RESUMEN
The K+ uptake system KtrAB is essential for bacterial survival in low K+ environments. The activity of KtrAB is regulated by nucleotides and Na+. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na+ activates KtrAB and the Na+ binding site remain unknown. Here we present the cryo-EM structures of ATP- and ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) both solved at 2.8 Å. A cryo-EM density at the intra-dimer interface of ATP-KtrA was identified as Na+, as supported by X-ray crystallography and ICP-MS. Thermostability assays and functional studies demonstrated that Na+ binding stabilizes the ATP-bound BsKtrAB complex and enhances its K+ flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that BsKtrB Arg417 and Phe91 serve as a channel gate. The synergism of ATP and Na+ in activating BsKtrAB is likely applicable to Na+-activated K+ channels in central nervous system.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Proteínas de Transporte de Catión , Potasio , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Moleculares , Potasio/metabolismo , Unión Proteica , Sodio/metabolismoRESUMEN
The theories for substrate recognition in enzyme catalysis have evolved from lock-key to induced fit, then conformational selection, and conformational selection followed by induced fit. However, the prevalence and consensus of these theories require further examination. Here we use cryogenic electron microscopy and African swine fever virus type 2 topoisomerase (AsfvTop2) to demonstrate substrate binding theories in a joint and ordered manner: catalytic selection by the enzyme, conformational selection by the substrates, then induced fit. The apo-AsfvTop2 pre-exists in six conformers that comply with the two-gate mechanism directing DNA passage and release in the Top2 catalytic cycle. The structures of AsfvTop2-DNA-inhibitor complexes show that substantial induced-fit changes occur locally from the closed apo-conformer that however is too far-fetched for the open apo-conformer. Furthermore, the ATPase domain of AsfvTop2 in the MgAMP-PNP-bound crystal structures coexist in reduced and oxidized forms involving a disulfide bond, which can regulate the AsfvTop2 function.
RESUMEN
Phycobilisomes (PBS) are antenna megacomplexes that transfer energy to photosystems II and I in thylakoids. PBS likely evolved from a basic, inefficient form into the predominant hemidiscoidal shape with radiating peripheral rods. However, it has been challenging to test this hypothesis because ancestral species are generally inaccessible. Here we use spectroscopy and cryo-electron microscopy to reveal a structure of a "paddle-shaped" PBS from a thylakoid-free cyanobacterium that likely retains ancestral traits. This PBS lacks rods and specialized ApcD and ApcF subunits, indicating relict characteristics. Other features include linkers connecting two chains of five phycocyanin hexamers (CpcN) and two core subdomains (ApcH), resulting in a paddle-shaped configuration. Energy transfer calculations demonstrate that chains are less efficient than rods. These features may nevertheless have increased light absorption by elongating PBS before multilayered thylakoids with hemidiscoidal PBS evolved. Our results provide insights into the evolution and diversification of light-harvesting strategies before the origin of thylakoids.
Asunto(s)
Cianobacterias , Tilacoides , Tilacoides/metabolismo , Ficobilisomas/metabolismo , Microscopía por Crioelectrón , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismoRESUMEN
Once thought to be non-naturally occurring, D-amino acids (DAAs) have in recent years been revealed to play a wide range of physiological roles across the tree of life, including in human systems. Synthetic biologists have since exploited DAAs' unique biophysical properties to generate peptides and proteins with novel or enhanced functions. However, while peptides and small proteins containing DAAs can be efficiently prepared in vitro, producing large-sized heterochiral proteins poses as a major challenge mainly due to absence of pre-existing DAA translational machinery and presence of endogenous chiral discriminators. Based on our previous work demonstrating pyrrolysyl-tRNA synthetase's (PylRS') remarkable substrate polyspecificity, this work attempts to increase PylRS' ability in directly charging tRNAPyl with D-phenylalanine analogs (DFAs). We here report a novel, polyspecific Methanosarcina mazei PylRS mutant, DFRS2, capable of incorporating DFAs into proteins via ribosomal synthesis in vivo. To validate its utility, in vivo translational DAA substitution were performed in superfolder green fluorescent protein and human heavy chain ferritin, successfully altering both proteins' physiochemical properties. Furthermore, aminoacylation kinetic assays further demonstrated aminoacylation of DFAs by DFRS2 in vitro.
RESUMEN
Regulation of CO2 fixation in cyanobacteria is important both for the organism and global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a distinct ATP-sensing mechanism, where a drop in ATP level allows SeXPK to divert precursors of the RuBisCO substrate away from the Calvin-Benson-Bassham cycle. Deleting the SeXPK gene increased CO2 fixation particularly during light-dark transitions. In high-density cultures, the Δxpk strain showed a 60% increase in carbon fixation and unexpectedly resulted in sucrose secretion without any pathway engineering. Using cryo-EM analysis, we discovered that these functions were enabled by a unique allosteric regulatory site involving two subunits jointly binding two ATP, which constantly suppresses the activity of SeXPK until the ATP level drops. This magnesium-independent ATP allosteric site is present in many species across all three domains of life, where it may also play important regulatory functions.
Asunto(s)
Dióxido de Carbono , Fotosíntesis , Dióxido de Carbono/metabolismo , Fotosíntesis/fisiología , Ciclo del Carbono , Adenosina Trifosfato/metabolismoRESUMEN
Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FADâ¢- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FADâ¢-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Protones , Arginina/metabolismo , Cristalografía , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Transporte de Electrón , Electrones , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Flavinas , Oxidación-ReducciónRESUMEN
Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly in solving the structures of large protein complexes or cellular machineries that play important biological functions. This review focuses on the contribution and future potential of cryo-EM in related emerging applications-enzymatic mechanisms and dynamic processes. Work on these subjects can benefit greatly from the capability of cryo-EM to solve the structures of specific protein complexes in multiple conditions, including variations in the buffer condition, ligands, and temperature, and to capture multiple conformational states, conformational change intermediates, and reaction intermediates. These studies can expand the structural landscape of specific proteins or protein complexes in multiple dimensions and drive new advances in the fields of enzymology and dynamic processes. The advantages and complementarity of cryo-EM relative to X-ray crystallography and nuclear magnetic resonance with regard to these applications are also addressed.
Asunto(s)
Proteínas , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X , Humanos , Ligandos , Conformación MolecularRESUMEN
The catalog of enzymes known to catalyze the nucleophile-assisted formation of C-C bonds is extremely small, and there is presently no definitive example of a biological Rauhut-Currier reaction. Biosynthesis of the polyketide insecticide spinosyn A in Saccharopolyspora spinosa involves a [4 + 2]-cycloaddition and a subsequent intramolecular C-C bond formation catalyzed by SpnF and SpnL, respectively. Isotope tracer experiments and kinetic isotope effects, however, imply that the SpnL-catalyzed reaction proceeds without initial deprotonation of the substrate. The crystal structure of SpnL exhibits high similarity to SAM-dependent methyltransferases as well as SpnF. The residue Cys60 is also shown to reside in the SpnL active site, and the Cys60Ala SpnL mutant is found to be devoid of activity. Moreover, SpnL is covalently modified at Cys60 and irreversibly inactivated when it is coincubated with a fluorinated substrate analogue designed as a suicide inactivator of nucleophile-assisted C-C bond formation. These results suggest that SpnL catalyzes a biological Rauhut-Currier reaction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Isomerasas/metabolismo , Macrólidos/metabolismo , Proteínas Bacterianas/química , Biocatálisis , Dominio Catalítico , Cisteína/química , Isomerasas/química , Modelos Químicos , Saccharopolyspora/enzimologíaRESUMEN
The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 °C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 °C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 °C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 °C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.
Asunto(s)
Sistemas de Computación , Proteínas , Microscopía por Crioelectrón , Temperatura , VitrificaciónRESUMEN
Tumor necrosis factor receptor-associated factor-interacting protein with a forkhead-associated domain (TIFA), a key regulator of inflammation, may be involved in the pathogenesis of pulmonary arterial hypertension (PAH). A total of 48 PAH patients (age 50.1 ± 13.1 years, 22.9% men), 25 hypertensive subjects, and 26 healthy controls were enrolled. TIFA protein expression in peripheral blood mononuclear cells (PBMCs) and plasma interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were measured. Pulmonary arterial hemodynamics were derived from right heart catheterization. PAH patients had the highest expression of TIFA, TNF-α, and IL-1ß. TIFA protein expression was significantly associated with IL-1ß (r = 0.94; P < 0.001), TNF-α (r = 0.93; P < 0.001), mean pulmonary artery pressure (r = 0.41; P = 0.006), and pulmonary vascular resistance (r = 0.41; P = 0.007). TIFA protein expression could independently predict the presence of PAH (odds ratio [95% confidence interval per-0.1 standard deviation]: 1.72 [1.37-2.16]; P < 0.001) and outperformed echocardiographic estimation. Ex vivo silencing of TIFA protein expression in PBMCs led to the suppression of the cellular expression of IL-1ß and TNF-α. IL-1ß and TNF-α mediated 80.4% and 56.6% of the causal relationship between TIFA and PAH, respectively, supporting the idea that TIFA protein is involved in the pathogenesis of PAH.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Interleucina-1beta/genética , Hipertensión Arterial Pulmonar/genética , Factor de Necrosis Tumoral alfa/genética , Adulto , Femenino , Regulación de la Expresión Génica/genética , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , FN-kappa B/genética , Hipertensión Arterial Pulmonar/sangre , Hipertensión Arterial Pulmonar/patología , Transducción de Señal/genéticaRESUMEN
d-glycero-d-manno-Heptose-1ß,7-bisphosphate (HBP) is a bacterial metabolite that can induce a TIFA-dependent innate immune response in mammals. It was recently discovered that after HBP enters into the cytoplasm of the host cell, it is transformed into ADP-heptose-7-phosphate, which then leads to ALPK1-TIFA-dependent inflammatory response. In order to provide a molecular tool allowing the discovery of the proteins involved in this novel inflammatory pathway, we designed and synthesized a biotinylated analogue of HBP. This chemical probe displays an anomeric ß-phosphate and a phosphonate at the 7-position, and a d-configured 6-position to which is attached the biotin moiety. To do so, different synthetic strategies were explored and described in this report. Moreover, we demonstrated that the biotinylated version of HBP is still biologically active and can activate the NF-κB pathway in HEK293T cells.
Asunto(s)
HeptosasRESUMEN
Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Microscopía por Crioelectrón , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Ratones , Conformación Proteica en Hélice alfa , Recombinasa Rad51/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which biofilm communities are considered to be environmental reservoirs. In endemic regions, and after algal blooms, which may result from phosphate enrichment following agricultural runoff, the bacterium is released from biofilms resulting in seasonal disease outbreaks. However, the molecular mechanism by which V. cholerae senses its environment and switches lifestyles from the biofilm-bound state to the planktonic state is largely unknown. Here, we report that the major biofilm scaffolding protein RbmA undergoes autocatalytic proteolysis via a phosphate-dependent induced proximity activation mechanism. Furthermore, we show that RbmA mutants that are defective in autoproteolysis cause V. cholerae biofilms to grow larger and mechanically stronger, correlating well with the observation that RbmA stability directly affects microbial community homeostasis and rheological properties. In conclusion, our biophysical study characterizes a novel phosphate-dependent breakdown pathway of RbmA, while microbiological data suggest a new, sensory role of this biofilm scaffolding element.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Compuestos de Magnesio/farmacología , Fosfatos/farmacología , Proteolisis , Vibrio cholerae/metabolismo , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/crecimiento & desarrolloRESUMEN
Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA have been evolved to generate genetically encoded noncanonical amino acids (ncAAs). Use of tryptophan (Trp) analogues with pyrrole ring modification for their spatial and polarity tuning in enzyme activity and substrate specificity is still limited. Herein, we report the application of an evolved PylRS, FOWRS2, for efficient incorporation of five Trp analogues into the deubiquitinase USP30 to decipher the role of W475 for diubiquitin selectivity. Structures of the five FOWRS-C/Trp analogue complexes at 1.7-2.5 Å resolution showed multiple ncAA binding modes. The W475 near the USP30 active site was replaced with Trp analogues, and the effect on the activity as well as the selectivity toward diubiquitin linkage types was examined. It was found that the Trp analogue with a formyl group attached to the nitrogen atom of the indole ring led to an improved activity of USP30 likely due to enhanced polar interactions and that another Trp analogue, 3-benzothienyl-l-alanine, induced a unique K6-specificity. Collectively, genetically encoded noncanonical Trp analogues by evolved PylRS·tRNACUAPyl pair unravel the spatial role of USP30-W475 in its diubiquitin selectivity.
Asunto(s)
Proteínas Mitocondriales/química , Tioléster Hidrolasas/química , Triptófano/análogos & derivados , Triptófano/química , Aminoacil-ARNt Sintetasas/química , Proteínas Arqueales/química , Dominio Catalítico , Humanos , Methanosarcina/enzimología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismo , Triptófano/metabolismoRESUMEN
d-Glycero-d-manno-heptose-1ß,7-bisphosphate (HBP) and d-glycero-d-manno-heptose-1ß-phosphate (H1P) are bacterial metabolites that were recently shown to stimulate inflammatory responses in host cells through the activation of the TIFA-dependent NF-κB pathway. To better understand structure-based activity in relation to this process, a family of nonhydrolyzable phosphonate analogues of HBP and H1P was synthesized. The inflammation modulation by which these molecules induce the TIFA-NF-κB signal axis was evaluated inâ vivo at a low-nanomolar concentration (6â nM) and compared to that of the natural metabolites. Our data showed that three phosphonate analogues had similar stimulatory activity to HBP, whereas two phosphonates antagonized HBP-induced TIFA-NF-κB signaling. These results open new horizons for the design of pro-inflammatory and innate immune modulators that could be used as vaccine adjuvant.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Heptosas/farmacología , Inflamación/inmunología , FN-kappa B/inmunología , Fosfatos/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Conformación de Carbohidratos , Diseño de Fármacos , Heptosas/síntesis química , Heptosas/química , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , FN-kappa B/genética , Fosfatos/síntesis química , Fosfatos/química , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
Antiviral drug resistance in influenza infections has been a major threat to public health. To develop a broad-spectrum inhibitor of influenza to combat the problem of drug resistance, we previously identified the highly conserved E339...R416 salt bridge of the nucleoprotein trimer as a target and compound 1 as an inhibitor disrupting the salt bridge with an EC50 = 2.7 µM against influenza A (A/WSN/1933). We have further modified this compound via a structure-based approach and performed antiviral activity screening to identify compounds 29 and 30 with EC50 values of 110 and 120 nM, respectively, and without measurable host cell cytotoxicity. Compared to the clinically used neuraminidase inhibitors, these two compounds showed better activity profiles against drug-resistant influenza A strains, as well as influenza B, and improved survival of influenza-infected mice.
Asunto(s)
Compuestos de Anilina/farmacología , Antivirales/farmacología , Virus de la Influenza A/química , Multimerización de Proteína/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Tiazoles/farmacología , Proteínas del Núcleo Viral/metabolismo , Compuestos de Anilina/síntesis química , Compuestos de Anilina/metabolismo , Animales , Antivirales/síntesis química , Antivirales/metabolismo , Sitios de Unión/efectos de los fármacos , Femenino , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Estructura Molecular , Proteínas de la Nucleocápside , Unión Proteica , Electricidad Estática , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/metabolismoRESUMEN
Protein functions are temperature-dependent, but protein structures are usually solved at a single (often low) temperature because of limitations on the conditions of crystal growth or protein vitrification. Here we demonstrate the feasibility of solving cryo-EM structures of proteins vitrified at high temperatures, solve 12 structures of an archaeal ketol-acid reductoisomerase (KARI) vitrified at 4-70 °C, and show that structures of both the Mg2+ form (KARI:2Mg2+) and its ternary complex (KARI:2Mg2+:NADH:inhibitor) are temperature-dependent in correlation with the temperature dependence of enzyme activity. Furthermore, structural analyses led to dissection of the induced-fit mechanism into ligand-induced and temperature-induced effects and to capture of temperature-resolved intermediates of the temperature-induced conformational change. The results also suggest that it is preferable to solve cryo-EM structures of protein complexes at functional temperatures. These studies should greatly expand the landscapes of protein structure-function relationships and enhance the mechanistic analysis of enzymatic functions.
Asunto(s)
Cetoácido Reductoisomerasa/metabolismo , Temperatura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Cetoácido Reductoisomerasa/química , Modelos Moleculares , Conformación Molecular , Sulfolobus solfataricus/enzimologíaRESUMEN
It was reported in 1995 that T7 and Taq DNA polymerases possess 3'-esterase activity, but without follow-up studies. Here we report that the 3'-esterase activity is intrinsic to the Thermococcus sp. 9°N DNA polymerase, and that it can be developed into a continuous method for DNA sequencing with dNTP analogs carrying a 3'-ester with a fluorophore. We first show that 3'-esterified dNTP can be incorporated into a template-primer DNA, and solve the crystal structures of the reaction intermediates and products. Then we show that the reaction can occur continuously, modulated by active site residues Tyr409 and Asp542. Finally, we use 5'-FAM-labeled primer and esterified dNTP with a dye to show that the reaction can proceed to ca. 450 base pairs, and that the intermediates of many individual steps can be identified. The results demonstrate the feasibility of a 3'-editing based DNA sequencing method that could find practical applications after further optimization.