RESUMEN

Imidazole is largely employed in recombinant protein purification, including GH1 ß-glucosidases, but its effect on the enzyme activity is rarely taken into consideration. Computational docking suggested that imidazole interacts with residues forming the active site of the GH1 ß-glucosidase from Spodoptera frugiperda (Sfßgly). We confirmed this interaction by showing that imidazole reduces the activity of Sfßgly, which does not result from enzyme covalent modification or promotion of transglycosylation reactions. Instead, this inhibition occurs through a partial competitive mechanism. Imidazole binds to the Sfßgly active site, reducing the substrate affinity by about threefold, whereas the rate constant of product formation remains unchanged. The binding of imidazole within the active site was further confirmed by enzyme kinetic experiments in which imidazole and cellobiose competed to inhibit the hydrolysis of p-nitrophenyl ß-glucoside. Finally, imidazole interaction in the active site was also demonstrated by showing that it hinders access of carbodiimide to the Sfßgly catalytic residues, protecting them from chemical inactivation. In conclusion, imidazole binds in the Sfßgly active site, generating a partial competitive inhibition. Considering that GH1 ß-glucosidases share conserved active sites, this inhibition phenomenon is probably widespread among these enzymes, and this should be taken into account when considering the characterization of their recombinant forms.

Asunto(s)

Glucósidos , beta-Glucosidasa , beta-Glucosidasa/química , beta-Glucosidasa/metabolismo , Dominio Catalítico , Hidrólisis , Imidazoles/farmacología

RESUMEN

Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effector proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl-terminal domain of ∼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translocation. However, the structural basis of the XVIPCD-VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.

Asunto(s)

Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Escherichia coli/química , Sistemas de Secreción Tipo IV/antagonistas & inhibidores , Sistemas de Secreción Tipo IV/química , Xanthomonas/química , Proteínas Bacterianas/genética , Escherichia coli/genética , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Relación Estructura-Actividad , Sistemas de Secreción Tipo IV/genética , Xanthomonas/genética

RESUMEN

Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Fimbrias Bacterianas/metabolismo , Oxidorreductasas/metabolismo , Xanthomonas/metabolismo , Cristalografía por Rayos X , Oxidorreductasas/química , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Virulencia , Xanthomonas/crecimiento & desarrollo

RESUMEN

Na+/Ca2+ exchangers (NCXs) are secondary active transporters that couple the translocation of Na+ with the transport of Ca2+ in the opposite direction. The exchanger is an essential Ca2+ extrusion mechanism in excitable cells. It consists of a transmembrane domain and a large intracellular loop that contains two Ca2+-binding domains, CBD1 and CBD2. The two CBDs are adjacent to each other and form a two-domain Ca2+ sensor called CBD12. Binding of intracellular Ca2+ to CBD12 activates the NCX but inhibits the NCX of Drosophila, CALX. NMR spectroscopy and SAXS studies showed that CALX and NCX CBD12 constructs display significant interdomain flexibility in the apo state but assume rigid interdomain arrangements in the Ca2+-bound state. However, detailed structure information on CBD12 in the apo state is missing. Structural characterization of proteins formed by two or more domains connected by flexible linkers is notoriously challenging and requires the combination of orthogonal information from multiple sources. As an attempt to characterize the conformational ensemble of CALX-CBD12 in the apo state, we applied molecular dynamics (MD) simulations, NMR (1H-15N residual dipolar couplings), and small-angle x-ray scattering (SAXS) data in a combined strategy to select an ensemble of conformations in agreement with the experimental data. This joint approach demonstrated that CALX-CBD12 preferentially samples closed conformations, whereas the wide-open interdomain arrangement characteristic of the Ca2+-bound state is less frequently sampled. These results are consistent with the view that Ca2+ binding shifts the CBD12 conformational ensemble toward extended conformers, which could be a key step in the NCXs' allosteric regulation mechanism. This strategy, combining MD with NMR and SAXS, provides a powerful approach to select ensembles of conformations that could be applied to other flexible multidomain systems.

Asunto(s)

Calcio , Simulación de Dinámica Molecular , Calcio/metabolismo , Conformación Proteica , Dispersión del Ángulo Pequeño , Intercambiador de Sodio-Calcio/metabolismo , Difracción de Rayos X

RESUMEN

The Na+/Ca2+ exchanger of Drosophila melanogaster, CALX, is the main Ca2+-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+ exchangers have two Ca2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologs, CALX is inhibited by Ca2+ binding to CALX-CBD1, whereas CALX-CBD2 does not bind Ca2+ at physiological concentrations. CALX-CBD1 consists of a ß-sandwich and displays four Ca2+-binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2+ binding to CALX-CBD1. We observed that this domain binds Ca2+ in the slow exchange regime at the NMR chemical shift timescale. Ca2+ binding restricts the dynamics in the Ca2+-binding region. Experiments of 15N chemical exchange saturation transfer and 15N R2 dispersion allowed the determination of Ca2+ dissociation rates (∼30 s-1). NMR titration curves of residues in the Ca2+-binding region were sigmoidal because of the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel, to our knowledge, approach to analyze NMR titration curves was proposed. Ca2+-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill ∼2.9, near maximal cooperativity (nHill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+ is approximately three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+ binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+ concentration in sensory neuronal cells.

Asunto(s)

Proteínas de Drosophila , Drosophila melanogaster , Animales , Antiportadores/metabolismo , Teorema de Bayes , Sitios de Unión , Calcio/metabolismo , Calorimetría , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Espectroscopía de Resonancia Magnética , Unión Proteica , Intercambiador de Sodio-Calcio/metabolismo

RESUMEN

Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I-IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitors.

RESUMEN

Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 Å-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein-protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution.

Asunto(s)

Bacterias/metabolismo , Proteínas Bacterianas/química , Microscopía por Crioelectrón/métodos , Complejos Multiproteicos/química , Sistemas de Secreción Tipo IV/química , Xanthomonas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Complejos Multiproteicos/genética , Mutación , Unión Proteica , Conformación Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Sistemas de Secreción Tipo IV/genética , Xanthomonas/genética

RESUMEN

Cathepsins L are the major digestive peptidases in the beetle Tenebrio molitor. Two digestive cathepsins L (TmCAL2 and TmCAL3) from it had their 3D structures solved. The aim of this paper was to study in details TmCAL3 specificity and properties and relate them to its 3D structure. Recombinant TmCAL3 was assayed with 64 oligopeptides with different amino acid replacements in positions P2, P1, P1' and P2'. Results showed that TmCAL3 S2 specificity differs from the human enzyme and that its specificities also explain why on autoactivation two propeptide residues remain in the enzyme. Data on free energy of binding and of activation showed that S1 and S2' are mainly involved in substrate binding, S1' acts in substrate binding and catalysis, whereas S2 is implied mainly in catalysis. Enzyme subsite residues were identified by docking with the same oligopeptide used for kinetics. The subsite hydrophobicities were calculated from the efficiency of hydrolysis of different amino acid replacements in the peptide and from docking data. The results were closer for S1 and S2' than for S1' and S2, indicating that the residue subsites that were more involved in transition state binding are different from those binding the substrate seen in docking. Besides TmCAL1-3, there are nine other cathepsins L, most of them more expressed at midgut. They are supposed to be directed to lysosomes by a Drosophila-like Lerp receptor and/or motifs in their prodomains. The mannose 6-phosphate lysosomal sorting machinery is absent from T. molitor transcriptome. Cathepsin L direction to midgut contents seems to depend on overexpression.

Asunto(s)

Catepsina L/metabolismo , Tenebrio/enzimología , Secuencia de Aminoácidos , Animales , Catálisis , Catepsina L/química , Tracto Gastrointestinal/enzimología , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Lisosomas/enzimología , Simulación del Acoplamiento Molecular , Señales de Clasificación de Proteína , Especificidad por Sustrato

RESUMEN

Cdc25B phosphatases are involved in cell cycle checkpoints and have become a possible target for developing new anticancer drugs. A more rational design of Cdc25B ligands would benefit from detailed knowledge of its tertiary structure. The conformational flexibility of the C-terminal region of the Cdc25B catalytic domain has been debated recently and suggested to play an important structural role. Here, a combination of experimental NMR measurements and molecular dynamics simulations for the complete catalytic domain of the Cdc25B phosphatase is presented. The stability of the C-terminal α-helix is confirmed, but the last 20 residues in the complete catalytic domain are very flexible, partially occlude the active site and may establish transient contacts with the protein core. This flexibility in the C-terminal tail may modulate the molecular recognition of natural substrates and competitive inhibitors by Cdc25B. Proteins 2016; 84:1567-1575. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Proteínas Recombinantes de Fusión/química , Fosfatasas cdc25/química , Secuencias de Aminoácidos , Dominio Catalítico , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Docilidad , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismo

RESUMEN

The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca(2+) concentrations. In Canis familiaris, Na(+) /Ca(2+) exchanger (NCX) activity is regulated by the binding of Ca(2+) to two cytosolic Ca(2+) -binding domains, CBD1 and CBD2, such that Ca(2+) -binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca(2+) -regulation remains unclear. It was found previously that for NCX in the absence of Ca(2+) the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca(2+) -binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca(2+) -binding to CBD1 inhibits Ca(2+) exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca(2+) modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca(2+) regulation of the Na(+) /Ca(2+) exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca(2+) -binding to CBD1 controlling ion transport across the plasma membrane.

Asunto(s)

Calcio/metabolismo , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Perros , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica

RESUMEN

Type IV secretion systems (T4SSs) are multiprotein complexes that transport effector proteins and protein-DNA complexes through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs. Here we show that the Xanthomonas citri T4SS provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. The secretion of one type IV bacterial effector protein is shown to require a conserved C-terminal domain and its bacteriolytic activity is neutralized by a cognate immunity protein whose 3D structure is similar to peptidoglycan hydrolase inhibitors. This is the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as a mediator of both antagonistic and cooperative interbacterial interactions.

Asunto(s)

Antibiosis/fisiología , Proteínas Bacterianas/metabolismo , Bacteriólisis/fisiología , Modelos Moleculares , Sistemas de Secreción Tipo IV/metabolismo , Xanthomonas/fisiología , Proteínas Bacterianas/inmunología , Clonación Molecular , Cristalización , Escherichia coli , Immunoblotting , Inmunoprecipitación , Microscopía Fluorescente , Conformación Proteica , Dispersión del Ángulo Pequeño , Sistemas de Secreción Tipo IV/química , Difracción de Rayos X , Xanthomonas/metabolismo

RESUMEN

Specific ion effects in surfactant solutions affect the properties of micelles. Dodecyltrimethylammonium chloride (DTAC), bromide (DTAB), and methanesulfonate (DTAMs) micelles are typically spherical, but some organic anions can induce shape or phase transitions in DTA(+) micelles. Above a defined concentration, sodium triflate (NaTf) induces a phase separation in dodecyltrimethylammonium triflate (DTATf) micelles, a phenomenon rarely observed in cationic micelles. This unexpected behavior of the DTATf/NaTf system suggests that DTATf aggregates have unusual properties. The structural properties of DTATf micelles were analyzed by time-resolved fluorescence quenching, small-angle X-ray scattering, nuclear magnetic resonance, and electron paramagnetic resonance and compared with those of DTAC, DTAB, and DTAMs micelles. Compared to the other micelle types, the DTATf micelles had a higher average number of monomers per aggregate, an uncommon disk-like shape, smaller interfacial hydration, and restricted monomer chain mobility. Molecular dynamic simulations supported these observations. Even small water-soluble salts can profoundly affect micellar properties; our data demonstrate that the -CF3 group in Tf(-) was directly responsible for the observed shape changes by decreasing interfacial hydration and increasing the degree of order of the surfactant chains in the DTATf micelles.

Asunto(s)

Mesilatos/química , Micelas , Compuestos de Amonio Cuaternario/química , Cationes/química , Modelos Moleculares , Simulación de Dinámica Molecular , Tensoactivos/química

RESUMEN

The Na(+)/Ca(2+) exchanger (NCX) is a membrane protein, which catalyzes the counter transport of Na(+) and Ca(2+) ions across the plasma membrane, playing a key role in the maintenance of the intracellular Ca(2+) homeostasis in various cell types. NCX consists of a transmembrane part and a large intracellular loop. The activation of the NCX transport function requires the binding of Ca(2+) to two tandem C2 domains, CBD1 and CBD2, which are an integral part of the exchanger's intracellular loop. Although high-resolution structures of individual CBD1 and CBD2 are available, their interdomain structure and dynamics and the atomic level mechanism of allosteric Ca(2+)-regulation remains unknown. Here, we use solution NMR spectroscopy to study the interdomain dynamics of CBD12, a 32 kDa construct that contains both the CBD1 and CBD2 domains connected by a short linker. Analysis of NMR residual dipolar couplings shows that CBD12 assumes on average an elongated shape both in the absence and in the presence of Ca(2+). NMR (15)N relaxation data of the Apo state indicate that the two domains sample a wide range of relative arrangements on the nanosecond time scale. These arrangements comprise significantly non-linear interdomain orientations. Binding of Ca(2+) to CBD1 significantly restricts the interdomain flexibility, stabilizing a more rigid elongated conformation. These findings suggest a molecular mechanism for the role of CBD12 in the function of NCX.

Asunto(s)

Calcio/química , Intercambiador de Sodio-Calcio/química , Regulación Alostérica/fisiología , Animales , Calcio/metabolismo , Perros , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Intercambiador de Sodio-Calcio/metabolismo , Relación Estructura-Actividad

RESUMEN

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7(XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Transporte de Membrana/química , Xanthomonas/química , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/metabolismo , Citrus sinensis/microbiología , Cristalografía por Rayos X/métodos , Prueba de Complementación Genética , Immunoblotting , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/aislamiento & purificación , Lipoproteínas/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/aislamiento & purificación , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Eliminación de Secuencia , Espectrometría de Fluorescencia , Factores de Virulencia/química , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Xanthomonas/genética , Xanthomonas/metabolismo

RESUMEN

High-resolution nuclear magnetic resonance is used to investigate the conformational dynamics of human glucokinase, a 52 kDa monomeric enzyme that displays kinetic cooperativity. (1)H-(15)N transverse relaxation optimized spectra of uniformly labeled glucokinase, recorded in the absence and presence of glucose, reveal significant cross-peak overlap and heterogeneous peak intensities that persist over a range of temperatures. (15)N-specific labeling of isoleucines and tryptophans, reporting on backbone and side chain dynamics, respectively, demonstrates that both unliganded and glucose-bound enzymes sample multiple conformations, although glucose stabilizes certain conformations. These results provide the first direct evidence of glucokinase conformational heterogeneity and hence shed light on the molecular basis of cooperativity.

Asunto(s)

Glucoquinasa/química , Glucoquinasa/metabolismo , Glucosa/química , Humanos , Imagen por Resonancia Magnética , Conformación Molecular , Páncreas/metabolismo

RESUMEN

Higher-rank correlation spectroscopy is introduced as an alternative to 3D Fourier-transform (FT) NMR spectroscopy for resonance assignment and molecular structure determination. The method combines standard 2D FT spectra that share a common frequency dimension, such as a 2D (13)C-(1)H HSQC and a 2D (1)H-(1)H TOCSY spectrum, and constructs higher-rank correlation spectra with ultra-high spectral resolution. Spectral overlap along a common dimension, in particular the (1)H dimension, is addressed by a spectral filtering method, which identifies mismatches between the 1(st) and 2(nd) moments of cross-peak profiles. The method, which provides a substantial speed-up over traditional 3D FT spectroscopy while effectively suppressing false peaks, is demonstrated for the triple-rank (13)C-(1)H HSQC-TOCSY spectrum of a cyclic decapeptide with different mixing times. Higher-rank correlation spectroscopy is usefully applicable to the analysis of a wide range of NMR spectra of synthetic and natural products.

RESUMEN

The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3'-->5')cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv citri (PilZ(XAC1133), this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ(XAC1133) shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which PilZ proteins regulate specific processes is not clear. In this study, we show that PilZ(XAC1133) binds to PilB, an ATPase required for T4P polymerization, and to the EAL domain of FimX(XAC2398), which regulates T4P biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimX(XAC2398)in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ(XAC1133). Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ(XAC1133) interactions with FimX(XAC2398) and PilB(XAC3239) are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of "degenerate" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Fimbrias Bacterianas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Movimiento Celular , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Oxidorreductasas/genética , Desnaturalización Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Xanthomonas axonopodis/química , Xanthomonas axonopodis/citología , Xanthomonas axonopodis/metabolismo , Xanthomonas campestris/química , Xanthomonas campestris/citología , Xanthomonas campestris/metabolismo

RESUMEN

Tropomyosin (Tm) is a dimeric coiled-coil protein that polymerizes through head-to-tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg(2+)-induced increase in stability of the C-terminal half of Tm is sensitive to mutations near the C-terminus. In the present report, we study the interaction between Mg(2+) and full-length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm(259-284(W269))) is flexible and to large extent unstructured, the larger Tm(220-284(W269)) fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg(2+). NMR analysis shows that Mg(2+) induces chemical shift perturbations in both Tm(220-284(W269)) and Tm(259-284(W269)) in the vicinity of His276, in which are located several negatively charged residues.

Asunto(s)

Magnesio/metabolismo , Músculo Esquelético/metabolismo , Tropomiosina/química , Tropomiosina/metabolismo , Secuencia de Aminoácidos , Aminoácidos , Animales , Sitios de Unión , Pollos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Docilidad , Desnaturalización Proteica , Estabilidad Proteica , Temperatura

Asunto(s)

Proteínas Fúngicas/química , Transactivadores/química , Trichoderma/química , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Terciaria de Proteína

RESUMEN

We present experimental evidence for a cooperative unfolding transition of an alpha-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations.

Asunto(s)

Proteínas de Unión al ADN/química , Medición de Intercambio de Deuterio , Regiones Operadoras Genéticas , Proteínas Represoras/química , Proteínas Bacterianas , Escherichia coli/genética , Proteínas de Escherichia coli , Concentración de Iones de Hidrógeno , Cinética , Represoras Lac , Espectroscopía de Resonancia Magnética , Unión Proteica