Expression and purification of human neutrophil proteinase 3 from insect cells and characterization of ligand binding.

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.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

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.

Structure and function of Plasmodium actin II in the parasite mosquito stages.

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.

Analysis of Plasmodium falciparum myosin B ATPase activity and structure in complex with the calmodulin-like domain of its light chain MLC-B.

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.

High-resolution structures of malaria parasite actomyosin and actin filaments.

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.

Structure and function of an atypical homodimeric actin capping protein from the malaria parasite.

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.

The XBI BioLab for life science experiments at the European XFEL.

The science of X-ray free-electron lasers (XFELs) critically depends on the performance of the X-ray laser and on the quality of the samples placed into the X-ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted.

Characterization of porphobilinogen deaminase mutants reveals that arginine-173 is crucial for polypyrrole elongation mechanism.

Porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis, catalyzes the sequential coupling of four porphobilinogen (PBG) molecules into a heme precursor. Mutations in PBGD are associated with acute intermittent porphyria (AIP), a rare metabolic disorder. We used Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to demonstrate that wild-type PBGD and AIP-associated mutant R167W both existed as holoenzymes (Eholo) covalently attached to the dipyrromethane cofactor, and three intermediate complexes, ES, ES2, and ES3, where S represents PBG. In contrast, only ES2 was detected in AIP-associated mutant R173W, indicating that the formation of ES3 is inhibited. The R173W crystal structure in the ES2-state revealed major rearrangements of the loops around the active site, compared to wild-type PBGD in the Eholo-state. These results contribute to elucidating the structural pathogenesis of two common AIP-associated mutations and reveal the important structural role of Arg173 in the polypyrrole elongation mechanism.

Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers.

Plasmodium falciparum causes the most lethal form of malaria. The cooperation of heat shock protein (Hsp) 70 and 90 is thought to facilitate folding of select group of cellular proteins that are crucial for cyto-protection and development of the parasites. Hsp70 and Hsp90 are brought into a functional complex that allows substrate exchange by stress inducible protein 1 (STI1), also known as Hsp70-Hsp90 organising protein (Hop). P. falciparum Hop (PfHop) co-localises and occurs in complex with the parasite cytosolic chaperones, PfHsp70-1 and PfHsp90. Here, we characterised the structure of recombinant PfHop using synchrotron radiation circular dichroism (SRCD) and small-angle X-ray scattering. Structurally, PfHop is a monomeric, elongated but folded protein, in agreement with its predicted TPR domain structure. Using SRCD, we established that PfHop is unstable at temperatures higher than 40°C. This suggests that PfHop is less stable at elevated temperatures compared to its functional partner, PfHsp70-1, that is reportedly stable at temperatures as high as 80°C. These findings contribute towards our understanding of the role of the Hop-mediated functional partnership between Hsp70 and Hsp90.

Functional homo- and heterodimeric actin capping proteins from the malaria parasite.

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.

A function of profilin in force generation during malaria parasite motility that is independent of actin binding.

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.

Atomic view into Plasmodium actin polymerization, ATP hydrolysis, and fragmentation.

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.

Genetic variation in metronidazole metabolism and oxidative stress pathways in clinical Giardia lamblia assemblage A and B isolates.

Purpose: Treatment-refractory Giardia cases have increased rapidly within the last decade. No markers of resistance nor a standardized susceptibility test have been established yet, but several enzymes and their pathways have been associated with metronidazole (MTZ) resistant Giardia. Very limited data are available regarding genetic variation in these pathways. We aimed to investigate genetic variation in metabolic pathway genes proposed to be involved in MTZ resistance in recently acquired, cultured clinical isolates. Methods: Whole genome sequencing of 12 assemblage A2 and 8 assemblage B isolates was done, to decipher genomic variation in Giardia. Twenty-nine genes were identified in a literature search and investigated for their single nucleotide variants (SNVs) in the coding/non-coding regions of the genes, either as amino acid changing (non-synonymous SNVs) or non-changing SNVs (synonymous). Results: In Giardia assemblage B, several genes involved in MTZ activation or oxidative stress management were found to have higher numbers of non-synonymous SNVs (thioredoxin peroxidase, nitroreductase 1, ferredoxin 2, NADH oxidase, nitroreductase 2, alcohol dehydrogenase, ferredoxin 4 and ferredoxin 1) than the average variation. For Giardia assemblage A2, the highest genetic variability was found in the ferredoxin 2, ferredoxin 6 and in nicotinamide adenine dinucleotide phosphate (NADPH) oxidoreductase putative genes. SNVs found in the ferredoxins and nitroreductases were analyzed further by alignment and homology modeling. SNVs close to the iron-sulfur cluster binding sites in nitroreductase-1 and 2 and ferredoxin 2 and 4 could potentially affect protein function. Flavohemoprotein seems to be a variable-copy gene, due to higher, but variable coverage compared to other genes investigated. Conclusion: In clinical Giardia isolates, genetic variability is common in important genes in the MTZ metabolizing pathway and in the management of oxidative and nitrosative stress and includes high numbers of non-synonymous SNVs. Some of the identified amino acid changes could potentially affect the respective proteins important in the MTZ metabolism.

The quaternary structure of human tyrosine hydroxylase: effects of dystonia-associated missense variants on oligomeric state and enzyme activity.

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.

Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases.

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.

A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.

Regulated exocytosis by secretory organelles is important for malaria parasite invasion and egress. Many parasite effector proteins, including perforins, adhesins, and proteases, are extensively proteolytically processed both pre- and postexocytosis. Here we report the multistage antiplasmodial activity of the aspartic protease inhibitor hydroxyl-ethyl-amine-based scaffold compound 49c. This scaffold inhibits the preexocytosis processing of several secreted rhoptry and microneme proteins by targeting the corresponding maturases plasmepsins IX (PMIX) and X (PMX), respectively. Conditional excision of PMIX revealed its crucial role in invasion, and recombinantly active PMIX and PMX cleave egress and invasion factors in a 49c-sensitive manner.

Near-atomic structure of jasplakinolide-stabilized malaria parasite F-actin reveals the structural basis of filament instability.

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.

Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility.

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.

Apicomplexan actin polymerization depends on nucleation.

Filamentous actin is critical for apicomplexan motility and host cell invasion. Yet, parasite actin filaments are short and unstable. Their kinetic characterization has been hampered by the lack of robust quantitative methods. Using a modified labeling method, we carried out thorough biochemical characterization of malaria parasite actin. In contrast to the isodesmic polymerization mechanism suggested for Toxoplasma gondii actin, Plasmodium falciparum actin I polymerizes via the classical nucleation-elongation pathway, with kinetics similar to canonical actins. A high fragmentation rate, governed by weak lateral contacts within the filament, is likely the main reason for the short filament length. At steady state, Plasmodium actin is present in equal amounts of short filaments and dimers, with a small proportion of monomers, representing the apparent critical concentration of ~0.1 µM. The dimers polymerize but do not serve as nuclei. Our work enhances understanding of actin evolution and the mechanistic details of parasite motility, serving as a basis for exploring parasite actin and actin nucleators as drug targets against malaria and other apicomplexan parasitic diseases.

Rapid cadmium SAD phasing at the standard wavelength (1†Å).

Cadmium ions can be effectively used to promote crystal growth and for experimental phasing. Here, the use of cadmium ions as a suitable anomalous scatterer at the standard wavelength of 1 Šis demonstrated. The structures of three different proteins were determined using cadmium single-wavelength anomalous dispersion (SAD) phasing. Owing to the strong anomalous signal, the structure of lysozyme could be automatically phased and built using a very low anomalous multiplicity (1.1) and low-completeness (77%) data set. Additionally, it is shown that cadmium ions can easily substitute divalent ions in ATP-divalent cation complexes. This property could be generally applied for phasing experiments of a wide range of nucleotide-binding proteins. Improvements in crystal growth and quality, good anomalous signal at standard wavelengths (i.e. no need to change photon energy) and rapid phasing and refinement using a single data set are benefits that should allow cadmium ions to be widely used for experimental phasing.