RESUMO
Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle. Egress is regulated by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and optimization of substrate-based peptidic boronic acids that inhibit Plasmodium falciparum SUB1 with low nanomolar potency. Structural optimization generated membrane-permeable, slow off-rate inhibitors that prevent Pfalciparum egress through direct inhibition of SUB1 activity and block parasite replication in vitro at submicromolar concentrations. Our results validate SUB1 as a potential target for a new class of antimalarial drugs designed to prevent parasite replication and disease progression.
Assuntos
Antimaláricos/farmacologia , Ácidos Borônicos/farmacologia , Peptídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/química , Subtilisinas/química , Antimaláricos/síntese química , Sítios de Ligação , Ácidos Borônicos/síntese química , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Expressão Gênica , Humanos , Cinética , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/fisiologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Peptídeos/síntese química , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Subtilisinas/antagonistas & inibidores , Subtilisinas/genética , Subtilisinas/metabolismo , TermodinâmicaRESUMO
Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACß) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.
Assuntos
AMP Cíclico/metabolismo , Interações Hospedeiro-Parasita , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Parasitos/metabolismo , Transdução de Sinais , Adenilil Ciclases/metabolismo , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Parasitos/enzimologia , Parasitos/crescimento & desenvolvimento , Parasitos/ultraestrutura , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/patogenicidade , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae (Sc_IGPD) and Arabidopsis thaliana (At_IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure-activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against Sc_IGPD than At_IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the At and Sc_IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at â¼3 Å are sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the At_IGPD/C348 complex. The structure of Sc_IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding.
Assuntos
Proteínas de Arabidopsis/antagonistas & inibidores , Arabidopsis/enzimologia , Inibidores Enzimáticos/química , Hidroliases/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , Arabidopsis/química , Arabidopsis/efeitos dos fármacos , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Herbicidas/química , Hidroliases/química , Hidroliases/ultraestrutura , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Programs of drug discovery generally exploit one enantiomer of a chiral compound for lead development following the principle that enantiomer recognition is central to biological specificity. However, chiral promiscuity has been identified for a number of enzyme families, which have shown that mirror-image packing can enable opposite enantiomers to be accommodated in an enzyme's active site. Reported here is a series of crystallographic studies of complexes between an enzyme and a potent experimental herbicide whose chiral center forms an essential part of the inhibitor pharmacophore. Initial studies with a racemate at 1.85â Å resolution failed to identify the chirality of the bound inhibitor, however, by extending the resolution to 1.1â Å and by analyzing high-resolution complexes with the enantiopure compounds, we determined that both enantiomers make equivalent pseudosymmetric interactions in the active site, thus mimicking an achiral reaction intermediate.
RESUMO
The organic polymer lignin is a component of plant cell walls, which like (hemi)-cellulose is highly abundant in nature and relatively resistant to degradation. However, extracellular enzymes released by natural microbial consortia can cleave the ß-aryl ether linkages in lignin, releasing monoaromatic phenylpropanoids that can be further catabolised by diverse species of bacteria. Biodegradation of lignin is therefore important in global carbon cycling, and its natural abundance also makes it an attractive biotechnological feedstock for the industrial production of commodity chemicals. Whilst the pathways for degradation of lignin-derived aromatics have been extensively characterised, much less is understood about how they are recognised and taken up from the environment. The purple phototrophic bacterium Rhodopseudomonas palustris can grow on a range of phenylpropanoid monomers and is a model organism for studying their uptake and breakdown. R. palustris encodes a tripartite ATP-independent periplasmic (TRAP) transporter (TarPQM) linked to genes encoding phenylpropanoid-degrading enzymes. The periplasmic solute-binding protein component of this transporter, TarP, has previously been shown to bind aromatic substrates. Here, we determine the high-resolution crystal structure of TarP from R. palustris as well as the structures of homologous proteins from the salt marsh bacterium Sagittula stellata and the halophile Chromohalobacter salexigens, which also grow on lignin-derived aromatics. In combination with tryptophan fluorescence ligand-binding assays, our ligand-bound co-crystal structures reveal the molecular basis for high-affinity recognition of phenylpropanoids by these TRAP transporters, which have potential for improving uptake of these compounds for biotechnological transformations of lignin.
Assuntos
Proteínas de Bactérias/genética , Biodegradação Ambiental , Lignina/genética , Proteínas de Ligação a RNA/genética , Rodopseudomonas/genética , Fatores de Transcrição/genética , Transporte Biológico/genética , Regulação Bacteriana da Expressão Gênica/genética , Ligantes , Lignina/química , Lignina/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Oxirredutases/genética , Periplasma/genética , Periplasma/microbiologia , Proteínas Periplásmicas de Ligação/genética , Proteobactérias/genética , Proteobactérias/crescimento & desenvolvimento , Rodopseudomonas/crescimento & desenvolvimentoRESUMO
Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use ß-phosphoglucomutase (ßPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. ßPGM catalyzes the isomerization of ß-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. ßPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported ß-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form.
RESUMO
Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.
Assuntos
Tomografia com Microscopia Eletrônica , Elétrons , Microscopia Crioeletrônica , Congelamento , Humanos , Microscopia Eletrônica de VarreduraRESUMO
Malaria parasites cause disease through repeated cycles of intraerythrocytic proliferation. Within each cycle, several rounds of DNA replication produce multinucleated forms, called schizonts, that undergo segmentation to form daughter merozoites. Upon rupture of the infected cell, the merozoites egress to invade new erythrocytes and repeat the cycle. In human malarial infections, an antibody response specific for the Plasmodium falciparum protein PF3D7_1021800 was previously associated with protection against malaria, leading to an interest in PF3D7_1021800 as a candidate vaccine antigen. Antibodies to the protein were reported to inhibit egress, resulting in it being named schizont egress antigen-1 (SEA1). A separate study found that SEA1 undergoes phosphorylation in a manner dependent upon the parasite cGMP-dependent protein kinase PKG, which triggers egress. While these findings imply a role for SEA1 in merozoite egress, this protein has also been implicated in kinetochore function during schizont development. Therefore, the function of SEA1 remains unclear. Here, we show that P. falciparum SEA1 localizes in proximity to centromeres within dividing nuclei and that conditional disruption of SEA1 expression severely impacts the distribution of DNA and formation of merozoites during schizont development, with a proportion of SEA1-null merozoites completely lacking nuclei. SEA1-null schizonts rupture, albeit with low efficiency, suggesting that neither SEA1 function nor normal segmentation is a prerequisite for egress. We conclude that SEA1 does not play a direct mechanistic role in egress but instead acts upstream of egress as an essential regulator required to ensure the correct packaging of nuclei within merozoites.IMPORTANCE Malaria is a deadly infectious disease. Rationally designed novel therapeutics will be essential for its control and eradication. The Plasmodium falciparum protein PF3D7_1021800, annotated as SEA1, is under investigation as a prospective component of a malaria vaccine, based on previous indications that antibodies to SEA1 interfere with parasite egress from infected erythrocytes. However, a consensus on the function of SEA1 is lacking. Here, we demonstrate that SEA1 localizes to dividing parasite nuclei and is necessary for the correct segregation of replicated DNA into individual daughter merozoites. In the absence of SEA1, merozoites develop defectively, often completely lacking a nucleus, and, consequently, egress is impaired and/or aberrant. Our findings provide insights into the divergent mechanisms by which intraerythrocytic malaria parasites develop and divide. Our conclusions regarding the localization and function of SEA1 are not consistent with the hypothesis that antibodies against it confer protective immunity to malaria by blocking merozoite egress.
Assuntos
Antígenos de Protozoários/genética , Eritrócitos/parasitologia , Merozoítos/genética , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/genética , Esquizontes/fisiologia , Antígenos de Protozoários/metabolismo , Divisão Celular , Humanos , Merozoítos/química , Fosforilação , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Estudos Prospectivos , Proteínas de Protozoários/metabolismoRESUMO
The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons.
Assuntos
Clorofila/biossíntese , Ativação Enzimática , Liases/metabolismo , Fotossíntese/fisiologia , Protoporfirinas/metabolismo , Thermosynechococcus/metabolismoRESUMO
The most severe form of human malaria is caused by Plasmodium falciparum. Its virulence is closely linked to the increase in rigidity of infected erythrocytes and their adhesion to endothelial receptors, obstructing blood flow to vital organs. Unlike other human-infecting Plasmodium species, P. falciparum exports a family of 18 FIKK serine/threonine kinases into the host cell, suggesting that phosphorylation may modulate erythrocyte modifications. We reveal substantial species-specific phosphorylation of erythrocyte proteins by P. falciparum but not by Plasmodium knowlesi, which does not export FIKK kinases. By conditionally deleting all FIKK kinases combined with large-scale quantitative phosphoproteomics we identified unique phosphorylation fingerprints for each kinase, including phosphosites on parasite virulence factors and host erythrocyte proteins. Despite their non-overlapping target sites, a network analysis revealed that some FIKKs may act in the same pathways. Only the deletion of the non-exported kinase FIKK8 resulted in reduced parasite growth, suggesting the exported FIKKs may instead support functions important for survival in the host. We show that one kinase, FIKK4.1, mediates both rigidification of the erythrocyte cytoskeleton and trafficking of the adhesin and key virulence factor PfEMP1 to the host cell surface. This establishes the FIKK family as important drivers of parasite evolution and malaria pathology.
Assuntos
Eritrócitos/metabolismo , Eritrócitos/parasitologia , Malária/metabolismo , Malária/parasitologia , Fosfotransferases/metabolismo , Plasmodium/fisiologia , Proteínas de Protozoários/metabolismo , Deleção de Genes , Técnicas de Silenciamento de Genes , Marcação de Genes , Humanos , Família Multigênica , Fosfoproteínas , Fosforilação , Fosfotransferases/genética , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Especificidade da Espécie , VirulênciaRESUMO
Phosphorus acquisition is critical for life. In low phosphate conditions, some species of bacteria have evolved mechanisms to import reduced phosphorus compounds, such as phosphite and hypophosphite, as alternative phosphorus sources. Uptake is facilitated by high-affinity periplasmic binding proteins (PBPs) that bind cargo in the periplasm and shuttle it to an ATP-binding cassette (ABC)-transporter in the bacterial inner membrane. PtxB and HtxB are the PBPs responsible for binding phosphite and hypophosphite, respectively. They recognize the P-H bond of phosphite/hypophosphite via a conserved P-H...π interaction, which confers nanomolar dissociation constants for their respective ligands. PtxB also has a low-level binding affinity for phosphate and hypophosphite, whilst HtxB can facilitate phosphite uptake in vivo. However, HtxB does not bind phosphate, thus the HtxBCDE transporter has recently been successfully exploited for biocontainment of genetically modified organisms by phosphite-dependent growth. Here we use a combination of X-ray crystallography, NMR and Microscale Thermophoresis to show that phosphite binding to HtxB depends on the protonation state of the ligand, suggesting that pH may effect the efficiency of phosphite uptake by HtxB in biotechnology applications.
Assuntos
Proteínas Periplásmicas de Ligação/metabolismo , Fosfitos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Cristalografia por Raios X/métodos , Ligantes , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/fisiologia , Fosfatos/química , Fosfatos/metabolismo , Fosfitos/química , Fósforo/química , Fósforo/metabolismo , Ligação ProteicaRESUMO
The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.
Assuntos
Aeromonas hydrophila/metabolismo , Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aeromonas hydrophila/química , Aeromonas hydrophila/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismoRESUMO
Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 µM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.
RESUMO
[This corrects the article DOI: 10.1038/s42003-019-0587-z.].
RESUMO
Acanthamoeba is normally free-living, but sometimes facultative and occasionally opportunistic parasites. Current therapies are, by necessity, arduous and yet poorly effective due to their inabilities to kill cyst stages or in some cases to actually induce encystation. Acanthamoeba can therefore survive as cysts and cause disease recurrence. Herein, in pursuit of better therapies and to understand the biochemistry of this understudied organism, we characterize its histidine biosynthesis pathway and explore the potential of targeting this with antimicrobials. We demonstrate that Acanthamoeba is a histidine autotroph, but with the ability to scavenge preformed histidine. It is able to grow in defined media lacking this amino acid, but is inhibited by 3-amino-1,2,4-triazole (3AT) that targets Imidazoleglycerol-Phosphate Dehydratase (IGPD) the rate limiting step of histidine biosynthesis. The structure of Acanthamoeba IGPD has also been determined in complex with 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate [(R)-C348], a recently described novel inhibitor of Arabidopsis thaliana IGPD. This compound inhibited the growth of four Acanthamoeba species, having a 50% inhibitory concentration (IC50) ranging from 250-526 nM. This effect could be ablated by the addition of 1 mM exogenous free histidine, but importantly not by physiological concentrations found in mammalian tissues. The ability of 3AT and (R)-C348 to restrict the growth of four strains of Acanthamoeba spp. including a recently isolated clinical strain, while not inducing encystment, demonstrates the potential therapeutic utility of targeting the histidine biosynthesis pathway in Acanthamoeba.
Assuntos
Acanthamoeba/enzimologia , Amitrol (Herbicida)/química , Antiprotozoários/química , Histidina/antagonistas & inibidores , Hidroliases/química , Acanthamoeba/efeitos dos fármacos , Acanthamoeba/genética , Acanthamoeba/crescimento & desenvolvimento , Amitrol (Herbicida)/farmacologia , Antiprotozoários/farmacologia , Processos Autotróficos/efeitos dos fármacos , Processos Autotróficos/genética , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/biossíntese , Hidroliases/antagonistas & inibidores , Hidroliases/genética , Hidroliases/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
In the version of this Letter originally published, Michele S. Y. Tan was incorrectly listed as Michele Y. S. Tan due to a technical error. This has now been amended in all online versions of the Letter.
RESUMO
Malaria parasites replicate within a parasitophorous vacuole in red blood cells (RBCs). Progeny merozoites egress upon rupture of first the parasitophorous vacuole membrane (PVM), then poration and rupture of the RBC membrane (RBCM). Egress is protease-dependent 1 , but none of the effector molecules that mediate membrane rupture have been identified and it is unknown how sequential rupture of the two membranes is controlled. Minutes before egress, the parasite serine protease SUB1 is discharged into the parasitophorous vacuole2-6 where it cleaves multiple substrates2,5,7-9 including SERA6, a putative cysteine protease10-12. Here, we show that Plasmodium falciparum parasites lacking SUB1 undergo none of the morphological transformations that precede egress and fail to rupture the PVM. In contrast, PVM rupture and RBCM poration occur normally in SERA6-null parasites but RBCM rupture does not occur. Complementation studies show that SERA6 is an enzyme that requires processing by SUB1 to function. RBCM rupture is associated with SERA6-dependent proteolytic cleavage within the actin-binding domain of the major RBC cytoskeletal protein ß-spectrin. We conclude that SUB1 and SERA6 play distinct, essential roles in a coordinated proteolytic cascade that enables sequential rupture of the two bounding membranes and culminates in RBCM disruption through rapid, precise, SERA6-mediated disassembly of the RBC cytoskeleton.
Assuntos
Cisteína Proteases/metabolismo , Eritrócitos/metabolismo , Malária Falciparum/patologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , Serina Proteases/metabolismo , Membrana Celular/metabolismo , Cisteína Proteases/genética , Citoesqueleto/metabolismo , Eritrócitos/parasitologia , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genéticaRESUMO
Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ácidos Fosfínicos/metabolismo , Fosfitos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Cinética , Ligantes , Modelos Moleculares , Filogenia , Prochlorococcus/genética , Prochlorococcus/metabolismo , Pseudomonas stutzeri/genética , Pseudomonas stutzeri/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Trichodesmium/genética , Trichodesmium/metabolismoRESUMO
Most organisms are exposed to the genotoxic chemical formaldehyde, either from endogenous or environmental sources. Therefore, biology has evolved systems to perceive and detoxify formaldehyde. The frmRA(B) operon that is present in many bacteria represents one such system. The FrmR protein is a transcriptional repressor that is specifically inactivated in the presence of formaldehyde, permitting expression of the formaldehyde detoxification machinery (FrmA and FrmB, when the latter is present). The X-ray structure of the formaldehyde-treated Escherichia coli FrmR (EcFrmR) protein reveals the formation of methylene bridges that link adjacent Pro2 and Cys35 residues in the EcFrmR tetramer. Methylene bridge formation has profound effects on the pattern of surface charge of EcFrmR and combined with biochemical/biophysical data suggests a mechanistic model for formaldehyde-sensing and derepression of frmRA(B) expression in numerous bacterial species.
Assuntos
Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/fisiologia , Formaldeído/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Formaldeído/farmacologia , Inativação Metabólica , Interferometria , Modelos Moleculares , Óperon , Regiões Promotoras Genéticas/genética , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Selenometionina/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Imidazoleglycerol-phosphate dehydratase (IGPD) catalyzes the Mn(II)-dependent dehydration of imidazoleglycerol phosphate (IGP) to 3-(1H-imidazol-4-yl)-2-oxopropyl dihydrogen phosphate during biosynthesis of histidine. As part of a program of herbicide design, we have determined a series of high-resolution crystal structures of an inactive mutant of IGPD2 from Arabidopsis thaliana in complex with IGP. The structures represent snapshots of the enzyme trapped at different stages of the catalytic cycle and show how substrate binding triggers a switch in the coordination state of an active site Mn(II) between six- and five-coordinate species. This switch is critical to prime the active site for catalysis, by facilitating the formation of a high-energy imidazolate intermediate. This work not only provides evidence for the molecular processes that dominate catalysis in IGPD, but also describes how the manipulation of metal coordination can be linked to discrete steps in catalysis, demonstrating one way that metalloenzymes exploit the unique properties of metal ions to diversify their chemistry.