RESUMEN
The evolution of the Collaborative Computational Project No. 4 (CCP4) has been described in a new article by Agirre et al. [(2023). Acta Cryst. D79, 449-461] that should provide the definitive reference for the CCP4 suite of programs.
Asunto(s)
Proteínas , Programas Informáticos , Proteínas/química , Cristalografía por Rayos X , Sustancias MacromolecularesRESUMEN
Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Homeostasis , Proteínas Serina-Treonina Quinasas/química , Estrés Fisiológico , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Clonación Molecular , Proteínas Quinasas Dependientes de AMP Cíclico/química , Eliminación de Gen , Regulación de la Expresión Génica de las Plantas , Concentración de Iones de Hidrógeno , Transporte Iónico , Litio/química , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Sodio/químicaRESUMEN
Separation of daughter cells during bacterial cell division requires splitting of the septal cross wall by peptidoglycan hydrolases. In Streptococcus pneumoniae, PcsB is predicted to perform this operation. Recent evidence shows that PcsB is recruited to the septum by the transmembrane FtsEX complex, and that this complex is required for cell division. However, PcsB lacks detectable catalytic activity in vitro, and while it has been proposed that FtsEX activates PcsB, evidence for this is lacking. Here we demonstrate that PcsB has muralytic activity, and report the crystal structure of full-length PcsB. The protein adopts a dimeric structure in which the V-shaped coiled-coil (CC) domain of each monomer acts as a pair of molecular tweezers locking the catalytic domain of each dimeric partner in an inactive configuration. This suggests that the release of the catalytic domains likely requires an ATP-driven conformational change in the FtsEX complex, conveyed towards the catalytic domains through coordinated movements of the CC domain.
Asunto(s)
Proteínas Bacterianas/metabolismo , División Celular/fisiología , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Streptococcus pneumoniae/fisiología , Proteínas Bacterianas/ultraestructura , Dominio Catalítico/fisiología , División Celular/genética , Pared Celular/fisiología , Cristalografía por Rayos X , Streptococcus pneumoniae/genéticaRESUMEN
Fosfomycin targets the first step of peptidoglycan biosynthesis in Streptococcus pneumoniae catalyzed by UDP-N-acetylglucosamine enolpyruvyltransferase (MurA1). We investigated whether heteroresistance to fosfomycin occurs in S. pneumoniae. We found that of 11 strains tested, all but 1 (Hungary(19A)) displayed heteroresistance and that deletion of murA1 abolished heteroresistance. Hungary(19A) differs from the other strains by a single amino acid substitution in MurA1 (Ala(364)Thr). To test whether this substitution is responsible for the lack of heteroresistance, it was introduced into strain D39. The heteroresistance phenotype of strain D39 was not changed. Furthermore, no relevant structural differences between the MurA1 crystal structures of heteroresistant strain D39 and nonheteroresistant strain Hungary(19A) were found. Our results reveal that heteroresistance to fosfomycin is the predominant phenotype of S. pneumoniae and that MurA1 is required for heteroresistance to fosfomycin but is not the only factor involved. The findings provide a caveat for any future use of fosfomycin in the treatment of pneumococcal infections.
Asunto(s)
Transferasas Alquil y Aril/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Fosfomicina/farmacología , Streptococcus pneumoniae/efectos de los fármacos , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Cristalización , Humanos , Hungría , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Infecciones Neumocócicas/tratamiento farmacológico , Infecciones Neumocócicas/microbiología , Análisis de Secuencia de ADN , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/genéticaRESUMEN
The Arabidopsis SOS2 family of twenty-six protein kinases (CIPKs), their interacting activators, the SOS3 family of ten calcium-binding proteins (CBLs) and protein phosphatases type 2C (PP2C), function together in decoding calcium signals elicited by different environmental stimuli. Biochemical data suggest that stable CBL-CIPK or CIPK-PP2C complexes may be regulating the activity of various substrates controlling ion homeostasis. The available structural information provides a general regulatory mechanism in which calcium perception by CBLs and kinase activation is coupled. The structural basis of this molecular mechanism and the specificity of the network is reviewed and discussed in detail.
RESUMEN
An uncharacterized protein from Arabidopsis thaliana consisting of a single C2 domain (At3g17980) was cloned into the pETM11 vector and expressed in Escherichia coli, allowing purification to homogeneity in a single chromatographic step. Good-quality diffracting crystals were obtained using vapour-diffusion techniques. The crystals diffracted to 2.2 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 35.3, b = 88.9, c = 110.6 Å. A promising molecular-replacement solution has been found using the structure of the C2 domain of Munc13-C2b (PDB entry 3kwt) as the search model.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas Portadoras/química , Cristalización , Cristalografía por Rayos XRESUMEN
Q88Y25_Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxylesterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-terminally His(6)-tagged Q88Y25_Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His(6)-tagged Q88Y25_Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 Å resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 Å.
Asunto(s)
Esterasas/química , Lactobacillus plantarum/enzimología , Cristalografía por Rayos XRESUMEN
SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Regulación de la Expresión Génica de las Plantas , Ósmosis , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Ácido Abscísico/farmacología , Proteínas de Arabidopsis/genética , Dicroismo Circular , Cristalografía por Rayos X , Modelos Moleculares , Mutación/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Conformación Proteica , Proteínas Quinasas/genética , Proteína Fosfatasa 2C , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , UltracentrifugaciónRESUMEN
AmpD is a cytoplasmic peptidoglycan (PG) amidase involved in bacterial cell-wall recycling and in induction of ß-lactamase, a key enzyme of ß-lactam antibiotic resistance. AmpD belongs to the amidase_2 family that includes zinc-dependent amidases and the peptidoglycan-recognition proteins (PGRPs), highly conserved pattern-recognition molecules of the immune system. Crystal structures of Citrobacter freundii AmpD were solved in this study for the apoenzyme, for the holoenzyme at two different pH values, and for the complex with the reaction products, providing insights into the PG recognition and the catalytic process. These structures are significantly different compared with the previously reported NMR structure for the same protein. The NMR structure does not possess an accessible active site and shows the protein in what is proposed herein as an inactive "closed" conformation. The transition of the protein from this inactive conformation to the active "open" conformation, as seen in the x-ray structures, was studied by targeted molecular dynamics simulations, which revealed large conformational rearrangements (as much as 17 Å) in four specific regions representing one-third of the entire protein. It is proposed that the large conformational change that would take the inactive NMR structure to the active x-ray structure represents an unprecedented mechanism for activation of AmpD. Analysis is presented to argue that this activation mechanism might be representative of a regulatory process for other intracellular members of the bacterial amidase_2 family of enzymes.
Asunto(s)
Amidohidrolasas/química , Citrobacter freundii/enzimología , N-Acetil Muramoil-L-Alanina Amidasa/química , Proteínas Bacterianas/química , Catálisis , Cristalografía por Rayos X , Activación Enzimática , Concentración de Iones de Hidrógeno , Peptidoglicano/metabolismo , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
The SnRK2.6 (SNF1-related kinase 2.6) gene from Arabidopsis thaliana encodes the serine/threonine protein kinase SnRK2.6/OST1 (OPEN STOMATA 1). It plays a central role in the drought-tolerance mechanism. OST1 is in fact the main positive effector in the hydric stress response. The SnRK2.6 gene was cloned into the pGEX4T1 plasmid, mutated and expressed in Escherichia coli, allowing purification to homogeneity in two chromatographic steps. Various OST1 mutants yielded crystals using vapour-diffusion techniques, but only one mutant showed a good diffraction pattern. Its crystals diffracted to 2.8â Å resolution and belonged to space group P222(1), with unit-cell parameters a=77.7, b=99.4, c=108.4â Å. A promising molecular-replacement solution was found using the structure of the kinase domain of the yeast AMP-activated protein kinase SNF1 (PDB entry 3hyh) as the search model.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Arabidopsis/química , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/aislamiento & purificación , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Difracción de Rayos XRESUMEN
The first structure of a pneumococcal autolysin, that of the LytC lysozyme, has been solved in ternary complex with choline and a pneumococcal peptidoglycan (PG) fragment. The active site of the hydrolase module is not fully exposed but is oriented toward the choline-binding module, which accounts for its unique in vivo features in PG hydrolysis, its activation and its regulatory mechanisms. Because of the unusual hook-shaped conformation of the multimodular protein, it is only able to hydrolyze non-cross-linked PG chains, an assertion validated by additional experiments. These results explain the activation of LytC by choline-binding protein D (CbpD) in fratricide, a competence-programmed mechanism of predation of noncompetent sister cells. The results provide the first structural insights to our knowledge into the critical and central function that LytC plays in pneumococcal virulence and explain a long-standing puzzle of how murein hydrolases can be controlled to avoid self-lysis during bacterial growth and division.
Asunto(s)
Colina/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Peptidoglicano/metabolismo , Streptococcus pneumoniae/química , Colina/química , Cristalografía por Rayos X , Modelos Moleculares , N-Acetil Muramoil-L-Alanina Amidasa/análisis , Peptidoglicano/química , Unión Proteica , Conformación Proteica , Streptococcus pneumoniae/citología , Streptococcus pneumoniae/metabolismo , Ácidos Teicoicos/metabolismoRESUMEN
LytC, one of the major autolysins from the human pathogen Streptococcus pneumoniae, has been crystallized as needles by the hanging-drop technique using 10%(w/v) PEG 3350 as precipitant and 10 mM HEPES pH 7.5. LytC crystals were quickly soaked in mother liquor containing 2 mM of the complex Gd-HPDO3A to produce derivatized crystals (LytC(Gd-HPDO3A)). Both native LytC and isomorphous LytC(Gd-HPDO3A) crystals were flash-cooled in a nitrogen flow at 120 K prior to X-ray data collection using an in-house Enraf-Nonius rotating-anode generator (lambda = 1.5418 A) and a MAR345 imaging-plate detector. In both cases, good-quality diffraction patterns were obtained at high resolution. LytC(Gd-HPDO3A) crystals allowed the collection of a SAD X-ray data set to 2.6 A resolution indexed in terms of a P2(1) monoclinic unit cell with parameters a = 59.37, b = 67.16, c = 78.85 A, beta = 105.69 degrees . The anomalous Patterson map allowed the identification of one heavy-atom binding site, which was sufficient for the calculation of an interpretable anomalous map at 2.6 A resolution.
Asunto(s)
Compuestos Heterocíclicos/química , N-Acetil Muramoil-L-Alanina Amidasa/química , Compuestos Organometálicos/química , Streptococcus pneumoniae/enzimología , Cristalización , Cristalografía por Rayos X , Gadolinio , Estructura MolecularRESUMEN
The bacterial thermoalkalophilic lipases that hydrolyze saturated fatty acids at 60-75 degrees C and pH 8-10 are grouped as the lipase family I.5. We report here the crystal structure of the lipase from Geobacillus thermocatenulatus, the first structure of a member of the lipase family I.5 showing an open configuration. Unexpectedly, enzyme activation involves large structural rearrangements of around 70 amino acids and the concerted movement of two lids, the alpha6- and alpha7-helices, unmasking the active site. Central in the restructuring process of the lids are both the transfer of bulky hydrophobic residues out of the N-terminal end of the alpha6-helix and the incorporation of short side chain residues to the alpha6 C-terminal end. All these structural changes are stabilized by the Zn(2+)-binding domain, which is characteristic of this family of lipases. Two detergent molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acids chains. The combination of structural and biochemical studies indicate that the lid opening is not mediated by temperature but triggered by interaction with lipid substrate.
Asunto(s)
Bacillaceae/enzimología , Proteínas Bacterianas/química , Ácidos Grasos/química , Lipasa/química , Zinc/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática , Ácidos Grasos/metabolismo , Calor , Interacciones Hidrofóbicas e Hidrofílicas , Lipasa/metabolismo , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Zinc/metabolismoRESUMEN
Bacillus thermocatenulatus lipase 2 (BTL2) is a thermoalkalophilic lipase that has been reported as an enantioselective biocatalyst for diverse reactions and that heads a group of enzymes that share high resistance towards many inactivation agents (heat, organic solvents, pH etc.). This makes BTL2 an important research target because of its potential industrial applications. BTL2 was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the sitting-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 13% MPD and 0.2 M ammonium acetate in 0.05 M sodium citrate pH 5.5-5.6. The crystals, which belonged to the orthorhombic space group I222 with unit-cell parameters a = 73.07, b = 129.08, c = 127.49 A, allowed the collection of an X-ray data set to 2.2 A resolution.
Asunto(s)
Bacillus/enzimología , Proteínas Bacterianas/química , Lipasa/química , Cristalización , Datos de Secuencia Molecular , Difracción de Rayos XRESUMEN
The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 A.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Arabidopsis/genética , Proteínas Serina-Treonina Quinasas/química , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Pneumococcal bacteriophage-encoded lysins are modular proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) in treatment of streptococcal infections. The first x-ray crystal structures of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1, in complex with three bacterial cell wall peptidoglycan (PG) analogues are reported herein. The Cpl-1 structure is folded in two well defined modules, one responsible for anchoring to the pneumococcal cell wall and the other, a catalytic module, that hydrolyzes the PG. Conformational rearrangement of Tyr-127 is a critical event in molecular recognition of a stretch of five saccharide rings of the polymeric peptidoglycan (cell wall). The PG is bound at a stretch of the surface that is defined as the peptidoglycan-binding sites 1 and 2, the juncture of which catalysis takes place. The peptidoglycan-binding site 1 binds to a stretch of three saccharides of the peptidoglycan in a conformation essentially identical to that of the peptidoglycan in solution. In contrast, binding of two peptidoglycan saccharides at the peptidoglycan-binding site 2 introduces a kink into the solution structure of the peptidoglycan, en route to catalytic turnover. These findings provide the first structural evidence on recognition of the peptidoglycan and shed light on the discrete events of cell wall degradation by Cpl-1.
Asunto(s)
Proteínas Bacterianas/fisiología , Pared Celular/metabolismo , Endopeptidasas/química , Endopeptidasas/fisiología , Streptococcus pneumoniae/metabolismo , Secuencia de Aminoácidos , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/química , Catálisis , Cristalografía por Rayos X , Ligandos , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
The plant SOS2 family of protein kinases and their interacting activators, the SOS3 family of calcium-binding proteins, function together in decoding calcium signals elicited by different environmental stimuli. SOS2 is activated by Ca-SOS3 and subsequently phosphorylates the ion transporter SOS1 to bring about cellular ion homeostasis under salt stress. In addition to possessing the kinase activity, members of the SOS2 family of protein kinases can bind to protein phosphatase 2Cs. The crystal structure of the binary complex of Ca-SOS3 with the C-terminal regulatory moiety of SOS2 resolves central questions regarding the dual function of SOS2 as a kinase and a phosphatase-binding protein. A comparison with the structure of unbound SOS3 reveals the basis of the molecular function of this family of kinases and their interacting calcium sensors. Furthermore, our study suggests that the structure of the phosphatase-interaction domain of SOS2 defines a scaffold module conserved from yeast to human.
Asunto(s)
Proteínas de Arabidopsis/química , Señalización del Calcio/fisiología , Calcio/metabolismo , Proteínas Sensoras del Calcio Intracelular/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencias de Aminoácidos/fisiología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cristalización , Activación Enzimática , Homeostasis/fisiología , Proteínas Sensoras del Calcio Intracelular/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Brotes de la Planta/metabolismo , Unión Proteica/fisiología , Proteína Fosfatasa 2C , Sales (Química)/químicaRESUMEN
Phosphorylcholine, a specific component of the pneumococcal cell wall, is crucial in pathogenesis. It directly binds to the human platelet-activating factor (PAF) receptor and acts as a docking station for the family of surface-located choline-binding proteins (CBP). The first structure of a complete pneumococcal CBP, Pce (or CbpE), has been solved in complex with the reaction product and choline analogs. Pce has a novel modular structure, with a globular N-terminal module containing a binuclear Zn(2+) catalytic center, and an elongated choline-binding module. Residues involved in substrate binding and catalysis are described and modular configuration of the active center accounts for in vivo features of teichoic acid hydrolysis. The hydrolysis of PAF by Pce and its regulatory role in phosphorylcholine decoration of the bacterial surface provide new insights into the critical function of Pce in pneumococcal adherence and invasiveness.