RESUMEN
Interferon-induced proteins, including the largely uncharacterized interferon-induced tetratricopeptide repeat (IFIT) protein family, provide defenses against pathogens. Differing from expectations for tetratricopeptide repeat (TPR) proteins and from human IFIT1, IFIT2, and IFIT3, we show that human IFIT5 recognizes cellular RNA instead of protein partners. In vivo and in vitro, IFIT5 bound to endogenous 5'-phosphate-capped RNAs, including transfer RNAs. The crystal structure of IFIT5 revealed a convoluted intramolecular packing of eight TPRs as a fold that we name the TPR eddy. Additional, non-TPR structural elements contribute to an RNA binding cleft. Instead of general cytoplasmic distribution, IFIT5 concentrated in actin-rich protrusions from the apical cell surface colocalized with the RNA-binding retinoic acid-inducible gene-I (RIG-I). These findings establish compartmentalized cellular RNA binding activity as a mechanism for IFIT5 function and reveal the TPR eddy as a scaffold for RNA recognition.
Asunto(s)
Proteínas de Neoplasias/metabolismo , ARN de Transferencia de Metionina/metabolismo , Actinas/metabolismo , Sustitución de Aminoácidos , Animales , Cristalografía por Rayos X , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/aislamiento & purificación , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/química , Proteínas de Neoplasias/aislamiento & purificación , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN de Transferencia de Metionina/química , Receptores InmunológicosRESUMEN
Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from Mycobacterium tuberculosis (Mtb). This enzyme transforms (E,E,E)-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the Mtb diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent Mycobacterium species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.
Asunto(s)
Transferasas Alquil y Aril/química , Diterpenos/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Ciclización/genética , Diterpenos/química , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/genéticaRESUMEN
Soybean lipoxygenase (SLO) has served as a prototype for understanding the molecular origin of enzymatic rate accelerations. The double mutant (DM) L546A/L754A is considered a dramatic outlier, due to the unprecedented size and near temperature-independence of its primary kinetic isotope effect, low catalytic efficiency, and elevated enthalpy of activation. To uncover the physical basis of these features, we herein apply three structural probes: hydrogen-deuterium exchange mass spectrometry, room-temperature X-ray crystallography and EPR spectroscopy on four SLO variants (wild-type (WT) enzyme, DM, and the two parental single mutants, L546A and L754A). DM is found to incorporate features of each parent, with the perturbation at position 546 predominantly influencing thermally activated motions that connect the active site to a protein-solvent interface, while mutation at position 754 disrupts the ligand field and solvation near the cofactor iron. However, the expanded active site in DM leads to more active site water molecules and their associated hydrogen bond network, and the individual features from L546A and L754A alone cannot explain the aggregate kinetic properties for DM. Using recently published QM/MM-derived ground-state SLO-substrate complexes for WT and DM, together with the thorough structural analyses presented herein, we propose that the impairment of DM is the combined result of a repositioning of the reactive carbon of linoleic acid substrate with regard to both the iron cofactor and a catalytically linked dynamic region of protein.
Asunto(s)
Coenzimas/metabolismo , Glycine max/enzimología , Lipooxigenasa/química , Lipooxigenasa/metabolismo , Metales/metabolismo , Mutación , Dominio Catalítico , Cinética , Lipooxigenasa/genética , Modelos Moleculares , Oxidación-Reducción , TermodinámicaRESUMEN
Recruitment of the P-TEFb kinase by HIV-1 Tat to the viral promoter triggers the phosphorylation and escape of RNA polymerase II from promoter-proximal pausing. It is unclear, however, if Tat recruits additional host factors that further stimulate HIV-1 transcription. Using a sequential affinity-purification scheme, we have identified human transcription factors/coactivators AFF4, ENL, AF9, and elongation factor ELL2 as components of the Tat-P-TEFb complex. Through the bridging functions of Tat and AFF4, P-TEFb and ELL2 combine to form a bifunctional elongation complex that greatly activates HIV-1 transcription. Without Tat, AFF4 can mediate the ELL2-P-TEFb interaction, albeit inefficiently. Tat overcomes this limitation by bringing more ELL2 to P-TEFb and stabilizing ELL2 in a process that requires active P-TEFb. The ability of Tat to enable two different classes of elongation factors to cooperate and coordinate their actions on the same polymerase enzyme explains why Tat is such a powerful activator of HIV-1 transcription.
Asunto(s)
VIH-1/genética , ARN Viral/biosíntesis , Proteínas Represoras/metabolismo , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión , Línea Celular , Quinasa 9 Dependiente de la Ciclina/metabolismo , VIH-1/metabolismo , Interacciones Huésped-Patógeno , Humanos , Complejos Multiproteicos , Proteínas Nucleares/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Interferencia de ARN , Proteínas Represoras/genética , Factores de Elongación Transcripcional/metabolismo , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å, binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a ßαßßß topology arranged radially in consecutive pairs to form two continuous eight-strand ß-sheets capped on both ends with an α-helix. The two ß-sheets intersect in the center at roughly a right angle and form two asymmetric deep "saddles" that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state {1H}-15N nuclear Overhauser effect, T1, and T2 values were generally uniform throughout the sequence with only a few modest pockets of differences. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.
Asunto(s)
Proteínas Bacterianas/metabolismo , Desoxiadenosinas/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Rojo Neutro/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Dicroismo Circular , Cristalografía por Rayos X , Desoxiadenosinas/química , Calor/efectos adversos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Ligandos , Conformación Molecular , Rojo Neutro/química , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Streptomyces coelicolor/metabolismo , Homología Estructural de ProteínaRESUMEN
Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Peptidoglicano/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Tuberculosis/metabolismo , Cristalografía por Rayos X , Humanos , Ligandos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/metabolismo , Tuberculosis/microbiologíaRESUMEN
Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with â¼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.
Asunto(s)
VIH-1/química , VIH-1/metabolismo , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/fisiología , Marcadores de Afinidad , Secuencia de Aminoácidos , Secuencia Conservada , Factor 3 de Iniciación Eucariótica/química , Factor 3 de Iniciación Eucariótica/metabolismo , Células HEK293 , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Proteasa del VIH/metabolismo , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/análisis , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/aislamiento & purificación , Humanos , Inmunoprecipitación , Células Jurkat , Espectrometría de Masas , Unión Proteica , Reproducibilidad de los Resultados , Replicación ViralRESUMEN
To increase the power of X-ray crystallography to determine not only the structures but also the motions of biomolecules, we developed methods to address two classic crystallographic problems: putting electron density maps on the absolute scale of e(-)/Å(3) and calculating the noise at every point in the map. We find that noise varies with position and is often six to eight times lower than thresholds currently used in model building. Analyzing the rescaled electron density maps from 485 representative proteins revealed unmodeled conformations above the estimated noise for 45% of side chains and a previously hidden, low-occupancy inhibitor of HIV capsid protein. Comparing the electron density maps in the free and nucleotide-bound structures of three human protein kinases suggested that substrate binding perturbs distinct intrinsic allosteric networks that link the active site to surfaces that recognize regulatory proteins. These results illustrate general approaches to identify and analyze alternative conformations, low-occupancy small molecules, solvent distributions, communication pathways, and protein motions.
Asunto(s)
Cristalografía por Rayos X/métodos , Electrones , Proteínas Quinasas/química , Proteínas/química , Sitio Alostérico , Anisotropía , Bacteriófago T4/química , Quinasa de la Caseína II/química , Dominio Catalítico , Simulación por Computador , Quinasa 2 Dependiente de la Ciclina/química , Proteínas Quinasas Asociadas a Muerte Celular/química , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Interleucina-1beta/química , Movimiento (Física) , Muramidasa/química , Conformación Proteica , Proteínas Tirosina Quinasas Receptoras/química , Receptor EphA3 , Reproducibilidad de los Resultados , Venenos de Escorpión/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
The positive transcription elongation factor b (P-TEFb) stimulates RNA polymerase elongation by inducing the transition of promoter proximally paused polymerase II into a productively elongating state. P-TEFb itself is regulated by reversible association with various transcription factors/cofactors to form several multisubunit complexes [e.g., the 7SK small nuclear ribonucleoprotein particle (7SK snRNP), the super elongation complexes (SECs), and the bromodomain protein 4 (Brd4)-P-TEFb complex] that constitute a P-TEFb network controlling cellular and HIV transcription. These complexes have been thought to share no components other than the core P-TEFb subunits cyclin-dependent kinase 9 (CDK9) and cyclin T (CycT, T1, T2a, and T2b). Here we show that the AF4/FMR2 family member 1 (AFF1) is bound to CDK9-CycT and is present in all major P-TEFb complexes and that the tripartite CDK9-CycT-AFF1 complex is transferred as a single unit within the P-TEFb network. By increasing the affinity of the HIV-encoded transactivating (Tat) protein for CycT1, AFF1 facilitates Tat's extraction of P-TEFb from 7SK snRNP and the formation of Tat-SECs for HIV transcription. Our data identify AFF1 as a ubiquitous P-TEFb partner and demonstrate that full Tat transactivation requires the complete SEC.
Asunto(s)
Ciclina T/química , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Factor B de Elongación Transcripcional Positiva/química , Ribonucleoproteínas Nucleares Pequeñas/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Alanina/genética , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Quinasa 9 Dependiente de la Ciclina/química , Células HeLa , Humanos , Proteínas Nucleares/química , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción/química , Activación Transcripcional , Factores de Elongación TranscripcionalRESUMEN
Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Factores de Virulencia/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Pared Celular/enzimología , Cristalografía por Rayos X , Activación Enzimática/fisiología , Hidrólisis , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/genética , Fenotipo , Conformación Proteica , Estructura Terciaria de Proteína , Transducción de Señal/fisiología , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.
Asunto(s)
Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Lípidos/biosíntesis , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/patogenicidad , Conformación Proteica , Transferasas Alquil y Aril/genética , Proteínas Bacterianas/genética , Cromatografía por Intercambio Iónico , Dimerización , Lípidos/química , Lípidos/genética , Espectrometría de Masas , Estructura Molecular , VirulenciaRESUMEN
Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative σ-factor SigF. Phosphorylation of Rv0516c regulated the abundance of EspA, a virulence-associated substrate of the type VII ESX-1 secretion system. These findings identify an osmosensory pathway orchestrated by PknD, Rv0516c, and SigF that enables adaptation to osmotic stress through cell wall remodeling and virulence factor production. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing.
Asunto(s)
Adaptación Biológica/fisiología , Regulación Bacteriana de la Expresión Génica/genética , Mycobacterium tuberculosis/fisiología , Presión Osmótica/fisiología , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Western Blotting , Proteínas Fluorescentes Verdes , Análisis por Micromatrices , Mycobacterium tuberculosis/enzimología , Concentración Osmolar , FosforilaciónRESUMEN
The HIV-1 Tat protein stimulates viral gene expression by recruiting human transcription elongation complexes containing P-TEFb, AFF4, ELL2, and ENL or AF9 to the viral promoter, but the molecular organization of these complexes remains unknown. To establish the overall architecture of the HIV-1 Tat elongation complex, we mapped the binding sites that mediate complex assembly in vitro and in vivo. The AFF4 protein emerges as the central scaffold that recruits other factors through direct interactions with short hydrophobic regions along its structurally disordered axis. Direct binding partners CycT1, ELL2, and ENL or AF9 act as bridging components that link this complex to two major elongation factors, P-TEFb and the PAF complex. The unique scaffolding properties of AFF4 allow dynamic and flexible assembly of multiple elongation factors and connect the components not only to each other but also to a larger network of transcriptional regulators.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , VIH-1 , Complejos Multiproteicos/metabolismo , Proteínas Represoras/metabolismo , Factores de Elongación Transcripcional/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión/genética , Western Blotting , Dicroismo Circular , Ciclina T/metabolismo , Electroforesis , Escherichia coli , Células HeLa , Humanos , Inmunoprecipitación , Luciferasas , Complejos Multiproteicos/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Proteínas Represoras/genética , Factores de Elongación Transcripcional/genéticaRESUMEN
Many Gram-positive bacteria coordinate cellular processes by signaling through Ser/Thr protein kinases (STPKs), but the architecture of these phosphosignaling cascades is unknown. To investigate the network structure of a prokaryotic STPK system, we comprehensively explored the pattern of signal transduction in the Mycobacterium tuberculosis Ser/Thr kinome. Autophosphorylation is the dominant mode of STPK activation, but the 11 M. tuberculosis STPKs also show a specific pattern of efficient cross-phosphorylation in vitro. The biochemical specificity intrinsic to each kinase domain was used to map the provisional signaling network, revealing a three-layer architecture that includes master regulators, signal transducers, and terminal substrates. Fluorescence microscopy revealed that the STPKs are specifically localized in the cell. Master STPKs are concentrated at the same subcellular sites as their substrates, providing additional support for the biochemically defined network. Together, these studies imply a branched functional architecture of the M. tuberculosis Ser/Thr kinome that could enable horizontal signal spreading. This systems-level approach provides a biochemical and spatial framework for understanding Ser/Thr phospho-signaling in M. tuberculosis, which differs fundamentally from previously defined linear histidine kinase cascades.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas Bacterianas/genética , Activación Enzimática/fisiología , Mycobacterium tuberculosis/genética , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo-protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.
Asunto(s)
Pared Celular/enzimología , Complejos Multienzimáticos/metabolismo , Mycobacterium smegmatis/enzimología , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , División Celular , ADN Bacteriano/análisis , Inhibidores Enzimáticos/farmacología , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/ultraestructura , N-Acetil Muramoil-L-Alanina Amidasa/antagonistas & inhibidores , ProteolisisRESUMEN
A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a timescale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme.
Asunto(s)
Cristalografía por Rayos X/métodos , Ciclofilina A/química , Modelos Moleculares , Catálisis , Ciclofilina A/genética , Humanos , Mutación , Estructura Terciaria de Proteína , TemperaturaRESUMEN
Peptidoglycan hydrolases are key enzymes in bacterial cell wall homeostasis. Understanding the substrate specificity and biochemical activity of peptidoglycan hydrolases in Mycobacterium tuberculosis is of special interest as it can aid in the development of new cell wall targeting therapeutics. In this study, we report biochemical and structural characterization of the mycobacterial N-acetylmuramyl-L-alanine amidase, Rv3717. The crystal structure of Rv3717 in complex with a dipeptide product shows that, compared with previously characterized peptidoglycan amidases, the enzyme contains an extra disulfide-bonded ß-hairpin adjacent to the active site. The structure of two intermediates in assembly reveal that Zn(2+) binding rearranges active site residues, and disulfide formation promotes folding of the ß-hairpin. Although Zn(2+) is required for hydrolysis of muramyl dipeptide, disulfide oxidation is not required for activity on this substrate. The orientation of the product in the active site suggests a role for a conserved glutamate (Glu-200) in catalysis; mutation of this residue abolishes activity. The product binds at the head of a closed tunnel, and the enzyme showed no activity on polymerized peptidoglycan. These results point to a potential role for Rv3717 in peptidoglycan fragment recycling.
Asunto(s)
Amidohidrolasas/química , Proteínas Bacterianas/química , Dipéptidos/química , Mycobacterium tuberculosis/enzimología , Peptidoglicano/química , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dipéptidos/genética , Dipéptidos/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Mycobacterium tuberculosis/genética , Peptidoglicano/genética , Peptidoglicano/metabolismo , Zinc/química , Zinc/metabolismoRESUMEN
The enzyme soybean lipoxygenase (SLO) has served as a prototype for hydrogen-tunneling reactions, as a result of its unusual kinetic isotope effects (KIEs) and their temperature dependencies. Using a synergy of kinetic, structural, and theoretical studies, we show how the interplay between donor-acceptor distance and active-site flexibility leads to catalytic behavior previously predicted by quantum tunneling theory. Modification of the size of two hydrophobic residues by site-specific mutagenesis in SLO reduces the reaction rate 10(4)-fold and is accompanied by an enormous and unprecedented room-temperature KIE. Fitting of the kinetic data to a non-adiabatic model implicates an expansion of the active site that cannot be compensated by donor-acceptor distance sampling. A 1.7 Å resolution X-ray structure of the double mutant further indicates an unaltered backbone conformation, almost identical side-chain conformations, and a significantly enlarged active-site cavity. These findings show the compelling property of room-temperature hydrogen tunneling within a biological context and demonstrate the very high sensitivity of such tunneling to barrier width.
Asunto(s)
Carbono/química , Hidrógeno/química , Lipooxigenasa/química , Lipooxigenasa/metabolismo , Sitios de Unión , Catálisis , Simulación por Computador , Transporte de Electrón , Activación Enzimática , Isótopos/química , Cinética , Ácidos Linoleicos/química , Lipooxigenasa/genética , Modelos Moleculares , Mutación , Conformación Proteica , Teoría Cuántica , Glycine max/enzimología , Especificidad por Sustrato , TemperaturaRESUMEN
The in planta association of the Hyaloperonospora arabidopsidis effector ATR1 with the cognate Arabidopsis thaliana RPP1 immune receptor activates a disease-resistance signaling pathway that inhibits pathogen growth. To define the molecular events specifying effector recognition by RPP1, we determined the crystal structure of ATR1 and assayed in planta the effects of surface polymorphisms that are critical to activating plant immunity. ATR1 adopts an elongated, all-helical, two-domain, seahorse-like structure with an overall architecture unlike any previously described fold. Structural comparisons highlight a tandemly duplicated, five-helix motif in the C-terminal domain that creates a structural framework for rapid diversification. Identification and mapping of critical recognition sites suggest that ATR1 detection by the RPP1 resistance protein is mediated by several distinct protein surfaces that allow the effectors to escape recognition through diverse surface polymorphisms. ATR1 gain-of-recognition mutants demonstrate that multiple amino acid substitutions are necessary for recognition and that surface polymorphisms exert additive effects. These results suggest that ATR1 is a modular repeat protein belonging to an ancient family of oomycete effectors that rapidly evolves to escape host detection and adopt diverse virulence functions.
Asunto(s)
Oomicetos/metabolismo , Proteínas/química , Proteínas/metabolismo , Secuencias Repetitivas de Aminoácido , Alelos , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Arabidopsis/parasitología , Proteínas de Arabidopsis , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The Super Elongation Complex (SEC), containing transcription elongation activators/coactivators P-TEFb, ELL2, AFF4/1, ENL, and AF9, is recruited by HIV-1 Tat and mixed lineage leukemia (MLL) proteins to activate the expression of HIV-1 and MLL-target genes, respectively. In the absence of Tat and MLL, however, it is unclear how SEC is targeted to RNA polymerase (Pol) II to stimulate elongation in general. Furthermore, although ENL and AF9 can bind the H3K79 methyltransferase Dot1L, it is unclear whether these bindings are required for SEC-mediated transcription. Here, we show that the homologous ENL and AF9 exist in separate SECs with similar but nonidentical functions. ENL/AF9 contacts the scaffolding protein AFF4 that uses separate domains to recruit different subunits into SEC. ENL/AF9 also exists outside SEC when bound to Dot1L, which is found to inhibit SEC function. The YEATS domain of ENL/AF9 targets SEC to Pol II on chromatin through contacting the human Polymerase-Associated Factor complex (PAFc) complex. This finding explains the YEATS domain's dispensability for leukemogenesis when ENL/AF9 is translocated to MLL, whose interactions with PAFc and DNA likely substitute for the PAFc/chromatin-targeting function of the YEATS domain.