RESUMEN
Bruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Agammaglobulinemia Tirosina Quinasa , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Xenoinjertos , Resistencia a Antineoplásicos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéuticoRESUMEN
Early efforts to broaden the spectrum and potency of cyclic boronic acid ß-lactamase inhibitor vaborbactam included a series of 7-membered ring boronates. Exploration of stereoisomers and incorporation of heteroatoms allowed identification of the all-carbon cyclic boronate with substituents trans as the preferred core structure, showing inhibition of Class A and C enzymes. Crystal structures of one analog bound to important ß-lactamase enzymes were obtained. When isolated under acidic conditions, these compounds spontaneously formed a neutral cyclic anhydride (intramolecular prodrug) which was shown to have much-improved oral bioavailability (52-69%) compared to the ring-opened carboxylate salt (9%).
Asunto(s)
Profármacos , Inhibidores de beta-Lactamasas , Antibacterianos/química , Antibacterianos/farmacología , Disponibilidad Biológica , Profármacos/farmacología , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/metabolismoRESUMEN
Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg(2+). We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg(2+), and VO4); a RbsAC complex in the presence of ADP and Mg(2+); and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Unión Periplasmáticas/metabolismo , Ribosa/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico/genética , Western Blotting , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Escherichia coli/genética , Magnesio/metabolismo , Proteínas de Transporte de Membrana/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Proteínas de Unión Periplasmáticas/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Ebolaviruses cause severe hemorrhagic fever. Central to the Ebola life cycle is the matrix protein VP40, which oligomerizes and drives viral budding. Here we present the crystal structure of the Sudan virus (SUDV) matrix protein. This structure is higher resolution (1.6 Å) than previously achievable. Despite differences in the protein purification, we find that it still forms a stable dimer in solution, as was noted for other Ebola VP40s. Although the N-terminal domain interface by which VP40 dimerizes is conserved between Ebola virus and SUDV, the C-terminal domain interface by which VP40 dimers may further assemble is significantly smaller in this SUDV assembly.
Asunto(s)
Ebolavirus/química , Proteínas de la Matriz Viral/química , Ebolavirus/metabolismo , Escherichia coli/metabolismo , Multimerización de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Soluciones/química , Sudán , Proteínas de la Matriz Viral/metabolismoRESUMEN
The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear. Here, we identify the M. tuberculosis protein Rv1496 and its homologs in M. smegmatis and M. thermoresistibile as MeaB. The crystal structures of all three homologs are highly similar to MeaB and MMAA structures and reveal a characteristic three-domain homodimer with GDP bound in the G domain active site. A structure of Rv1496 obtained from a crystal grown in the presence of GTP exhibited electron density for GDP, suggesting GTPase activity. These structures identify the mycobacterial MeaB and provide a structural framework for therapeutic targeting of M. tuberculosis MeaB.
Asunto(s)
Proteínas Bacterianas/química , GTP Fosfohidrolasas/química , Mycobacterium tuberculosis/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Mycobacterium tuberculosis/genéticaRESUMEN
The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an â¼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD+ and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.
Asunto(s)
Proteínas Bacterianas , Mycobacterium smegmatis , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Modelos Moleculares , Secuencia de Aminoácidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, is an essential effector of B-cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematological malignancies, which makes it an attractive therapeutic target. Pharmacological inhibition of BTK enzymatic function is now a well-proven strategy for the treatment of patients with these malignancies. We report the discovery and characterization of NX-2127, a BTK degrader with concomitant immunomodulatory activity. By design, NX-2127 mediates the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interactions with the cereblon E3 ubiquitin ligase complex. NX-2127 degrades common BTK resistance mutants, including BTKC481S. NX-2127 is orally bioavailable, exhibits in vivo degradation across species, and demonstrates efficacy in preclinical oncology models. NX-2127 has advanced into first-in-human clinical trials and achieves deep and sustained degradation of BTK following daily oral dosing at 100 mg.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas Tirosina Quinasas , Humanos , Agammaglobulinemia Tirosina Quinasa , Inhibidores de Proteínas Quinasas/efectos adversos , Transducción de SeñalRESUMEN
Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.
Asunto(s)
Anemia de Células Falciformes , Antidrepanocíticos , Hemoglobina Fetal , Factores de Transcripción de Tipo Kruppel , Proteínas del Tejido Nervioso , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/metabolismo , Antidrepanocíticos/química , Antidrepanocíticos/farmacología , Antidrepanocíticos/uso terapéutico , Cristalografía por Rayos X , Descubrimiento de Drogas , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Macaca fascicularis , Proteínas del Tejido Nervioso/metabolismo , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The design of cereblon-binding molecular glues (MGs) that selectively recruit a desired protein while excluding teratogenic SALL4 is an area of significant interest when designing therapeutic agents. Previous studies show that SALL4 is degraded in the presence of IKZF1 degraders pomalidomide, and to a lesser extent by CC-220. To expand our understanding of the molecular basis for the interaction of SALL4 with cereblon, we performed biophysical and structural studies demonstrating that SALL4 zinc finger domains one and two (ZF1-2) interact with cereblon (CRBN) in a unique manner. ZF1 interacts with the N-terminal domain of cereblon and ZF2 binds as expected in the C-terminal IMiD-binding domain. Both ZF1 and ZF2 contribute to the potency of the interaction of ZF1-2 with CRBN:MG complexes and the affinities of SALL4 ZF1-2 for the cereblon:CC-220 complex are less potent than for the corresponding pomalidomide complex. Structural analysis provides a rationale for understanding the reduced affinity of SALL4 for cereblon in the presence of CC-220, which engages both ZF1 and ZF2. These studies further our understanding of the molecular glue-mediated interactions of zinc finger-based proteins with cereblon and may provide structural tools for the prospective design of compounds with reduced binding and degradation of SALL4.
Asunto(s)
Talidomida , Dedos de Zinc , Talidomida/farmacología , Talidomida/química , Teratógenos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Malignant tumors can evade destruction by the immune system by attracting immune-suppressive regulatory T cells (Treg) cells. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Treg cells, and IKZF2 deficiency reduces tumor growth in mice. Here we report the discovery of NVP-DKY709, a selective molecular glue degrader of IKZF2 that spares IKZF1/3. We describe the recruitment-guided medicinal chemistry campaign leading to NVP-DKY709 that redirected the degradation selectivity of cereblon (CRBN) binders from IKZF1 toward IKZF2. Selectivity of NVP-DKY709 for IKZF2 was rationalized by analyzing the DDB1:CRBN:NVP-DKY709:IKZF2(ZF2 or ZF2-3) ternary complex X-ray structures. Exposure to NVP-DKY709 reduced the suppressive activity of human Treg cells and rescued cytokine production in exhausted T-effector cells. In vivo, treatment with NVP-DKY709 delayed tumor growth in mice with a humanized immune system and enhanced immunization responses in cynomolgus monkeys. NVP-DKY709 is being investigated in the clinic as an immune-enhancing agent for cancer immunotherapy.
Asunto(s)
Neoplasias , Factores de Transcripción , Animales , Humanos , Ratones , Factor de Transcripción Ikaros , Inmunoterapia , Neoplasias/terapia , Neoplasias/metabolismo , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Here, we report that lipocalin 2 (Lcn2) promotes breast cancer progression, and we identify the mechanisms underlying this function. We first found that Lcn2 levels were consistently associated with invasive breast cancer in human tissue and urine samples. To investigate the function of Lcn2 in breast cancer progression, Lcn2 was overexpressed in human breast cancer cells and was found to up-regulate mesenchymal markers, including vimentin and fibronectin, down-regulate the epithelial marker E-cadherin, and significantly increase cell motility and invasiveness. These changes in marker expression and cell motility are hallmarks of an epithelial to mesenchymal transition (EMT). In contrast, Lcn2 silencing in aggressive breast cancer cells inhibited cell migration and the mesenchymal phenotype. Furthermore, reduced expression of estrogen receptor (ER) alpha and increased expression of the key EMT transcription factor Slug were observed with Lcn2 expression. Overexpression of ERalpha in Lcn2-expressing cells reversed the EMT and reduced Slug expression, suggesting that ERalpha negatively regulates Lcn2-induced EMT. Finally, orthotopic studies demonstrated that Lcn2-expressing breast tumors displayed a poorly differentiated phenotype and showed increased local tumor invasion and lymph node metastasis. Taken together, these in vitro, in vivo, and human studies demonstrate that Lcn2 promotes breast cancer progression by inducing EMT through the ERalpha/Slug axis and may be a useful biomarker of breast cancer.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Neoplasias de la Mama/patología , Lipocalinas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Fase Aguda/orina , Animales , Neoplasias de la Mama/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Endotelio/patología , Receptor alfa de Estrógeno/metabolismo , Femenino , Silenciador del Gen , Humanos , Lipocalina 2 , Lipocalinas/orina , Metástasis Linfática , Mesodermo/patología , Ratones , Invasividad Neoplásica , Estadificación de Neoplasias , Fenotipo , Proteínas Proto-Oncogénicas/orina , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis. RESULTS: Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD) phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB. CONCLUSION: The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Coccidioides/enzimología , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Yoduros/química , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulosafosfatos/química , Ribulosafosfatos/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Antimicrobial proteins comprise a significant component of the acute innate immune response to infection. They are induced by pattern recognition receptors as well as by cytokines of the innate and adaptive immune pathways and play important roles in infection control and immunomodulatory homeostasis. Lipocalin 2 (siderocalin, NGAL, 24p3), a siderophore-binding antimicrobial protein, is critical for control of systemic infection with Escherichia coli; however, its role in mucosal immunity in the respiratory tract is unknown. In this study, we found that lipocalin 2 is rapidly and robustly induced by Klebsiella pneumoniae infection and is TLR4 dependent. IL-1beta and IL-17 also individually induce lipocalin 2. Mucosal administration of IL-1beta alone could reconstitute the lipocalin 2 deficiency in TLR4 knockout animals and rescue them from infection. Lipocalin 2-deficient animals have impaired lung bacterial clearance in this model and mucosal reconstitution of lipocalin 2 protein in these animals resulted in rescue of this phenotype. We conclude that lipocalin 2 is a crucial component of mucosal immune defense against pulmonary infection with K. pneumoniae.
Asunto(s)
Proteínas de Fase Aguda/inmunología , Proteínas de Fase Aguda/metabolismo , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/metabolismo , Lipocalinas/inmunología , Lipocalinas/metabolismo , Proteínas Oncogénicas/inmunología , Proteínas Oncogénicas/metabolismo , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Fase Aguda/genética , Animales , Bronquios/metabolismo , Línea Celular , Epitelio/metabolismo , Humanos , Interleucina-17/farmacología , Interleucina-1beta/farmacología , Infecciones por Klebsiella/genética , Infecciones por Klebsiella/patología , Lipocalina 2 , Lipocalinas/genética , Ratones , Ratones Noqueados , Proteínas Oncogénicas/genética , Neumonía Bacteriana/genética , Neumonía Bacteriana/patología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Cystathionine γ-synthase (CGS) is a transulfurication enzyme that catalyzes the first specific step in L-methionine biosynthesis by the reaction of O(4)-succinyl-L-homoserine and L-cysteine to produce L-cystathionine and succinate. Controlling the first step in L-methionine biosythesis, CGS is an excellent potential drug target. Mycobacterium ulcerans is a slow-growing mycobacterium that is the third most common form of mycobacterial infection, mainly infecting people in Africa, Australia and Southeast Asia. Infected patients display a variety of skin ailments ranging from indolent non-ulcerated lesions as well as ulcerated lesions. Here, the crystal structure of CGS from M. ulcerans covalently linked to the cofactor pyridoxal phosphate (PLP) is reported at 1.9 Šresolution. A second structure contains PLP as well as a highly ordered HEPES molecule in the active site acting as a pseudo-ligand. These results present the first structure of a CGS from a mycobacterium and allow comparison with other CGS enzymes. This is also the first structure reported from the pathogen M. ulcerans.
Asunto(s)
Liasas de Carbono-Oxígeno/química , Mycobacterium ulcerans/enzimología , Dominio Catalítico , Modelos Moleculares , Estructura Cuaternaria de Proteína , Electricidad EstáticaRESUMEN
Recent studies have revealed that the mammalian immune system directly interferes with siderophore-mediated iron acquisition through siderophore-binding proteins and that the association of certain siderophores, or siderophore modifications, with virulence is a direct response of pathogens to evade these defenses.
Asunto(s)
Inmunidad Innata/fisiología , Lipocalinas/química , Lipocalinas/metabolismo , Sideróforos/metabolismo , Animales , Humanos , Lipocalinas/fisiología , Unión Proteica/genética , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Siderocalin/Lipocalin 2/Neutrophil Gelatinase Associated Lipocalin/24p3 is an innate immune system protein with bacteriostatic activity, acting by tightly binding and sequestering diverse catecholate and mixed-type ferric siderophores from enteric bacteria and mycobacteria. Bacterial virulence achieved through siderophore modifications, or utilization of alternate siderophores, can be explained by evasion of Siderocalin binding. Siderocalin has also been implicated in a wide variety of disease processes, though often in seemingly contradictory ways, and has been proposed to bind to a broader array of ligands beyond siderophores. Using structural, directed mutational, and binding studies, we have sought to rigorously test, and fully elucidate, the Siderocalin recognition mechanism. Several proposed ligands fail to meet rigorous binding criteria, including the bacterial siderophore pyochelin, the iron-chelating catecholamine hormone norepinephrine, and the bacterial second messenger cyclic diguanylate monophosphate. While possessing a remarkably rigid structure, in principle simplifying analyses of ligand recognition, understanding Scn recognition is complicated by the observed conformational and stoichiometric plasticity, and instability, of its bona fide siderophore ligands. Since the role of Siderocalin at the early host/pathogen interface is to compete for bacterial ferric siderophores, we also analyzed how bacterial siderophore binding proteins and enzymes alternately recognize siderophores that efficiently bind to, or evade, Siderocalin sequestration - including determining the crystal structure of Bacillus cereus YfiY bound to schizokinen. These studies combine to refine the potential physiological functions of Siderocalin by defining its multiplexed recognition mechanism.
RESUMEN
The siderophore enterobactin (Ent) is produced by enteric bacteria to mediate iron uptake. Ent scavenges iron and is taken up by the bacteria as the highly stable ferric complex [Fe (III)(Ent)] (3-). This complex is also a specific target of the mammalian innate immune system protein, Siderocalin (Scn), which acts as an antibacterial agent by specifically sequestering siderophores and their ferric complexes during infection. Recent literature suggesting that Scn may also be involved in cellular iron transport has increased the importance of understanding the mechanism of siderophore interception and clearance by Scn; Scn is observed to release iron in acidic endosomes and [Fe (III)(Ent)] (3-) is known to undergo a change from catecholate to salicylate coordination in acidic conditions, which is predicted to be sterically incompatible with the Scn binding pocket (also referred to as the calyx). To investigate the interactions between the ferric Ent complex and Scn at different pH values, two recombinant forms of Scn with mutations in three residues lining the calyx were prepared: Scn-W79A/R81A and Scn-Y106F. Binding studies and crystal structures of the Scn-W79A/R81A:[Fe (III)(Ent)] (3-) and Scn-Y106F:[Fe (III)(Ent)] (3-) complexes confirm that such mutations do not affect the overall conformation of the protein but do weaken significantly its affinity for [Fe (III)(Ent)] (3-). Fluorescence, UV-vis, and EXAFS spectroscopies were used to determine Scn/siderophore dissociation constants and to characterize the coordination mode of iron over a wide pH range, in the presence of both mutant proteins and synthetic salicylate analogues of Ent. While Scn binding hinders salicylate coordination transformation, strong acidification results in the release of iron and degraded siderophore. Iron release may therefore result from a combination of Ent degradation and coordination change.
Asunto(s)
Enterobacteriaceae/química , Enterobactina/química , Hierro/metabolismo , Mamíferos/inmunología , Sideróforos/química , Animales , Sitios de Unión , Transporte Biológico , Enterobacteriaceae/metabolismo , Enterobactina/metabolismo , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Concentración de Iones de Hidrógeno , Mamíferos/metabolismo , Estructura Molecular , Salicilatos/química , Salicilatos/metabolismo , Sideróforos/metabolismo , Análisis EspectralRESUMEN
The Bas-Congo virus (BASV) is the first rhabdovirus associated with a human outbreak of acute hemorrhagic fever. The single-stranded, negative-sense RNA genome of BASV contains the five core genes present in all rhabdoviral genomes plus an additional three genes, annotated U1, U2, and U3, with weak (<21%) sequence similarity only to a handful of genes observed in a few other rhabdoviral genomes. The function of the rhabdoviral U proteins is unknown, but, they are hypothesized to play a role in viral infection or replication. To better understand this unique family of proteins, a construct containing residues 27-203 of the 216-residue U1 protein (BASV-U1*) was prepared. By collecting data in 0.5 M urea it was possible to eliminate transient association enough to enable the assignment of most of the observable 1HN, 1Hα, 15N, 13Cα, 13Cß, and 13C´ chemical shifts for BASV-U1* that will provide a foundation to study its solution properties. The analyses of these chemical shifts along with 15N-edited NOESY data enabled the identification of the elements of secondary structure present in BASV-U1*.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Rhabdoviridae , Proteínas Virales/química , Estructura Secundaria de Proteína , Proteínas Virales/metabolismoRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) charge tRNAs with their cognate amino acid, an essential precursor step to loading of charged tRNAs onto the ribosome and addition of the amino acid to the growing polypeptide chain during protein synthesis. Because of this important biological function, aminoacyl-tRNA synthetases have been the focus of anti-infective drug development efforts and two aaRS inhibitors have been approved as drugs. Several researchers in the scientific community requested aminoacyl-tRNA synthetases to be targeted in the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure determination pipeline. Here we investigate thirty-one aminoacyl-tRNA synthetases from infectious disease organisms by co-crystallization in the presence of their cognate amino acid, ATP, and/or inhibitors. Crystal structures were determined for a CysRS from Borrelia burgdorferi bound to AMP, GluRS from Borrelia burgdorferi and Burkholderia thailandensis bound to glutamic acid, a TrpRS from the eukaryotic pathogen Encephalitozoon cuniculi bound to tryptophan, a HisRS from Burkholderia thailandensis bound to histidine, and a LysRS from Burkholderia thailandensis bound to lysine. Thus, the presence of ligands may promote aaRS crystallization and structure determination. Comparison with homologous structures shows conformational flexibility that appears to be a recurring theme with this enzyme class.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Borrelia burgdorferi/enzimología , Burkholderia/enzimología , Encephalitozoon cuniculi/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Enfermedades Transmisibles/microbiología , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.