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1.
PLoS One ; 15(7): e0235643, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32735615

RESUMEN

BACKGROUND: Pyrazinamide is an important drug against the latent stage of tuberculosis and is used in both first- and second-line treatment regimens. Pyrazinamide-susceptibility test usually takes a week to have a diagnosis to guide initial therapy, implying a delay in receiving appropriate therapy. The continued increase in multi-drug resistant tuberculosis and the prevalence of pyrazinamide resistance in several countries makes the development of assays for prompt identification of resistance necessary. The main cause of pyrazinamide resistance is the impairment of pyrazinamidase function attributed to mutations in the promoter and/or pncA coding gene. However, not all pncA mutations necessarily affect the pyrazinamidase function. OBJECTIVE: To develop a methodology to predict pyrazinamidase function from detected mutations in the pncA gene. METHODS: We measured the catalytic constant (kcat), KM, enzymatic efficiency, and enzymatic activity of 35 recombinant mutated pyrazinamidase and the wild type (Protein Data Bank ID = 3pl1). From all the 3D modeled structures, we extracted several predictors based on three categories: structural stability (estimated by normal mode analysis and molecular dynamics), physicochemical, and geometrical characteristics. We used a stepwise Akaike's information criterion forward multiple log-linear regression to model each kinetic parameter with each category of predictors. We also developed weighted models combining the three categories of predictive models for each kinetic parameter. We tested the robustness of the predictive ability of each model by 6-fold cross-validation against random models. RESULTS: The stability, physicochemical, and geometrical descriptors explained most of the variability (R2) of the kinetic parameters. Our models are best suited to predict kcat, efficiency, and activity based on the root-mean-square error of prediction of the 6-fold cross-validation. CONCLUSIONS: This study shows a quick approach to predict the pyrazinamidase function only from the pncA sequence when point mutations are present. This can be an important tool to detect pyrazinamide resistance.


Asunto(s)
Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , Amidohidrolasas/química , Amidohidrolasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clonación Molecular , Cinética , Modelos Lineales , Simulación de Dinámica Molecular , Mutagénesis , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación
2.
Nat Commun ; 11(1): 4135, 2020 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-32811817

RESUMEN

Complex I is the first and the largest enzyme of respiratory chains in bacteria and mitochondria. The mechanism which couples spatially separated transfer of electrons to proton translocation in complex I is not known. Here we report five crystal structures of T. thermophilus enzyme in complex with NADH or quinone-like compounds. We also determined cryo-EM structures of major and minor native states of the complex, differing in the position of the peripheral arm. Crystal structures show that binding of quinone-like compounds (but not of NADH) leads to a related global conformational change, accompanied by local re-arrangements propagating from the quinone site to the nearest proton channel. Normal mode and molecular dynamics analyses indicate that these are likely to represent the first steps in the proton translocation mechanism. Our results suggest that quinone binding and chemistry play a key role in the coupling mechanism of complex I.


Asunto(s)
Complejo I de Transporte de Electrón/química , Simulación de Dinámica Molecular , Quinonas/química , Thermus thermophilus/enzimología , Regulación Alostérica , Proteínas Bacterianas/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , Transporte de Electrón/genética , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/ultraestructura , Modelos Moleculares , NAD/química , NAD/metabolismo , Redes Neurales de la Computación , Conformación Proteica , Protones , Quinonas/metabolismo , Thermus thermophilus/genética
3.
PLoS Biol ; 18(8): e3000790, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32776918

RESUMEN

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.


Asunto(s)
Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Uridina/química , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Transporte Biológico , Clonación Molecular , Microscopía por Crioelectrón , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Homología Estructural de Proteína , Especificidad por Sustrato , Uridina/metabolismo
4.
Nat Commun ; 11(1): 4248, 2020 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-32843623

RESUMEN

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X/métodos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Proteínas Bacterianas/efectos de la radiación , Cinética , Luz , Estructura Molecular , Procesos Fotoquímicos , Fotorreceptores Microbianos/efectos de la radiación , Conformación Proteica , Análisis Espectral
5.
Nat Commun ; 11(1): 4245, 2020 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-32843629

RESUMEN

Diheme-containing succinate:menaquinone oxidoreductases (Sdh) are widespread in Gram-positive bacteria but little is known about the catalytic mechanisms they employ for succinate oxidation by menaquinone. Here, we present the 2.8 Å cryo-electron microscopy structure of a Mycobacterium smegmatis Sdh, which forms a trimer. We identified the membrane-anchored SdhF as a subunit of the complex. The 3 kDa SdhF forms a single transmembrane helix and this helix plays a role in blocking the canonically proximal quinone-binding site. We also identified two distal quinone-binding sites with bound quinones. One distal binding site is formed by neighboring subunits of the complex. Our structure further reveals the electron/proton transfer pathway for succinate oxidation by menaquinone. Moreover, this study provides further structural insights into the physiological significance of a trimeric respiratory complex II. The structure of the menaquinone binding site could provide a framework for the development of Sdh-selective anti-mycobacterial drugs.


Asunto(s)
Proteínas Bacterianas/química , Mycobacterium smegmatis/enzimología , Succinato Deshidrogenasa/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Catálisis , Microscopía por Crioelectrón , Transporte de Electrón , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mycobacterium smegmatis/química , Oxidación-Reducción , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Succinato Deshidrogenasa/metabolismo , Ácido Succínico/metabolismo , Vitamina K 2/metabolismo
6.
Nat Commun ; 11(1): 4196, 2020 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-32826907

RESUMEN

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.


Asunto(s)
Proteínas Bacterianas/química , ADN Primasa/química , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , ADN/química , Mycobacterium/genética , Mycobacterium/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , ADN/metabolismo , ADN Primasa/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas
7.
Nat Commun ; 11(1): 3974, 2020 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-32769995

RESUMEN

Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.


Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/ultraestructura , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Tripsina/metabolismo
8.
PLoS Comput Biol ; 16(8): e1007898, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32797038

RESUMEN

New treatments for diseases caused by antimicrobial-resistant microorganisms can be developed by identifying unexplored therapeutic targets and by designing efficient drug screening protocols. In this study, we have screened a library of compounds to find ligands for the flavin-adenine dinucleotide synthase (FADS) -a potential target for drug design against tuberculosis and pneumonia- by implementing a new and efficient virtual screening protocol. The protocol has been developed for the in silico search of ligands of unexplored therapeutic targets, for which limited information about ligands or ligand-receptor structures is available. It implements an integrative funnel-like strategy with filtering layers that increase in computational accuracy. The protocol starts with a pharmacophore-based virtual screening strategy that uses ligand-free receptor conformations from molecular dynamics (MD) simulations. Then, it performs a molecular docking stage using several docking programs and an exponential consensus ranking strategy. The last filter, samples the conformations of compounds bound to the target using MD simulations. The MD conformations are scored using several traditional scoring functions in combination with a newly-proposed score that takes into account the fluctuations of the molecule with a Morse-based potential. The protocol was optimized and validated using a compound library with known ligands of the Corynebacterium ammoniagenes FADS. Then, it was used to find new FADS ligands from a compound library of 14,000 molecules. A small set of 17 in silico filtered molecules were tested experimentally. We identified five inhibitors of the activity of the flavin adenylyl transferase module of the FADS, and some of them were able to inhibit growth of three bacterial species: C. ammoniagenes, Mycobacterium tuberculosis, and Streptococcus pneumoniae, where the last two are human pathogens. Overall, the results show that the integrative VS protocol is a cost-effective solution for the discovery of ligands of unexplored therapeutic targets.


Asunto(s)
Antibacterianos , Proteínas Bacterianas , Nucleotidiltransferasas , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Corynebacterium/efectos de los fármacos , Corynebacterium/enzimología , Diseño de Fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Ligandos , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(29): 17011-17018, 2020 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-32636271

RESUMEN

Few antibiotics are effective against Acinetobacter baumannii, one of the most successful pathogens responsible for hospital-acquired infections. Resistance to chlorhexidine, an antiseptic widely used to combat A. baumannii, is effected through the proteobacterial antimicrobial compound efflux (PACE) family. The prototype membrane protein of this family, AceI (Acinetobacter chlorhexidine efflux protein I), is encoded for by the aceI gene and is under the transcriptional control of AceR (Acinetobacter chlorhexidine efflux protein regulator), a LysR-type transcriptional regulator (LTTR) protein. Here we use native mass spectrometry to probe the response of AceI and AceR to chlorhexidine assault. Specifically, we show that AceI forms dimers at high pH, and that binding to chlorhexidine facilitates the functional form of the protein. Changes in the oligomerization of AceR to enable interaction between RNA polymerase and promoter DNA were also observed following chlorhexidine assault. Taken together, these results provide insight into the assembly of PACE family transporters and their regulation via LTTR proteins on drug recognition and suggest potential routes for intervention.


Asunto(s)
Acinetobacter baumannii , Antibacterianos , Proteínas Bacterianas , Clorhexidina , Proteínas de Transporte de Membrana , Acinetobacter baumannii/química , Acinetobacter baumannii/enzimología , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Clorhexidina/química , Clorhexidina/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Farmacorresistencia Microbiana , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo
10.
Nat Commun ; 11(1): 3703, 2020 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-32710080

RESUMEN

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.


Asunto(s)
Acilcoenzima A/química , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Mycobacterium tuberculosis/metabolismo , Factores de Transcripción/química , Acilcoenzima A/metabolismo , Sitio Alostérico , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Cristalografía por Rayos X , ADN Bacteriano/química , Proteínas de Unión al ADN/metabolismo , Ácidos Grasos/metabolismo , Ligandos , Modelos Moleculares , Conformación Proteica , Factores de Transcripción/metabolismo
11.
Nat Commun ; 11(1): 3740, 2020 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-32719356

RESUMEN

Mfd couples transcription to nucleotide excision repair, and acts on RNA polymerases when elongation is impeded. Depending on impediment severity, this action results in either transcription termination or elongation rescue, which rely on ATP-dependent Mfd translocation on DNA. Due to its role in antibiotic resistance, Mfd is also emerging as a prime target for developing anti-evolution drugs. Here we report the structure of DNA-bound Mfd, which reveals large DNA-induced structural changes that are linked to the active site via ATPase motif VI. These changes relieve autoinhibitory contacts between the N- and C-termini and unmask UvrA recognition determinants. We also demonstrate that translocation relies on a threonine in motif Ic, widely conserved in translocases, and a family-specific histidine near motif IVa, reminiscent of the "arginine clamp" of RNA helicases. Thus, Mfd employs a mode of DNA recognition that at its core is common to ss/ds translocases that act on DNA or RNA.


Asunto(s)
Proteínas Bacterianas/metabolismo , Reparación del ADN , ADN/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , ADN/química , ADN/ultraestructura , Escherichia coli/metabolismo , Modelos Moleculares , Unión Proteica , Dominios Proteicos , ARN Helicasas/metabolismo , Factores de Transcripción/química
12.
PLoS One ; 15(7): e0235687, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32678825

RESUMEN

Lactobacillus amylolyticus L6, a gram-positive amylolytic bacterium isolated from naturally fermented tofu whey (NFTW), was able to hydrolyze raffinose and stachyose for the production of α-galactosidase. The cell-free extract of L. amylolyticus L6 was found to exhibit glycosyltransferase activity to synthesize α-galacto-oligosaccharides (GOS) with melibiose as substrate. The coding genes of α-galactosidase were identified in the genome of L. amylolyticus L6. The α-galactosidase (AglB) was placed into GH36 family by amino acid sequence alignments with other α-galactosidases from lactobacilli. The optimal reaction conditions of pH and temperature for AglB were pH 6.0 and 37°C, respectively. Besides, potassium ion was found to improve the activity of AglB while divalent mercury ion, copper ion and zinc ion displayed different degrees of inhibition effect. Under the optimum reaction condition, AglB could catalyze the synthesis of GOS with degree of polymerization (DP) ≥5 by using 300 mM melibiose concentration as substrate. The maximum yield of GOS with (DP) ≥3 could reach 31.56% (w/w). Transgalactosyl properties made AglB a potential candidate for application in the production of GOS.


Asunto(s)
Proteínas Bacterianas/metabolismo , Clonación Molecular , Lactobacillus/enzimología , alfa-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Estabilidad de Enzimas , Glicosilación , Concentración de Iones de Hidrógeno , Hidrólisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Temperatura , alfa-Galactosidasa/química , alfa-Galactosidasa/genética
13.
PLoS One ; 15(6): e0230652, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32603331

RESUMEN

Toxin-antitoxin systems (TAS) are commonly found on bacterial plasmids and are generally involved in plasmid maintenance. In addition to plasmid maintenance, several plasmid-mediated TAS are also involved in bacterial stress response and virulence. Even though the same TAS are present in a variety of plasmid types and bacterial species, differences in their sequences, expression and functions are not well defined. Here, we aimed to identify commonly occurring plasmid TAS in Escherichia coli and Klebsiella pneumoniae and compare the sequence, expression and plasmid stability function of their variants. 27 putative type II TAS were identified from 1063 plasmids of Klebsiella pneumoniae in GenBank. Among these, ccdAB and pemIK were found to be most common, also occurring in plasmids of E. coli. Comparisons of ccdAB variants, taken from E. coli and K. pneumoniae, revealed sequence differences, while pemIK variants from IncF and IncL/M plasmids were almost identical. Similarly, the expression and plasmid stability functions of ccdAB variants varied according to the host strain and species, whereas the expression and functions of pemIK variants were consistent among host strains. The specialised functions of some TAS may determine the host specificity and epidemiology of major antibiotic resistance plasmids.


Asunto(s)
Enterobacteriaceae/genética , Plásmidos/genética , Sistemas Toxina-Antitoxina/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia Conservada , Farmacorresistencia Bacteriana/genética , Enterobacteriaceae/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica
14.
Proc Natl Acad Sci U S A ; 117(25): 14187-14193, 2020 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-32513729

RESUMEN

NaChBac, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Nav channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ∼6 to ∼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Nav channels in a membrane environment.


Asunto(s)
Proteínas Bacterianas/química , Microscopía por Crioelectrón/métodos , Nanoestructuras/química , Canales de Sodio Activados por Voltaje/química , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Proteínas Bacterianas/genética , Humanos , Membrana Dobles de Lípidos/química , Modelos Moleculares , Conformación Proteica , Canales de Sodio , Venenos de Araña/química , Canales de Sodio Activados por Voltaje/genética
15.
Nat Commun ; 11(1): 3210, 2020 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-32587243

RESUMEN

The bacterial flagellum is a remarkable molecular motor, whose primary function in bacteria is to facilitate motility through the rotation of a filament protruding from the bacterial cell. A cap complex, consisting of an oligomer of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex, which reveals that FliD is pentameric, with the N-terminal region of the protomer forming an extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded, contrary to a previously published cryo-EM structure, and propose a molecular model for the filament-cap interaction.


Asunto(s)
Proteínas Bacterianas/química , Campylobacter jejuni , Flagelos , Campylobacter jejuni/fisiología , Campylobacter jejuni/ultraestructura , Microscopía por Crioelectrón , Flagelos/fisiología , Flagelos/ultraestructura , Modelos Moleculares , Estructura Molecular
16.
PLoS One ; 15(6): e0234958, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32574185

RESUMEN

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/ß sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.


Asunto(s)
Bacillales/enzimología , Proteínas Bacterianas/química , Péptido Hidrolasas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Clonación Molecular , Pruebas de Enzimas , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Simulación de Dinámica Molecular , Peso Molecular , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/ultraestructura , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/ultraestructura , Especificidad por Sustrato
17.
Proc Natl Acad Sci U S A ; 117(24): 13437-13446, 2020 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-32482881

RESUMEN

Pentameric ligand-gated ion channels (pLGICs) are allosteric receptors that mediate rapid electrochemical signal transduction in the animal nervous system through the opening of an ion pore upon binding of neurotransmitters. Orthologs have been found and characterized in prokaryotes and they display highly similar structure-function relationships to eukaryotic pLGICs; however, they often encode greater architectural diversity involving additional amino-terminal domains (NTDs). Here we report structural, functional, and normal-mode analysis of two conformational states of a multidomain pLGIC, called DeCLIC, from a Desulfofustis deltaproteobacterium, including a periplasmic NTD fused to the conventional ligand-binding domain (LBD). X-ray structure determination revealed an NTD consisting of two jelly-roll domains interacting across each subunit interface. Binding of Ca2+ at the LBD subunit interface was associated with a closed transmembrane pore, with resolved monovalent cations intracellular to the hydrophobic gate. Accordingly, DeCLIC-injected oocytes conducted currents only upon depletion of extracellular Ca2+; these were insensitive to quaternary ammonium block. Furthermore, DeCLIC crystallized in the absence of Ca2+ with a wide-open pore and remodeled periplasmic domains, including increased contacts between the NTD and classic LBD agonist-binding sites. Functional, structural, and dynamical properties of DeCLIC paralleled those of sTeLIC, a pLGIC from another symbiotic prokaryote. Based on these DeCLIC structures, we would reclassify the previous structure of bacterial ELIC (the first high-resolution structure of a pLGIC) as a "locally closed" conformation. Taken together, structures of DeCLIC in multiple conformations illustrate dramatic conformational state transitions and diverse regulatory mechanisms available to ion channels in pLGICs, particularly involving Ca2+ modulation and periplasmic NTDs.


Asunto(s)
Proteínas Bacterianas/química , Canales Iónicos Activados por Ligandos/química , Regulación Alostérica , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Calcio/metabolismo , Cristalografía por Rayos X , Deltaproteobacteria/química , Deltaproteobacteria/metabolismo , Canales Iónicos Activados por Ligandos/genética , Canales Iónicos Activados por Ligandos/metabolismo , Ligandos , Modelos Moleculares , Oocitos/metabolismo , Periplasma/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de Proteína , Relación Estructura-Actividad , Xenopus laevis
18.
Nat Commun ; 11(1): 2877, 2020 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-32513917

RESUMEN

Mycoplasma genitalium is a human pathogen adhering to host target epithelial cells and causing urethritis, cervicitis and pelvic inflammatory disease. Essential for infectivity is a transmembrane adhesion complex called Nap comprising proteins P110 and P140. Here we report the crystal structure of P140 both alone and in complex with the N-terminal domain of P110. By cryo-electron microscopy (cryo-EM) and tomography (cryo-ET) we find closed and open Nap conformations, determined at 9.8 and 15 Å, respectively. Both crystal structures and the cryo-EM structure are found in a closed conformation, where the sialic acid binding site in P110 is occluded. By contrast, the cryo-ET structure shows an open conformation, where the binding site is accessible. Structural information, in combination with functional studies, suggests a mechanism for attachment and release of M. genitalium to and from the host cell receptor, in which Nap conformations alternate to sustain motility and guarantee infectivity.


Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Mycoplasma genitalium/metabolismo , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Cristalografía por Rayos X , Humanos , Mutación/genética , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Relación Estructura-Actividad
19.
Nat Commun ; 11(1): 2864, 2020 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-32513920

RESUMEN

Legionella pneumophila is a bacterial pathogen that utilises a Type IV secretion (T4S) system to inject effector proteins into human macrophages. Essential to the recruitment and delivery of effectors to the T4S machinery is the membrane-embedded T4 coupling complex (T4CC). Here, we purify an intact T4CC from the Legionella membrane. It contains the DotL ATPase, the DotM and DotN proteins, the chaperone module IcmSW, and two previously uncharacterised proteins, DotY and DotZ. The atomic resolution structure reveals a DotLMNYZ hetero-pentameric core from which the flexible IcmSW module protrudes. Six of these hetero-pentameric complexes may assemble into a 1.6-MDa hexameric nanomachine, forming an inner membrane channel for effectors to pass through. Analysis of multiple cryo EM maps, further modelling and mutagenesis provide working models for the mechanism for binding and delivery of two essential classes of Legionella effectors, depending on IcmSW or DotM, respectively.


Asunto(s)
Proteínas Bacterianas/metabolismo , Legionella pneumophila/metabolismo , Sistemas de Secreción Tipo IV/metabolismo , Animales , Proteínas Bacterianas/química , Células CHO , Cricetulus , Modelos Moleculares , Mutación/genética , Mapas de Interacción de Proteínas , Multimerización de Proteína , Reproducibilidad de los Resultados , Especificidad por Sustrato , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/aislamiento & purificación
20.
Nat Commun ; 11(1): 2991, 2020 06 12.
Artículo en Inglés | MEDLINE | ID: mdl-32532989

RESUMEN

Biocatalysts that perform C-H hydroxylation exhibit exceptional substrate specificity and site-selectivity, often through the use of high valent oxidants to activate these inert bonds. Rieske oxygenases are examples of enzymes with the ability to perform precise mono- or dioxygenation reactions on a variety of substrates. Understanding the structural features of Rieske oxygenases responsible for control over selectivity is essential to enable the development of this class of enzymes for biocatalytic applications. Decades of research has illuminated the critical features common to Rieske oxygenases, however, structural information for enzymes that functionalize diverse scaffolds is limited. Here, we report the structures of two Rieske monooxygenases involved in the biosynthesis of paralytic shellfish toxins (PSTs), SxtT and GxtA, adding to the short list of structurally characterized Rieske oxygenases. Based on these structures, substrate-bound structures, and mutagenesis experiments, we implicate specific residues in substrate positioning and the divergent reaction selectivity observed in these two enzymes.


Asunto(s)
Variación Genética , Proteínas con Hierro-Azufre/genética , Oxigenasas de Función Mixta/genética , Oxigenasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Biocatálisis , Dominio Catalítico , Cianobacterias/enzimología , Cianobacterias/genética , Hidroxilación , Proteínas con Hierro-Azufre/química , Proteínas con Hierro-Azufre/metabolismo , Cinética , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Modelos Moleculares , Oxigenasas/química , Oxigenasas/metabolismo , Conformación Proteica , Multimerización de Proteína , Especificidad por Sustrato
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