of the conformational propensity of the SH4-U region and how this affects the association to the membrane surface are likely to lead to the discovery of new intermediate states and alternate strategies for inhibition of kinase activity for drug discovery. The existence of a multitude of KD conformations poses a great challenge aimed at the design of specific inhibitors. One promising computational strategy to explore the conformational flexibility of the KD is to construct Markov state models from aggregated MD data.
Probing protein flexibility reveals a mechanism for selective promiscuity
Apr 22, 2017   ELife
Pabon NA, Camacho CJ
Probing protein flexibility reveals a mechanism for selective promiscuity
Apr 22, 2017
ELife
Many eukaryotic regulatory proteins adopt distinct bound and unbound conformations, and use this structural flexibility to bind specifically to multiple partners. However, we lack an understanding of how an interface can select some ligands, but not others. Here, we present a molecular dynamics approach to identify and quantitatively evaluate the interactions responsible for this selective promiscuity. We apply this approach to the anti-cancer target PD-1 and its ligands PD-L1 and PD-L2. We discover that while unbound PD-1 exhibits a hard-to-drug hydrophilic interface, conserved specific triggers encoded in the cognate ligands activate a promiscuous binding pathway that reveals a flexible hydrophobic binding cavity. Specificity is then established by additional contacts that stabilize the PD-1 cavity into distinct bound-like modes. Collectively, our studies provide insight into the structural basis and evolution of multiple binding partners, and also suggest a biophysical approach to exploit innate binding pathways to drug seemingly undruggable targets.

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