RESUMO
With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.
Assuntos
Antimaláricos , Malária Falciparum , Humanos , Plasmodium falciparum/metabolismo , Actinas/genética , Actinas/metabolismo , Profilinas/genética , Profilinas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Malária Falciparum/tratamento farmacológico , Malária Falciparum/prevenção & controle , Malária Falciparum/genética , Eritrócitos/parasitologia , Antimaláricos/farmacologiaRESUMO
Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and also have nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the oocyst. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We confirm expression in male gametocytes and zygotes and show that actin II is associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filaments in vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. The function of actin II was investigated through mutational analysis, suggesting that long and stable filaments are necessary for male gametogenesis, while a second function in the oocyst stage also requires fine-tuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 µM at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium.
Assuntos
Culicidae , Parasitos , Plasmodium , Animais , Masculino , Actinas/metabolismo , Parasitos/metabolismo , Citoesqueleto de Actina/metabolismo , Culicidae/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium/metabolismoRESUMO
Malaria is responsible for half a million deaths annually and poses a huge economic burden on the developing world. The mosquito-borne parasites (Plasmodium spp.) that cause the disease depend upon an unconventional actomyosin motor for both gliding motility and host cell invasion. The motor system, often referred to as the glideosome complex, remains to be understood in molecular terms and is an attractive target for new drugs that might block the infection pathway. Here, we present the high-resolution structure of the actomyosin motor complex from Plasmodium falciparum. The complex includes the malaria parasite actin filament (PfAct1) complexed with the class XIV myosin motor (PfMyoA) and its two associated light-chains. The high-resolution core structure reveals the PfAct1:PfMyoA interface in atomic detail, while at lower-resolution, we visualize the PfMyoA light-chain binding region, including the essential light chain (PfELC) and the myosin tail interacting protein (PfMTIP). Finally, we report a bare PfAct1 filament structure at improved resolution.
Assuntos
Malária , Parasitos , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animais , Malária/metabolismo , Miosinas/metabolismo , Parasitos/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Myosin B (MyoB) is a class 14 myosin expressed in all invasive stages of the malaria parasite, Plasmodium falciparum. It is not associated with the glideosome complex that drives motility and invasion of host cells. During red blood cell invasion, MyoB remains at the apical tip of the merozoite but is no longer observed once invasion is completed. MyoB is not essential for parasite survival, but when it is knocked out, merozoites are delayed in the initial stages of red blood cell invasion, giving rise to a growth defect that correlates with reduced invasion success. Therefore, further characterization is needed to understand how MyoB contributes to parasite invasion. Here, we have expressed and purified functional MyoB with the help of parasite-specific chaperones Hsp90 and Unc45, characterized its binding to actin and its known light chain MLC-B using biochemical and biophysical methods and determined its low-resolution structure in solution using small angle X-ray scattering. In addition to MLC-B, we found that four other putative regulatory light chains bind to the MyoB IQ2 motif in vitro. The purified recombinant MyoB adopted the overall shape of a myosin, exhibited actin-activated ATPase activity, and moved actin filaments in vitro. Additionally, we determined that the ADP release rate was faster than the ATP turnover number, and thus, does not appear to be rate limiting. This, together with the observed high affinity to actin and the specific localization of MyoB, may point toward a role in tethering and/or force sensing during early stages of invasion.
Assuntos
Miosina não Muscular Tipo IIB , Plasmodium falciparum , Proteínas de Protozoários , Actinas/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Miosinas/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Apicomplexan parasites, such as Plasmodium spp., rely on an unusual actomyosin motor, termed glideosome, for motility and host cell invasion. The actin filaments are maintained by a small set of essential regulators, which provide control over actin dynamics in the different stages of the parasite life cycle. Actin filament capping proteins (CPs) are indispensable heterodimeric regulators of actin dynamics. CPs have been extensively characterized in higher eukaryotes, but their role and functional mechanism in Apicomplexa remain enigmatic. Here, we present the first crystal structure of a homodimeric CP from the malaria parasite and compare the homo- and heterodimeric CP structures in detail. Despite retaining several characteristics of a canonical CP, the homodimeric Plasmodium berghei (Pb)CP exhibits crucial differences to the canonical heterodimers. Both homo- and heterodimeric PbCPs regulate actin dynamics in an atypical manner, facilitating rapid turnover of parasite actin, without affecting its critical concentration. Homo- and heterodimeric PbCPs show partially redundant activities, possibly to rescue actin filament capping in life cycle stages where the ß-subunit is downregulated. Our data suggest that the homodimeric PbCP also influences actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a ß-subunit, as the asymmetric PbCP homodimer lacks structural elements essential for canonical barbed end interactions suggesting a novel CP binding mode. These findings will facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium.
Assuntos
Proteínas de Capeamento de Actina , Antígenos de Protozoários , Malária/parasitologia , Plasmodium/metabolismo , Proteínas de Protozoários , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
Toxoplasma gondii aspartyl protease 3 (TgASP3) phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X (PfPMIX, PfPMX). These proteases are essential for parasite survival, acting as key maturases for secreted proteins implicated in invasion and egress. A potent antimalarial peptidomimetic inhibitor (49c) originally developed against Plasmepsin II selectively targets TgASP3, PfPMIX, and PfPMX To unravel the molecular basis for the selectivity of 49c, we constructed homology models of PfPMIX, PfPMX, and TgASP3 that were first validated by identifying the determinants of microneme and rhoptry substrate recognition. The flap and flap-like structures of several reported Plasmepsins are highly flexible and critically modulate the access to the binding cavity. Molecular docking of 49c to TgASP3, PfPMIX, and PfPMX models predicted that the conserved phenylalanine residues in the flap, F344, F291, and F305, respectively, account for the sensitivity toward 49c. Concordantly, phenylalanine mutations in the flap of the three proteases increase twofold to 15-fold the IC50 values of 49c. Compellingly the selection of mutagenized T. gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue.
Assuntos
Antimaláricos/farmacologia , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/metabolismo , Resistência a Medicamentos/fisiologia , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/química , Antimaláricos/química , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Cisteína , Relação Dose-Resposta a Droga , Resistência a Medicamentos/efeitos dos fármacos , Concentração Inibidora 50 , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Testes de Sensibilidade Parasitária , Fenilalanina/efeitos dos fármacos , Fenilalanina/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Alinhamento de Sequência , Toxoplasma/efeitos dos fármacos , Toxoplasma/genéticaRESUMO
During transmission of malaria-causing parasites from mosquito to mammal, Plasmodium sporozoites migrate at high speed within the skin to access the bloodstream and infect the liver. This unusual gliding motility is based on retrograde flow of membrane proteins and highly dynamic actin filaments that provide short tracks for a myosin motor. Using laser tweezers and parasite mutants, we previously suggested that actin filaments form macromolecular complexes with plasma membrane-spanning adhesins to generate force during migration. Mutations in the actin-binding region of profilin, a near ubiquitous actin-binding protein, revealed that loss of actin binding also correlates with loss of force production and motility. Here, we show that different mutations in profilin, that do not affect actin binding in vitro, still generate lower force during Plasmodium sporozoite migration. Lower force generation inversely correlates with increased retrograde flow suggesting that, like in mammalian cells, the slow down of flow to generate force is the key underlying principle governing Plasmodium gliding motility.
Assuntos
Malária , Parasitos , Actinas/genética , Animais , Plasmodium berghei , Profilinas/genética , Proteínas de Protozoários/genéticaRESUMO
Plasmodium actins form very short filaments and have a noncanonical link between ATP hydrolysis and polymerization. Long filaments are detrimental to the parasites, but the structural factors constraining Plasmodium microfilament lengths have remained unknown. Using high-resolution crystallography, we show that magnesium binding causes a slight flattening of the Plasmodium actin I monomer, and subsequent phosphate release results in a more twisted conformation. Thus, the Mg-bound monomer is closer in conformation to filamentous (F) actin than the Ca form, and this likely facilitates polymerization. A coordinated potassium ion resides in the active site during hydrolysis and leaves together with the phosphate, a process governed by the position of the Arg178/Asp180-containing A loop. Asp180 interacts with either Lys270 or His74, depending on the protonation state of the histidine, while Arg178 links the inner and outer domains (ID and OD) of the actin protomer. Hence, the A loop acts as a switch between stable and unstable filament conformations, the latter leading to fragmentation. Our data provide a comprehensive model for polymerization, ATP hydrolysis and phosphate release, and fragmentation of parasite microfilaments. Similar mechanisms may well exist in canonical actins, although fragmentation is much less favorable due to several subtle sequence differences as well as the methylation of His73, which is absent on the corresponding His74 in Plasmodium actin I.
Assuntos
Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Plasmodium/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Difosfato de Adenosina/metabolismo , Animais , Citoesqueleto/metabolismo , Hidrólise , Cinética , Magnésio/metabolismo , Fosfatos/metabolismo , PolimerizaçãoRESUMO
Actin capping proteins belong to the core set of proteins minimally required for actin-based motility and are present in virtually all eukaryotic cells. They bind to the fast-growing barbed end of an actin filament, preventing addition and loss of monomers, thus restricting growth to the slow-growing pointed end. Actin capping proteins are usually heterodimers of two subunits. The Plasmodium orthologs are an exception, as their α subunits are able to form homodimers. We show here that, while the ß subunit alone is unstable, the α subunit of the Plasmodium actin capping protein forms functional homo- and heterodimers. This implies independent functions for the αα homo- and αß heterodimers in certain stages of the parasite life cycle. Structurally, the homodimers resemble canonical αß heterodimers, although certain rearrangements at the interface must be required. Both homo- and heterodimers bind to actin filaments in a roughly equimolar ratio, indicating they may also bind other sites than barbed ends.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Malária/parasitologia , Parasitos/metabolismo , Multimerização Proteica , Proteínas de Protozoários/metabolismo , Proteínas de Capeamento de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Plasmodium/metabolismo , Ligação Proteica , Dobramento de Proteína , Soluções , TemperaturaRESUMO
During their life cycle, apicomplexan parasites, such as the malaria parasite Plasmodium falciparum, use actomyosin-driven gliding motility to move and invade host cells. For this process, actin filament length and stability are temporally and spatially controlled. In contrast to canonical actin, P. falciparum actin 1 (PfAct1) does not readily polymerize into long, stable filaments. The structural basis of filament instability, which plays a pivotal role in host cell invasion, and thus infectivity, is poorly understood, largely because high-resolution structures of PfAct1 filaments were missing. Here, we report the near-atomic structure of jasplakinolide (JAS)-stabilized PfAct1 filaments determined by electron cryomicroscopy. The general filament architecture is similar to that of mammalian F-actin. The high resolution of the structure allowed us to identify small but important differences at inter- and intrastrand contact sites, explaining the inherent instability of apicomplexan actin filaments. JAS binds at regular intervals inside the filament to three adjacent actin subunits, reinforcing filament stability by hydrophobic interactions. Our study reveals the high-resolution structure of a small molecule bound to F-actin, highlighting the potential of electron cryomicroscopy for structure-based drug design. Furthermore, our work serves as a strong foundation for understanding the structural design and evolution of actin filaments and their function in motility and host cell invasion of apicomplexan parasites.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Depsipeptídeos/química , Modelos Moleculares , Plasmodium falciparum/química , Proteínas de Protozoários/química , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Microscopia Crioeletrônica , Plasmodium falciparum/metabolismo , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/metabolismoRESUMO
Tyrosine hydroxylase (TH) is a multi-domain, homo-oligomeric enzyme that catalyses the rate-limiting step of catecholamine neurotransmitter biosynthesis. Missense variants of human TH are associated with a recessive neurometabolic disease with low levels of brain dopamine and noradrenaline, resulting in a variable clinical picture, from progressive brain encephalopathy to adolescent onset DOPA-responsive dystonia (DRD). We expressed isoform 1 of human TH (hTH1) and its dystonia-associated missense variants in E. coli, analysed their quaternary structure and thermal stability using size-exclusion chromatography, circular dichroism, multi-angle light scattering, transmission electron microscopy, small-angle X-ray scattering and assayed hydroxylase activity. Wild-type (WT) hTH1 was a mixture of enzymatically stable tetramers (85.6%) and octamers (14.4%), with little interconversion between these species. We also observed small amounts of higher order assemblies of long chains of enzyme by transmission electron microscopy. To investigate the role of molecular assemblies in the pathogenesis of DRD, we compared the structure of WT hTH1 with the DRD-associated variants R410P and D467G that are found in vicinity of the predicted subunit interfaces. In contrast to WT hTH1, R410P and D467G were mixtures of tetrameric and dimeric species. Inspection of the available structures revealed that Arg-410 and Asp-467 are important for maintaining the stability and oligomeric structure of TH. Disruption of the normal quaternary enzyme structure by missense variants is a new molecular mechanism that may explain the loss of TH enzymatic activity in DRD. Unstable missense variants could be targets for pharmacological intervention in DRD, aimed to re-establish the normal oligomeric state of TH.
Assuntos
Distúrbios Distônicos/genética , Tirosina 3-Mono-Oxigenase/química , Tirosina 3-Mono-Oxigenase/genética , Humanos , Mutação de Sentido Incorreto , Estrutura Quaternária de ProteínaRESUMO
Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like ß-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.
Assuntos
Actinas/metabolismo , Malária/parasitologia , Plasmodium berghei/citologia , Plasmodium berghei/metabolismo , Profilinas/metabolismo , Proteínas de Protozoários/metabolismo , Actinas/genética , Animais , Movimento Celular , Feminino , Humanos , Camundongos Endogâmicos C57BL , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Profilinas/genética , Ligação Proteica , Proteínas de Protozoários/genética , Esporozoítos/citologia , Esporozoítos/crescimento & desenvolvimento , Esporozoítos/metabolismoRESUMO
Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion.
Assuntos
Estágios do Ciclo de Vida/fisiologia , Proteínas de Membrana , Miosinas , Plasmodium berghei , Plasmodium falciparum , Proteínas de Protozoários , Animais , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Miosinas/genética , Miosinas/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Rhoptries are club-shaped, regulated secretory organelles that cluster at the apical pole of apicomplexan parasites. Their discharge is essential for invasion and the establishment of an intracellular lifestyle. Little is known about rhoptry biogenesis and recycling during parasite division. In Toxoplasma gondii, positioning of rhoptries involves the armadillo repeats only protein (ARO) and myosin F (MyoF). Here, we show that two ARO partners, ARO-interacting protein (AIP) and adenylate cyclase ß (ACß) localize to a rhoptry subcompartment. In absence of AIP, ACß disappears from the rhoptries. By assessing the contribution of each ARO armadillo (ARM) repeat, we provide evidence that ARO is multifunctional, participating not only in positioning but also in clustering of rhoptries. Structural analyses show that ARO resembles the myosin-binding domain of the Caenorhabditis elegans myosin chaperone UNC-45. A conserved patch of aromatic and acidic residues denotes the putative MyoF-binding site, and the overall arrangement of the ARM repeats explains the dramatic consequences of deleting each of them. Finally, Plasmodium falciparum ARO functionally complements ARO depletion and interacts with the same partners, highlighting the conservation of rhoptry biogenesis in Apicomplexa.
Assuntos
Proteínas do Domínio Armadillo/fisiologia , Proteínas de Protozoários/fisiologia , Toxoplasma/metabolismo , Sequência de Aminoácidos , Proteínas do Domínio Armadillo/química , Sequência Conservada , Modelos Moleculares , Organelas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Toxoplasma/ultraestruturaRESUMO
BACKGROUND: Eukaryotic tRNA splicing is an essential process in the transformation of a primary tRNA transcript into a mature functional tRNA molecule. 5'-phosphate ligation involves two steps: a healing reaction catalyzed by polynucleotide kinase (PNK) in association with cyclic phosphodiesterase (CPDase), and a sealing reaction catalyzed by an RNA ligase. The enzymes that catalyze tRNA healing in yeast and higher eukaryotes are homologous to the members of the 2H phosphoesterase superfamily, in particular to the vertebrate myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). RESULTS: We employed different biophysical and biochemical methods to elucidate the overall structural and functional features of the tRNA healing enzymes yeast Trl1 PNK/CPDase and lancelet PNK/CPDase and compared them with vertebrate CNPase. The yeast and the lancelet enzymes have cyclic phosphodiesterase and polynucleotide kinase activity, while vertebrate CNPase lacks PNK activity. In addition, we also show that the healing enzymes are structurally similar to the vertebrate CNPase by applying synchrotron radiation circular dichroism spectroscopy and small-angle X-ray scattering. CONCLUSIONS: We provide a structural analysis of the tRNA healing enzyme PNK and CPDase domains together. Our results support evolution of vertebrate CNPase from tRNA healing enzymes with a loss of function at its N-terminal PNK-like domain.
Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/química , Evolução Molecular , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/fisiologia , Animais , Dicroísmo Circular , Células Eucarióticas/enzimologia , Anfioxos , Camundongos , Bainha de Mielina/enzimologia , Polinucleotídeo 5'-Hidroxiquinase/química , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Splicing de RNA , RNA de Transferência/genética , Saccharomyces cerevisiae , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Plasmodium berghei/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Gene fusion is a common mechanism of protein evolution that has mainly been discussed in the context of multidomain or symmetric proteins. Less is known about fusion of ancestral genes to produce small single-domain proteins. Here, we show with a domain-swapped mutant Plasmodium profilin that this small, globular, apparently single-domain protein consists of two foldons. The separation of binding sites for different protein ligands in the two halves suggests evolution via an ancient gene fusion event, analogous to the formation of multidomain proteins. Finally, the two fragments can be assembled together after expression as two separate gene products. The possibility to engineer both domain-swapped dimers and half-profilins that can be assembled back to a full profilin provides perspectives for engineering of novel protein folds, e.g., with different scaffolding functions.
Assuntos
Evolução Molecular , Fusão Gênica , Plasmodium falciparum/química , Profilinas/química , Profilinas/genética , Dicroísmo Circular , Cristalografia por Raios X , Éxons , Íntrons , Modelos Moleculares , Mutação , Dobramento de Proteína , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Coronins are involved in the regulation of actin dynamics in a multifaceted way, participating in cell migration and vesicular trafficking. Apicomplexan parasites, which exhibit an actin-dependent gliding motility that is essential for traversal through tissues, as well as invasion of and egress from host cells, express only a single coronin, whereas higher eukaryotes possess several isoforms. We set out to characterize the 3-D structure, biochemical function, subcellular localization, and genetic ablation of Toxoplasma gondii coronin (TgCOR), to shed light on its biological role. A combination of X-ray crystallography, small-angle scattering of X-rays, and light scattering revealed the atomic structure of the conserved WD40 domain and the dimeric arrangement of the full-length protein. TgCOR binds to F-actin and increases the rate and extent of actin polymerization. In vivo, TgCOR relocalizes transiently to the posterior pole of motile and invading parasites, independent of actin dynamics, but concomitant to microneme secretory organelle discharge. TgCOR contributes to, but is not essential for, invasion and egress. Taken together, our data point toward a role for TgCOR in stabilizing newly formed, short filaments and F-actin cross-linking, as well as functions linked to endocytosis and recycling of membranes.
Assuntos
4-Butirolactona/análogos & derivados , Actinas/metabolismo , Citoesqueleto/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Toxoplasma/química , Toxoplasma/metabolismo , 4-Butirolactona/metabolismo , Animais , Modelos Moleculares , Organelas/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , SuínosRESUMO
P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93â Å allows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.
Assuntos
Proteína P2 de Mielina/química , Proteína P2 de Mielina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation.