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1.
IUCrJ ; 8(Pt 5): 732-746, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34584735

RESUMO

Many pathogenic gram-negative bacteria have developed mechanisms to increase resistance to cationic antimicrobial peptides by modifying the lipid A moiety. One modification is the addition of phospho-ethano-lamine to lipid A by the enzyme phospho-ethano-lamine transferase (EptA). Previously we reported the structure of EptA from Neisseria, revealing a two-domain architecture consisting of a periplasmic facing soluble domain and a transmembrane domain, linked together by a bridging helix. Here, the conformational flexibility of EptA in different detergent environments is probed by solution scattering and intrinsic fluorescence-quenching studies. The solution scattering studies reveal the enzyme in a more compact state with the two domains positioned close together in an n-do-decyl-ß-d-maltoside micelle environment and an open extended structure in an n-do-decyl-phospho-choline micelle environment. Intrinsic fluorescence quenching studies localize the domain movements to the bridging helix. These results provide important insights into substrate binding and the molecular mechanism of endotoxin modification by EptA.

2.
Nat Commun ; 12(1): 2889, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001871

RESUMO

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Clatrina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endocitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/ultraestrutura , Sítios de Ligação/genética , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Endocitose/genética , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
3.
J Appl Crystallogr ; 54(Pt 1): 169-179, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33833646

RESUMO

Small-angle X-ray scattering (SAXS) is widely utilized to study soluble macromolecules, including those embedded into lipid carriers and delivery systems such as surfactant micelles, phospho-lipid vesicles and bilayered nanodiscs. To adequately describe the scattering from such systems, one needs to account for both the form factor (overall structure) and long-range-order Bragg reflections emerging from the organization of bilayers, which is a non-trivial task. Presently existing methods separate the analysis of lipid mixtures into distinct procedures using form-factor fitting and the fitting of the Bragg peak regions. This article describes a general approach for the computation and analysis of SAXS data from lipid mixtures over the entire angular range of an experiment. The approach allows one to restore the electron density of a lipid bilayer and simultaneously recover the corresponding size distribution and multilamellar organization of the vesicles. The method is implemented in a computer program, LIPMIX, and its performance is demonstrated on an aqueous solution of layered lipid vesicles undergoing an extrusion process. The approach is expected to be useful for the analysis of various types of lipid-based systems, e.g. for the characterization of interactions between target drug molecules and potential carrier/delivery systems.

4.
J Appl Crystallogr ; 54(Pt 1): 343-355, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33833657

RESUMO

The ATSAS software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small-angle scattering data, with a focus on the data measured from biological macromolecules. Here, new developments in the ATSAS 3.0 package are described. They include IMSIM, for simulating isotropic 2D scattering patterns; IMOP, to perform operations on 2D images and masks; DATRESAMPLE, a method for variance estimation of structural invariants through parametric resampling; DATFT, which computes the pair distance distribution function by a direct Fourier transform of the scattering data; PDDFFIT, to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in DATMW for Bayesian consensus-based concentration-independent molecular weight estimation; DATMIF, an ab initio shape analysis method that optimizes the search model directly against the scattering data; DAMEMB, an application to set up the initial search volume for multiphase modelling of membrane proteins; ELLLIP, to perform quasi-atomistic modelling of liposomes with elliptical shapes; NMATOR, which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; DAMMIX, which reconstructs the shape of an unknown intermediate in an evolving system; and LIPMIX and BILMIX, for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the PRIMUS graphical interface to Qt5, updating SASpy - a PyMOL plugin to run a subset of ATSAS tools - to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future ATSAS releases. All these features are implemented in ATSAS 3.0, freely available for academic users at https://www.embl-hamburg.de/biosaxs/software.html.

5.
Commun Biol ; 3(1): 568, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33051581

RESUMO

Gliding, a type of motility based on an actin-myosin motor, is specific to apicomplexan parasites. Myosin A binds two light chains which further interact with glideosome associated proteins and assemble into the glideosome. The role of individual glideosome proteins is unclear due to the lack of structures of larger glideosome assemblies. Here, we investigate the role of essential light chains (ELCs) in Toxoplasma gondii and Plasmodium falciparum and present their crystal structures as part of trimeric sub-complexes. We show that although ELCs bind a conserved MyoA sequence, P. falciparum ELC adopts a distinct structure in the free and MyoA-bound state. We suggest that ELCs enhance MyoA performance by inducing secondary structure in MyoA and thus stiffen its lever arm. Structural and biophysical analysis reveals that calcium binding has no influence on the structure of ELCs. Our work represents a further step towards understanding the mechanism of gliding in Apicomplexa.


Assuntos
Apicomplexa , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Sequência de Aminoácidos , Apicomplexa/metabolismo , Cálcio/química , Cálcio/metabolismo , Sequência Conservada , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Termodinâmica
6.
Sci Rep ; 10(1): 8081, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415234

RESUMO

The application of small angle X-ray scattering (SAXS) to the structural characterization of transmembrane proteins (MPs) in detergent solutions has become a routine procedure at synchrotron BioSAXS beamlines around the world. SAXS provides overall parameters and low resolution shapes of solubilized MPs, but is also meaningfully employed in hybrid modeling procedures that combine scattering data with information provided by high-resolution techniques (eg. macromolecular crystallography, nuclear magnetic resonance and cryo-electron microscopy). Structural modeling of MPs from SAXS data is non-trivial, and the necessary computational procedures require further formalization and facilitation. We propose an automated pipeline integrated with the laboratory-information management system ISPyB, aimed at preliminary SAXS analysis and the first-step reconstruction of MPs in detergent solutions, in order to streamline high-throughput studies, especially at synchrotron beamlines. The pipeline queries an ISPyB database for available a priori information via dedicated services, estimates model-free SAXS parameters and generates preliminary models utilizing either ab initio, high-resolution-based, or mixed/hybrid methods. The results of the automated analysis can be inspected online using the standard ISPyB interface and the estimated modeling parameters may be utilized for further in-depth modeling beyond the pipeline. Examples of the pipeline results for the modelling of the tetrameric alpha-helical membrane channel Aquaporin0 and mechanosensitive channel T2, solubilized by n-Dodecyl ß-D-maltoside are presented. We demonstrate how increasing the amount of a priori information improves model resolution and enables deeper insights into the molecular structure of protein-detergent complexes.


Assuntos
Aquaporinas/química , Detergentes/química , Processamento Eletrônico de Dados/métodos , Proteínas do Olho/química , Processamento de Imagem Assistida por Computador/métodos , Proteínas de Membrana/química , Automação , Humanos , Modelos Moleculares , Conformação Proteica , Espalhamento a Baixo Ângulo
7.
Proc Natl Acad Sci U S A ; 117(8): 4337-4346, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32034094

RESUMO

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.


Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Lipase Lipoproteica/química , Triglicerídeos/sangue , Motivos de Aminoácidos , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Anticorpos Monoclonais/metabolismo , Dimerização , Humanos , Hipertrigliceridemia/enzimologia , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Desdobramento de Proteína , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismo
9.
Sci Rep ; 9(1): 13615, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541188

RESUMO

Human phenylalanine hydroxylase (hPAH) hydroxylates L-phenylalanine (L-Phe) to L-tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. To maintain physiological L-Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. L-Phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH L-Phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of L-Phe. Binding of L-Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Fenilalanina Hidroxilase/ultraestrutura , Domínio Catalítico , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenilalanina/metabolismo , Fenilcetonúrias/genética , Fenilcetonúrias/fisiopatologia , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Raios X
10.
J Biol Chem ; 294(18): 7403-7418, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30894413

RESUMO

The urokinase receptor (uPAR) is a founding member of a small protein family with multiple Ly6/uPAR (LU) domains. The motif defining these LU domains contains five plesiotypic disulfide bonds stabilizing its prototypical three-fingered fold having three protruding loops. Notwithstanding the detailed knowledge on structure-function relationships in uPAR, one puzzling enigma remains unexplored. Why does the first LU domain in uPAR (DI) lack one of its consensus disulfide bonds, when the absence of this particular disulfide bond impairs the correct folding of other single LU domain-containing proteins? Here, using a variety of contemporary biophysical methods, we found that reintroducing the two missing half-cystines in uPAR DI caused the spontaneous formation of the corresponding consensus 7-8 LU domain disulfide bond. Importantly, constraints due to this cross-link impaired (i) the binding of uPAR to its primary ligand urokinase and (ii) the flexible interdomain assembly of the three LU domains in uPAR. We conclude that the evolutionary deletion of this particular disulfide bond in uPAR DI may have enabled the assembly of a high-affinity urokinase-binding cavity involving all three LU domains in uPAR. Of note, an analogous neofunctionalization occurred in snake venom α-neurotoxins upon loss of another pair of the plesiotypic LU domain half-cystines. In summary, elimination of the 7-8 consensus disulfide bond in the first LU domain of uPAR did have significant functional and structural consequences.


Assuntos
Evolução Biológica , Deleção de Sequência , Sulfetos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Fenômenos Biofísicos , Quimotripsina/metabolismo , Glicosilação , Cinética , Ligantes , Dobramento de Proteína , Proteólise , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Ativador de Plasminogênio Tipo Uroquinase/química
11.
Proc Natl Acad Sci U S A ; 116(5): 1723-1732, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30559189

RESUMO

Lipoprotein lipase (LPL) is responsible for the intravascular processing of triglyceride-rich lipoproteins. The LPL within capillaries is bound to GPIHBP1, an endothelial cell protein with a three-fingered LU domain and an N-terminal intrinsically disordered acidic domain. Loss-of-function mutations in LPL or GPIHBP1 cause severe hypertriglyceridemia (chylomicronemia), but structures for LPL and GPIHBP1 have remained elusive. Inspired by our recent discovery that GPIHBP1's acidic domain preserves LPL structure and activity, we crystallized an LPL-GPIHBP1 complex and solved its structure. GPIHBP1's LU domain binds to LPL's C-terminal domain, largely by hydrophobic interactions. Analysis of electrostatic surfaces revealed that LPL contains a large basic patch spanning its N- and C-terminal domains. GPIHBP1's acidic domain was not defined in the electron density map but was positioned to interact with LPL's large basic patch, providing a likely explanation for how GPIHBP1 stabilizes LPL. The LPL-GPIHBP1 structure provides insights into mutations causing chylomicronemia.


Assuntos
Lipase Lipoproteica/metabolismo , Plasma/metabolismo , Receptores de Lipoproteínas/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Animais , Células CHO , Capilares/metabolismo , Linhagem Celular , Cricetulus , Cristalografia por Raios X/métodos , Células Endoteliais/metabolismo , Humanos , Hidrólise , Hipertrigliceridemia/metabolismo
12.
Sci Rep ; 8(1): 4326, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29531348

RESUMO

Human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), essential for DNA integrity, acts as a survival factor for tumor cells and is a target for cancer chemotherapy. Here we report that the Staphylococcal repressor protein StlSaPIBov1 (Stl) forms strong complex with human dUTPase. Functional analysis reveals that this interaction results in significant reduction of both dUTPase enzymatic activity and DNA binding capability of Stl. We conducted structural studies to understand the mechanism of this mutual inhibition. Small-angle X-ray scattering (SAXS) complemented with hydrogen-deuterium exchange mass spectrometry (HDX-MS) data allowed us to obtain 3D structural models comprising a trimeric dUTPase complexed with separate Stl monomers. These models thus reveal that upon dUTPase-Stl complex formation the functional homodimer of Stl repressor dissociates, which abolishes the DNA binding ability of the protein. Active site forming dUTPase segments were directly identified to be involved in the dUTPase-Stl interaction by HDX-MS, explaining the loss of dUTPase activity upon complexation. Our results provide key novel structural insights that pave the way for further applications of the first potent proteinaceous inhibitor of human dUTPase.


Assuntos
Proteínas de Bactérias/metabolismo , Pirofosfatases/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Domínio Catalítico , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Pirofosfatases/química , Proteínas Repressoras/química , Espalhamento a Baixo Ângulo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Difração de Raios X
13.
Neuron ; 97(6): 1261-1267.e4, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29503192

RESUMO

Axon guidance involves the spatiotemporal interplay between guidance cues and membrane-bound cell-surface receptors, present on the growth cone of the axon. Netrin-1 is a prototypical guidance cue that binds to deleted in colorectal cancer (DCC), and it has been proposed that the guidance cue Draxin modulates this interaction. Here, we present structural snapshots of Draxin/DCC and Draxin/Netrin-1 complexes, revealing a triangular relationship that affects Netrin-mediated haptotaxis and fasciculation. Draxin interacts with DCC through the N-terminal four immunoglobulin domains, and Netrin-1 through the EGF-3 domain, in the same region where DCC binds. Netrin-1 and DCC bind to adjacent sites on Draxin, which appears to capture Netrin-1 and tether it to the DCC receptor. We propose the conformational flexibility of the single-pass membrane receptor DCC is used to promote fasciculation and regulate axon guidance through concerted Netrin-1/Draxin binding. VIDEO ABSTRACT.


Assuntos
Orientação de Axônios/fisiologia , Receptor DCC/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Netrina-1/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Receptor DCC/química , Receptor DCC/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Netrina-1/química , Netrina-1/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
Structure ; 26(2): 345-355.e5, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29413323

RESUMO

Saposin-derived lipid nanoparticles (SapNPs) are a new alternative tool for membrane protein reconstitution. Here we demonstrate the potential and advantages of SapNPs. We show that SapA has the lowest lipid specificity for SapNP formation. These nanoparticles are modular and offer a tunable range of size and composition depending on the stoichiometric ratio of lipid and saposin components. They are stable and exhibit features typical of lipid-bilayer systems. Our data suggest that SapNPs are versatile and can adapt to membrane proteins of various sizes and architectures. Using SapA and various types of lipids we could reconstitute membrane proteins of different transmembrane cross-sectional areas (from 14 to 56 transmembrane α helices). SapNP-reconstituted proteins bound their respective ligands and were more heat stable compared with the detergent-solubilized form. Moreover, SapNPs encircle membrane proteins in a compact way, allowing structural investigations of small membrane proteins in a detergent-free environment using small-angle X-ray scattering.


Assuntos
Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Nanopartículas/metabolismo , Saposinas/metabolismo , Modelos Moleculares , Conformação Proteica
15.
Nat Commun ; 9(1): 328, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29362354

RESUMO

In clathrin-mediated endocytosis, adapter proteins assemble together with clathrin through interactions with specific lipids on the plasma membrane. However, the precise mechanism of adapter protein assembly at the cell membrane is still unknown. Here, we show that the membrane-proximal domains ENTH of epsin and ANTH of Sla2 form complexes through phosphatidylinositol 4,5-bisphosphate (PIP2) lipid interfaces. Native mass spectrometry reveals how ENTH and ANTH domains form assemblies by sharing PIP2 molecules. Furthermore, crystal structures of epsin Ent2 ENTH domain from S. cerevisiae in complex with PIP2 and Sla2 ANTH domain from C. thermophilum illustrate how allosteric phospholipid binding occurs. A comparison with human ENTH and ANTH domains reveal only the human ENTH domain can form a stable hexameric core in presence of PIP2, which could explain functional differences between fungal and human epsins. We propose a general phospholipid-driven multifaceted assembly mechanism tolerating different adapter protein compositions to induce endocytosis.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Fúngicas/química , Fosfatidilinositol 4,5-Difosfato/química , Domínios Proteicos , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Membrana Celular/metabolismo , Chaetomium/genética , Chaetomium/metabolismo , Cristalografia por Raios X , Endocitose , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
16.
Arch Biochem Biophys ; 628: 33-41, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28501583

RESUMO

Small-angle X-ray scattering (SAXS) and Nuclear Magnetic Resonance (NMR) are established methods to analyze the structure and structural transitions of biological macromolecules in solution. Both methods are directly applicable to near-native macromolecular solutions and allow one to study structural responses to physical and chemical changes or ligand additions. Whereas SAXS is applied to elucidate overall structure, interactions and flexibility over a wide range of particle sizes, NMR yields atomic resolution detail for moderately sized macromolecules. NMR is arguably the most powerful technique for the experimental analysis of dynamics. The joint application of these two highly complementary techniques provides an extremely useful approach that facilitates comprehensive characterization of biomacromolecular solutions.


Assuntos
Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Estatística como Assunto
17.
Nat Microbiol ; 2: 17047, 2017 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-28394313

RESUMO

Mycobacteria are characterized by their impermeable outer membrane, which is rich in mycolic acids1. To transport substrates across this complex cell envelope, mycobacteria rely on type VII (also known as ESX) secretion systems2. In Mycobacterium tuberculosis, these ESX systems are essential for growth and full virulence and therefore represent an attractive target for anti-tuberculosis drugs3. However, the molecular details underlying type VII secretion are largely unknown, due to a lack of structural information. Here, we report the molecular architecture of the ESX-5 membrane complex from Mycobacterium xenopi determined at 13 Šresolution by electron microscopy. The four core proteins of the ESX-5 complex (EccB5, EccC5, EccD5 and EccE5) assemble with equimolar stoichiometry into an oligomeric assembly that displays six-fold symmetry. This membrane-associated complex seems to be embedded exclusively in the inner membrane, which indicates that additional components are required to translocate substrates across the mycobacterial outer membrane. Furthermore, the extended cytosolic domains of the EccC ATPase, which interact with secretion effectors, are highly flexible, suggesting an as yet unseen mode of substrate interaction. Comparison of our results with known structures of other bacterial secretion systems demonstrates that the architecture of type VII secretion system is fundamentally different, suggesting an alternative secretion mechanism.


Assuntos
Membrana Celular/metabolismo , Mycobacterium tuberculosis/química , Sistemas de Secreção Tipo VII/química , Sistemas de Secreção Tipo VII/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Parede Celular/metabolismo , Tomografia com Microscopia Eletrônica , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Sistemas de Secreção Tipo VII/ultraestrutura
18.
Structure ; 24(6): 851-61, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27133022

RESUMO

The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas Quinases Associadas com Morte Celular/química , Proteínas Quinases Associadas com Morte Celular/metabolismo , Sítios de Ligação , Proteínas Quinases Associadas com Morte Celular/genética , Dimerização , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Transdução de Sinais
19.
Lab Chip ; 16(7): 1161-70, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26931639

RESUMO

We present a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering (SAXS) on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) features automatic generation of 20 different measurement conditions from three input liquids and (2) requires only 2.5 µl of protein solution, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. Total hands on time for preparation of 120 different measurement conditions is less than 5 min. Read-out is performed on disk within the synchrotron beamline P12 at EMBL Hamburg (PETRA III, DESY). We demonstrate: (1) aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirm fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7-4.4% CV of protein concentration). We apply the LabDisk for SAXS for basic analysis methods, such as measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. The suitability of the LabDisk for SAXS for protein structure analysis under different environmental conditions is demonstrated for glucose isomerase under varying protein and NaCl concentrations. We show that the apparent radius of gyration of the negatively charged glucose isomerase decreases with increasing protein concentration at low salt concentration. At high salt concentration the radius of gyration (Rg) does not change with protein concentrations. Such experiments can be performed by a non-expert, since the LabDisk for SAXS does not require attachment of tubings or pumps and can be filled with regular pipettes. The new platform has the potential to introduce routine high-throughput SAXS screening of protein structures with minimal input volumes to the regular operation of synchrotron beamlines.


Assuntos
Técnicas Analíticas Microfluídicas , Proteínas/análise , Proteínas/química , Espalhamento a Baixo Ângulo , Difração de Raios X/instrumentação , Centrifugação , Teoria Quântica
20.
J Biol Chem ; 291(10): 4882-93, 2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26683375

RESUMO

Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections.


Assuntos
Endopeptidases/química , Sequência de Aminoácidos , Bacteriófagos/enzimologia , Bacteriófagos/genética , Clostridium tyrobutyricum/efeitos dos fármacos , Códon de Iniciação , Endopeptidases/genética , Endopeptidases/metabolismo , Endopeptidases/toxicidade , Dados de Sequência Molecular , Mutação , Ligação Proteica , Multimerização Proteica
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