RESUMO
Single-particle cryo-electron microscopy (cryo-EM) has been shown to be effective in defining the structure of macromolecules, including protein complexes. Complexes adopt different conformations and compositions to perform their biological functions. In cryo-EM, the protein complexes are observed in solution, enabling the recording of images of the protein in multiple conformations. Various methods exist for capturing the conformational variability through analysis of cryo-EM data. Here, we analyzed the conformational variability in the hexameric AAA + ATPase p97, a complex with a six-fold rotational symmetric core surrounded by six flexible N-domains. We compared the performance of discrete classification methods with our recently developed method, MDSPACE, which uses 3D-to-2D flexible fitting of an atomic structure to images based on molecular dynamics (MD) simulations. Our analysis detected a novel conformation adopted by approximately 2% of the particles in the dataset and determined that the N-domains of p97 sway by up to 60° around a central position. This study demonstrates the application of MDSPACE in analyzing the continuous conformational changes in partially symmetrical protein complexes, systems notoriously difficult to analyze due to the alignment errors caused by their partial symmetry.
Assuntos
Adenosina Trifosfatases , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Estrutura Terciária de Proteína , Modelos Moleculares , Microscopia Crioeletrônica/métodos , Adenosina Trifosfatases/metabolismoRESUMO
The Escherichia coli MalE-MalFGK2 complex is one of the best characterized members of the large and ubiquitous family of ATP-binding cassette (ABC) transporters. It is composed of a membrane-spanning heterodimer, MalF-MalG; a homodimeric ATPase, MalK2; and a periplasmic maltose receptor, MalE. Opening and closure of MalK2 is coupled to conformational changes in MalF-MalG and the alternate exposition of the substrate-binding site to either side of the membrane. To further define this alternate access mechanism and the impact of ATP, MalE, and maltose on the conformation of the transporter during the transport cycle, we have reconstituted MalFGK2 in nanodiscs and analyzed its conformations under 10 different biochemical conditions using negative stain single-particle EM. EM map results (at 15-25 Å resolution) indicate that binding of ATP to MalK2 promotes an asymmetric, semi-closed conformation in accordance with the low ATPase activity of MalFGK2 In the presence of MalE, the MalK dimer becomes fully closed, gaining the ability to hydrolyze ATP. In the presence of ADP or maltose, MalE·MalFGK2 remains essentially in a semi-closed symmetric conformation, indicating that release of these ligands is required for the return to the initial state. Taken together, this structural information provides a rationale for the stimulation of MalK ATPase activity by MalE as well as by maltose.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Escherichia coli/química , Microscopia Eletrônica/métodos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligantes , Conformação ProteicaRESUMO
Arp2/3 complex initiates the growth of branched actin-filament networks by inducing actin polymerization from the sides of pre-existing filaments. Nucleation promoting factors (NPFs) are essential for the branching reaction through interactions with the Arp2/3 complex prior to branch formation. The modes by which NPFs bind Arp2/3 complex and associated conformational changes have remained elusive. Here, we used electron microscopy to determine three-dimensional structures at ~2 nm resolution of Arp2/3 complex with three different bound NPFs: N-WASp, Scar-VCA and cortactin. All of these structures adopt a conformation with the two actin-related proteins in an actin-filament-like dimer and the NPF bound to the pointed end. Distance constraints derived by fluorescence resonance energy transfer independently verified the NPF location. Furthermore, all bound NPFs partially occlude the actin-filament binding site, suggesting that additional local structural rearrangements are required in the pathway of Arp2/3 complex activation to allow branch formation.
Assuntos
Proteína 2 Relacionada a Actina/química , Proteína 3 Relacionada a Actina/química , Proteínas Fúngicas/química , Proteínas de Protozoários/química , Família de Proteínas da Síndrome de Wiskott-Aldrich/química , Acanthamoeba , Sítios de Ligação , Microscopia Eletrônica , Modelos Moleculares , Conformação Proteica , SaccharomycetalesRESUMO
Single amino acid mutations in valosin containing protein (VCP/p97), a highly conserved member of the ATPases associated with diverse cellular activities (AAA) family of ATPases has been linked to a severe degenerative disease affecting brain, muscle and bone tissue. Previous studies have demonstrated the role of VCP mutations in altering the ATPase activity of the D2 ring; however the structural consequences of these mutations remain unclear. In this study, we report the three-dimensional (3D) map of the pathogenic VCP variant, R155P, as revealed by single-particle Cryo-Electron Microscopy (EM) analysis at 14 Å resolution. We show that the N-terminal R155P mutation induces a large structural reorganisation of the D2 ATPase ring. Results from docking studies using crystal structure data of available wild-type VCP in the EM density maps indicate that the major difference is localized at the interface between two protomers within the D2 ring. Consistent with a conformational change, the VCP R155P variant shifted the isoelectric point of the protein and reduced its interaction with its well-characterized cofactor, nuclear protein localization-4 (Npl4). Together, our results demonstrate that a single amino acid substitution in the N-terminal domain can relay long-range conformational changes to the distal D2 ATPase ring. Our results provide the first structural clues of how VCP mutations may influence the activity and function of the D2 ATPase ring.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestrutura , Microscopia Crioeletrônica/métodos , Adenosina Trifosfatases/química , Substituição de Aminoácidos , Proteínas de Ciclo Celular/química , Variação Genética/genética , Mutação/genética , Conformação Proteica , Estrutura Terciária de Proteína , Proteína com ValosinaRESUMO
The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Maltose/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas Periplásmicas de Ligação/química , Dobramento de Proteína , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Dicroísmo Circular , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação/genética , Nanotecnologia , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismoRESUMO
Non-homologous end-joining (NHEJ) stands as a pivotal DNA repair pathway crucial for the survival and persistence of Mycobacterium tuberculosis (Mtb) during its dormant, non-replicating phase, a key aspect of its long-term resilience. Mycobacterial NHEJ is a remarkably simple two-component system comprising the rate-limiting DNA binding protein Ku (mKu) and Ligase D. To elucidate mKu's role in NHEJ, we conducted a series of in silico and in vitro experiments. Molecular dynamics simulations and in vitro assays revealed that mKu's DNA binding stabilizes both the protein and DNA, while also shielding DNA ends from exonuclease degradation. Surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA) demonstrated mKu's robust affinity for linear double-stranded DNA (dsDNA), showing positive cooperativity for DNA substrates of 40 base pairs or longer, and its ability to slide along DNA strands. Moreover, analytical ultracentrifugation, size exclusion chromatography, and negative stain electron microscopy (EM) unveiled mKu's unique propensity to form higher-order oligomers exclusively with DNA, suggesting a potential role in mycobacterial NHEJ synapsis. This comprehensive characterization sheds new light on mKu's function within the Mtb NHEJ repair pathway. Targeting this pathway may thus impede the pathogen's ability to persist in its latent state within the host for prolonged periods.
RESUMO
The phenylacetate degradation pathway is present in a wide range of microbes. A key component of this pathway is the four-subunit phenylacetyl-coenzyme A monooxygenase complex (PA-CoA MO, PaaACBE) that catalyzes the insertion of an oxygen in the aromatic ring of PA. This multicomponent enzyme represents a new family of monooxygenases. We have previously determined the structure of the PaaAC subcomplex of catalytic (A) and structural (C) subunits and shown that PaaACB form a stable complex. The PaaB subunit is unrelated to the small subunits of homologous monooxygenases and its role and organization of the PaaACB complex is unknown. From low-resolution crystal structure, electron microscopy and small angle X-ray scattering we show that the PaaACB complex forms heterohexamers, with a homodimer of PaaB bridging two PaaAC heterodimers. Modeling the interactions of reductase subunit PaaE with PaaACB suggested that a unique and conserved 'lysine bridge' constellation near the Fe-binding site in the PaaA subunit (Lys68, Glu49, Glu72 and Asp126) may form part of the electron transfer path from PaaE to the iron center. The crystal structure of the PaaA(K68Q/E49Q)-PaaC is very similar to the wild-type enzyme structure, but when combined with the PaaE subunit the mutant showed 20-50 times reduced activity, supporting the functional importance of the 'lysine bridge'.
Assuntos
Proteínas de Bactérias/química , Klebsiella pneumoniae/enzimologia , Oxigenases de Função Mista/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/ultraestrutura , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Tioléster HidrolasesRESUMO
The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFß and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Células COS , Adesão Celular/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Fibroblastos/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Mutação , Fosfatos de Fosfatidilinositol/genética , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Microbial anaerobic and so-called hybrid pathways for degradation of aromatic compounds contain ß-oxidation-like steps. These reactions convert the product of the opening of the aromatic ring to common metabolites. The hybrid phenylacetate degradation pathway is encoded in Escherichia coli by the paa operon containing genes for 10 enzymes. Previously, we have analyzed protein-protein interactions among the enzymes catalyzing the initial oxidation steps in the paa pathway (Grishin, A. M., Ajamian, E., Tao, L., Zhang, L., Menard, R., and Cygler, M. (2011) J. Biol. Chem. 286, 10735-10743). Here we report characterization of interactions between the remaining enzymes of this pathway and show another stable complex, PaaFG, an enoyl-CoA hydratase and enoyl-Coa isomerase, both belonging to the crotonase superfamily. These steps are biochemically similar to the well studied fatty acid ß-oxidation, which can be catalyzed by individual monofunctional enzymes, multifunctional enzymes comprising several domains, or enzymatic complexes such as the bacterial fatty acid ß-oxidation complex. We have determined the structure of the PaaFG complex and determined that although individually PaaF and PaaG are similar to enzymes from the fatty acid ß-oxidation pathway, the structure of the complex is dissimilar from bacterial fatty acid ß-oxidation complexes. The PaaFG complex has a four-layered structure composed of homotrimeric discs of PaaF and PaaG. The active sites of PaaF and PaaG are adapted to accept the intermediary components of the Paa pathway, different from those of the fatty acid ß-oxidation. The association of PaaF and PaaG into a stable complex might serve to speed up the steps of the pathway following the conversion of phenylacetyl-CoA to a toxic and unstable epoxide-CoA by PaaABCE monooxygenase.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/química , Enoil-CoA Hidratase/química , Proteínas de Escherichia coli/química , Fenilacetatos/química , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Dodecenoil-CoA Isomerase , Enoil-CoA Hidratase/genética , Enoil-CoA Hidratase/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Microscopia Eletrônica , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Óperon/genética , Oxirredução , Fenilacetatos/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Água/química , Água/metabolismoRESUMO
A method for the study of conjugated polyelectrolyte (CPE) photophysics in solution at the single-molecule level is described. Extended observation times of single polymer molecules are enabled by the encapsulation of the CPEs within 200-nm lipid vesicles, which are in turn immobilized on a surface. When combined with a molecular-level visualization of vesicles and CPE via cryo-transmission electron microscopy, these single-molecule spectroscopy studies on CPEs enable us to directly correlate the polymer conformation with its spectroscopic features. These studies are conducted with poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylene-vinylene] (MPS-PPV, a negatively charged CPE), when encapsulated in neutral and in negatively charged lipid vesicles. MPS-PPV exists as a freely diffusing polymer when confined in negatively charged vesicles. Individual MPS-PPV molecules adopt a collapsed-chain conformation leading to efficient energy migration over multiple chromophores. Both the presence of stepwise photobleaching in fluorescence intensity-time trajectories and emission from low-energy chromophores along the chain are observed. These results correlate with the amplified sensing potential reported for MPS-PPV in aqueous solution. When confined within neutral vesicles, single MPS-PPV molecules adopt an extended conformation upon insertion in the lipid bilayer. In this case emission arises from multiple chromophores within the isolated polymer chains, leading to an exponential decay of the intensity over time and a broad blue-shifted emission spectrum.
Assuntos
Eletrólitos/química , Polímeros/química , Polivinil/química , Microscopia Crioeletrônica , Fluorescência , Bicamadas Lipídicas/química , FotodegradaçãoRESUMO
Intracellular magnetite crystal formation by magnetotactic bacteria has emerged as a powerful model for investigating the cellular and molecular mechanisms of biomineralization, a process common to all branches of life. Although magnetotactic bacteria are phylogenetically diverse and their crystals morphologically diverse, studies to date have focused on a few, closely related species with similar crystal habits. Here, we investigate the process of magnetite biomineralization in Desulfovibrio magneticus sp. RS-1, the only reported species of cultured magnetotactic bacteria that is outside of the alpha-Proteobacteria and that forms bullet-shaped crystals. Using a variety of high-resolution imaging and analytical tools, we show that RS-1 cells form amorphous, noncrystalline granules containing iron and phosphorus before forming magnetite crystals. Using NanoSIMS (dynamic secondary ion mass spectroscopy), we show that the iron-phosphorus granules and the magnetite crystals are likely formed through separate cellular processes. Analysis of the cellular ultrastructure of RS-1 using cryo-ultramicrotomy, cryo-electron tomography, and tomography of ultrathin sections reveals that the magnetite crystals are not surrounded by membranes but that the iron-phosphorus granules are surrounded by membranous compartments. The varied cellular paths for the formation of these two minerals lead us to suggest that the iron-phosphorus granules constitute a distinct bacterial organelle.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Desulfovibrio/metabolismo , Ferro/metabolismo , Fósforo/metabolismo , Microscopia Crioeletrônica , Cristalização , Grânulos Citoplasmáticos/química , Desulfovibrio/química , Desulfovibrio/ultraestrutura , Tomografia com Microscopia Eletrônica , Óxido Ferroso-Férrico/química , Magnetossomos/metabolismo , Magnetossomos/ultraestrutura , Microscopia Eletrônica de Transmissão , Minerais/química , Periplasma/metabolismo , Periplasma/ultraestruturaRESUMO
Cryo electron tomography (cryo-ET) allows observing macromolecular complexes in their native environment. The common routine of subtomogram averaging (STA) allows obtaining the three-dimensional (3D) structure of abundant macromolecular complexes, and can be coupled with discrete classification to reveal conformational heterogeneity of the sample. However, the number of complexes extracted from cryo-ET data is usually small, which restricts the discrete-classification results to a small number of enough populated states and, thus, results in a largely incomplete conformational landscape. Alternative approaches are currently being investigated to explore the continuity of the conformational landscapes that in situ cryo-ET studies could provide. In this article, we present MDTOMO, a method for analyzing continuous conformational variability in cryo-ET subtomograms based on Molecular Dynamics (MD) simulations. MDTOMO allows obtaining an atomic-scale model of conformational variability and the corresponding free-energy landscape, from a given set of cryo-ET subtomograms. The article presents the performance of MDTOMO on a synthetic ABC exporter dataset and an in situ SARS-CoV-2 spike dataset. MDTOMO allows analyzing dynamic properties of molecular complexes to understand their biological functions, which could also be useful for structure-based drug discovery.
Assuntos
COVID-19 , Simulação de Dinâmica Molecular , Humanos , Elétrons , SARS-CoV-2 , Descoberta de DrogasRESUMO
p97/VCP, a highly conserved type II ATPase associated with diverse cellular activities (AAA+ ATPase), is an important therapeutic target in the treatment of neurodegenerative diseases and cancer. p97 performs a variety of functions in the cell and facilitates virus replication. It is a mechanochemical enzyme that generates mechanical force from ATP-binding and hydrolysis to perform several functions, including unfolding of protein substrates. Several dozens of cofactors/adaptors interact with p97 and define the multifunctionality of p97. This review presents the current understanding of the molecular mechanism of p97 during the ATPase cycle and its regulation by cofactors and small-molecule inhibitors. We compare detailed structural information obtained in different nucleotide states in the presence and absence of substrates and inhibitors. We also review how pathogenic gain-of-function mutations modify the conformational changes of p97 during the ATPase cycle. Overall, the review highlights how the mechanistic knowledge of p97 helps in designing pathway-specific modulators and inhibitors.
Assuntos
Doenças Transmissíveis , Neoplasias , Humanos , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Nucleotídeos/metabolismo , Adenosina Trifosfatases/metabolismo , Neoplasias/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Proteína com Valosina/genéticaRESUMO
This article presents an original approach for extracting atomic-resolution landscapes of continuous conformational variability of biomolecular complexes from cryo electron microscopy (cryo-EM) single particle images. This approach is based on a new 3D-to-2D flexible fitting method, which uses molecular dynamics (MD) simulation and is embedded in an iterative conformational-landscape refinement scheme. This new approach is referred to as MDSPACE, which stands for Molecular Dynamics simulation for Single Particle Analysis of Continuous Conformational hEterogeneity. The article describes the MDSPACE approach and shows its performance using synthetic and experimental datasets.
Assuntos
Simulação de Dinâmica Molecular , Imagem Individual de Molécula , Microscopia Crioeletrônica/métodos , Conformação ProteicaRESUMO
The multifunctional AAA+ ATPase p97 is an unfoldase/segregase involved in various cellular processes and present in all kingdoms of life. In mammals and yeast, p97 functions upstream of the proteasome. Interestingly, proteasome inhibitors targeting pathogenic microorganisms display efficacy in overcoming drug-resistant strains. Homologues of p97 have been found in disease-causing parasites and mycobacteria. Here, we review the current knowledge on the structure, function, and conservation of p97 in pathogens. We discuss the potential of parasite and mycobacterial p97 as a drug target against these pathogens and explore strategies in designing novel inhibitors. A successful strategy for inhibiting pathogenic p97 should lead to effectively killing the pathogen, minimising toxic and off-target effects, and providing specificity to avoid interfering with human p97.
Assuntos
Parasitos , Tuberculose , ATPases Associadas a Diversas Atividades Celulares , Animais , Humanos , Mamíferos , Parasitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Tuberculose/tratamento farmacológicoRESUMO
BACKGROUND: The AAA + ATPase p97 is an essential unfoldase/segragase involved in a multitude of cellular processes. It functions as a molecular machine critical for protein homeostasis, homotypic membrane fusion events and organelle biogenesis during mitosis in which it acts in concert with cofactors p47 and p37. Cofactors assist p97 in extracting and unfolding protein substrates through ATP hydrolysis. In contrast to other p97's cofactors, p37 uniquely increases the ATPase activity of p97. Disease-causing mutations in p97, including mutations that cause neurodegenerative diseases, increase cofactor association with its N-domain, ATPase activity and improper substrate processing. Upregulation of p97 has also been observed in various cancers. This study aims towards the characterization of the protein-protein interaction between p97 and p37 at the atomic level. We defined the interacting residues in p97 and p37. The knowledge will facilitate the design of unique small molecules inhibiting this interaction with insights into cancer therapy and drug design. RESULTS: The homology model of human p37 UBX domain was built from the X-ray crystal structure of p47 C-terminus from rat (PDB code:1S3S, G) as a template and assessed by model validation analysis. According to the HDOCK, HAWKDOCK, MM-GBSA binding free energy calculations and Arpeggio, we found that there are several hydrophobic and two hydrogen-bonding interactions between p37 UBX and p97 N-D1 domain. Residues of p37 UBX predicted to be involved in the interactions with p97 N-D1 domain interface are highly conserved among UBX cofactors. CONCLUSION: This study provides a reliable structural insight into the p37-p97 complex binding sites at the atomic level though molecular docking coupled with molecular dynamics simulation. This can guide the rational design of small molecule drugs for inhibiting mutant p97 activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Adenosina Trifosfatases , Simulação de Dinâmica Molecular , Proteína com Valosina/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Ratos , Proteína com Valosina/metabolismoRESUMO
Atomic models of cryo electron microscopy (cryo-EM) maps of biomolecular conformations are often obtained by flexible fitting of the maps with available atomic structures of other conformations (e.g., obtained by X-ray crystallography). This article presents a new flexible fitting method, NMMD, which combines normal mode analysis (NMA) and molecular dynamics simulation (MD). Given an atomic structure and a cryo-EM map to fit, NMMD simultaneously estimates global atomic displacements based on NMA and local displacements based on MD. NMMD was implemented by modifying EMfit, a flexible fitting method using MD only, in GENESIS 1.4. As EMfit, NMMD can be run with replica exchange umbrella sampling procedure. The new method was tested using a variety of EM maps (synthetic and experimental, with different noise levels and resolutions). The results of the tests show that adding normal modes to MD-based fitting makes the fitting faster (40% in average) and, in the majority of cases, more accurate.
Assuntos
Simulação de Dinâmica Molecular , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X , Conformação Molecular , Conformação ProteicaRESUMO
Human immunodeficiency virus type 1 (HIV-1) vaccination of cows has elicited broadly neutralizing antibodies (bNAbs). In this study, monoclonal antibodies (mAbs) are isolated from a clade A (KNH1144 and BG505) vaccinated cow using a heterologous clade B antigen (AD8). CD4 binding site (CD4bs) bNAb (MEL-1872) is more potent than a majority of CD4bs bNAbs isolated so far. MEL-1872 mAb with CDRH3 of 57 amino acids shows more potency (geometric mean half-maximal inhibitory concentration [IC50]: 0.009 µg/mL; breadth: 66%) than VRC01 against clade B viruses (29-fold) and than CHO1-31 against tested clade A viruses (21-fold). It also shows more breadth and potency than NC-Cow1, the only other reported anti-HIV-1 bovine bNAb, which has 60% breadth with geometric mean IC50 of 0.090 µg/mL in this study. Using successive different stable-structured SOSIP trimers in bovines can elicit bNAbs focusing on epitopes ubiquitous across subtypes. Furthermore, the cross-clade selection strategy also results in ultra-potent bNAbs.
Assuntos
Infecções por HIV , HIV-1 , Vacinas , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes/química , Sítios de Ligação , Anticorpos Amplamente Neutralizantes , Antígenos CD4 , Bovinos , Feminino , Anticorpos Anti-HIV , Infecções por HIV/prevenção & controle , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
We report the discovery of exceptionally large biogenic magnetite crystals in clay-rich sediments spanning the Paleocene-Eocene Thermal Maximum (PETM) in a borehole at Ancora, NJ. Aside from previously described abundant bacterial magnetofossils, electron microscopy reveals novel spearhead-like and spindle-like magnetite up to 4 microm long and hexaoctahedral prisms up to 1.4 microm long. Similar to magnetite produced by magnetotactic bacteria, these single-crystal particles exhibit chemical composition, lattice perfection, and oxygen isotopes consistent with an aquatic origin. Electron holography indicates single-domain magnetization despite their large crystal size. We suggest that the development of a thick suboxic zone with high iron bioavailability--a product of dramatic changes in weathering and sedimentation patterns driven by severe global warming--drove diversification of magnetite-forming organisms, likely including eukaryotes.
Assuntos
Óxido Ferroso-Férrico/química , Sedimentos Geológicos/química , Silicatos de Alumínio/análise , Argila , Meio Ambiente , Óxido Ferroso-Férrico/metabolismo , Sedimentos Geológicos/microbiologia , História Antiga , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Isótopos de Oxigênio , Fatores de TempoRESUMO
The structure of the TriABC inner membrane component of the triclosan/SDS-specific efflux pump from Pseudomonas aeruginosa was determined by cryoelectron microscopy to 4.5 Å resolution. The complete structure of the inner membrane transporter TriC of the resistance-nodulation-division (RND) superfamily was solved, including a partial structure of the fused periplasmic membrane fusion subunits, TriA and TriB. The substrate-free conformation of TriABC represents an intermediate step in efflux complex assembly before the engagement of the outer membrane channel. Structural analysis identified a tunnel network whose constriction impedes substrate efflux, indicating inhibition of TriABC in the unengaged state. Blind docking studies revealed binding to TriC at the same loci by substrates and bulkier non-substrates. Together with functional analyses, we propose that selective substrate translocation involves conformational gating at the tunnel narrowing that, together with conformational ordering of TriA and TriB, creates an engaged state capable of mediating substrate efflux.