Biology subjects

Kursula, I.

Publications and source records attributed to Kursula, I..

12 recordsLinked to original sources

bioRxiv2026

Dynamic phosphorylation of the Plasmodium falciparum Myosin A motor regulates motor speed and is important for asexual replication

Malaria parasite invasion of red blood cells is powered by Myosin A (MyoA), the actomyosin motor which is a core component of the glideosome. Our previous phosphoproteomic study identified a cyclic GMP-dependent phosphorylation site on MyoA at Ser19, suggesting that cyclic nucleotide signalling may directly regulate motor activity during invasion. However, the functional significance of this phosphorylation site remains unclear. Here, we show that Plasmodium falciparum MyoA phosphorylation at Ser19 is dynamically regulated during the intraerythrocytic cycle, peaking in extracellular merozoites and rapidly declining following invasion. Using conditional depletion of MyoA, we show that MyoA is essential for red blood cell invasion but dispensable for gametocyte development. Conditional complementation with mutant versions of MyoA that either mimic (S19D) or ablate phosphorylation (S19A) reveal markedly impaired asexual parasite proliferation, highlighting the importance of precise regulation of MyoA phosphorylation at Ser19. In vitro motility assays using parasite-derived MyoA showed that ablating phosphorylation either pharmacologically or by introducing an S19A mutation, reduces motor speed. Conversely, introducing a phosphomimetic S19D mutation restores motility levels comparable to phosphorylated wild type MyoA. Collectively, these results establish MyoA Ser19 phosphorylation as a regulatory switch for actomyosin motor activity.

cell biology↗

bioRxiv2024

Functional insights into Plasmodium actin depolymerizing factor interactions with phosphoinositides

Malaria is caused by protozoan parasites, Plasmodium spp., that belong to the phylum Apicomplexa. The life cycle of these parasites depends on two different hosts; the definitive host, or vector, is a mosquito, and the intermediate host is a vertebrate, such as human. Malaria parasites use a unique form of substrate-dependent motility for host cell invasion and egress, which is dependent on an actomyosin motor complex called the glideosome. Apicomplexa have a small set of actin regulators, which are poorly conserved compared to their equivalents in higher eukaryotes. Actin depolymerizing factors (ADFs) are key regulators responsible for accelerating actin turnover in eukaryotic cells. The activity of ADFs is regulated by membrane phosphoinositides. Malaria parasites express two ADF isoforms at different life stages. ADF1 differs substantially from canonical ADF/cofilins and from Plasmodium ADF2 in terms of both structure and function. Here, we studied the interaction of both Plasmodium ADFs with phosphoinositides using biochemical and biophysical methods and mapped their binding sites on ADF1. Both Plasmodium ADFs bind to different phosphoinositides, and binding in vitro requires the formation of vesicles or micelles. Interaction with phosphoinositides increases the -helical content of the parasite ADFs, and the affinities are in the micromolar range. The binding site for PI(4,5)P2 in PfADF1 involves a small, positively charged surface patch.

biochemistry↗

bioRxiv2024

Crystal structure of Anopheles gambiae actin depolymerizing factor explains high affinity to monomeric actin

Actin is an intrinsically dynamic protein, the function and state of which are modulated by actin-binding proteins. Actin depolymerizing factors (ADF)/cofilins are ubiquitous actin-binding proteins that accelerate actin turnover. Malaria is an infectious disease caused by parasites of the genus Plasmodium, which belong to the phylum Apicomplexa. The parasites require two hosts to complete their life cycle: the definitive host, or the vector, which is an Anopheles spp. mosquito, and a vertebrate intermediate host, such as humans. Here, the crystal structure of the malaria vector Anopheles gambiae ADF (AgADF) is reported. AgADF has a conserved ADF/cofilin fold with six central {beta}-strands surrounded by five -helices with a long {beta}-hairpin loop protruding out of the structure. The G and F-actin binding sites of AgADF are conserved, and the structure shows features of potential importance for regulation by membrane binding and redox state. AgADF binds monomeric ATP- and ADP-actin with a high affinity, having a nanomolar Kd, and binds to and effectively destabilizes actin filaments.

biochemistry↗

bioRxiv2023

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.

biochemistry↗

bioRxiv2022

Structure of Toxoplasma gondii glideosome-associated connector suggests a role as an elastic element in actomyosin force generation for gliding motility

Toxoplasma gondii glideosome-associated connector (GAC) is a giant armadillo-repeat protein, essential for parasite motility and conserved across Apicomplexa. It connects actin filaments to the plasma membrane via interactions with phosphatidic acid and membrane-spanning adhesins. It is unclear how GAC contributes to gliding motility and invasion and why such a large connector is needed. We determined the crystal structure of full-length T. gondii GAC at 2.3 [A] resolution and explored its conformational space in solution using small-angle X-ray scattering and cryogenic electron microscopy. The crystal structure reveals a compact conformation but, in solution, GAC adopts both compact and extended forms. The PH domain stabilizes the compact form and may act as a switch triggered by membrane sensing. Based on its spring-like architecture, we suggest a role for GAC as an elastic element in actomyosin force generation during gliding motility and invasion.

biochemistry↗

bioRxiv2022

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 have also 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 zygote. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We show that it is expressed in male gametocytes and zygotes and 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. Mutational analyses suggest that long and stable filaments are necessary for male gametogenesis, while the second function in the zygote stage also requires finetuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 M at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium. Significance statementMalaria is a parasitic infection caused by Plasmodium spp., which belong to the phylum Apicomplexa. In 2020, approximately 627000 people died from nearly 241 million malaria cases registered worldwide. In contrast to other apicomplexan parasites, Plasmodium spp. encode two actin isoforms. While actin I is part of the glideosome complex, essential for locomotion, actin II is present in the mosquito stages, where it has functions in gametogenesis and in the zygote. The exact function of actin II is still unclear, and information at the molecular level is limited. We show here that actin II is associated with the nucleus in gametocytes and zygotes and performs specific functions requiring both long filaments and fine-tuning by methylation of histidine 73. We determined the structures of the filamentous form of actin II at near-atomic resolution and characterized its polymerization properties in vitro. Our study provides a molecular basis for the differences between actins of the malaria parasite and humans, as well as between the two parasite actin isoforms. Furthermore, the in vivo studies provide insights into the function of actin II in the parasite.

biochemistry↗

bioRxiv2022

The sulfonylpiperazine MMV020291 prevents red blood cell invasion by the malaria parasite Plasmodium falciparum 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 invasion, and have generated analogues with improved potency. Here, we identify actin and profilin as putative targets of the MMV020291 series through resistance selection and whole genome sequencing of three MMV020291 resistant populations. This revealed three non-synonymous single nucleotide polymorphisms in two genes; two in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wildtype parasites which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade red blood cells. Additionally, the series inhibits the actin-1 dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro co-sedimentation experiments using recombinant P. falciparum actin-1 and profilin proteins indicate that potent MMV020291 analogues amplify the actin-monomer sequestering effect of profilin, thereby reducing the formation of filamentous actin. Altogether, this study identifies the first compound series targeting the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

molecular biology↗

bioRxiv2022

Characterization of Plasmodium falciparum myosin B 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, it 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. 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, four other putative regulatory light chains were found to 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. 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 towards a role in tethering and/or force sensing during early stages of invasion.

biochemistry↗

bioRxiv2020

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

Actin capping proteins (CPs) are essential regulators of actin dynamics in all eukaryotes. Their structure and function have been extensively characterized in higher eukaryotes but their role and mechanism of action in apicomplexan parasites remain enigmatic. Here, we present a crystal structure of a unique homodimeric CP from the rodent malaria parasite Plasmodium berghei. In addition, we compare homo- and heterodimeric arrangements of P. berghei CPs (PbCPs) in solution. We complement our findings by describing the regulatory effects of PbCPs on heterologous skeletal muscle -actin as well as parasite actin. Comprehensive kinetic and steadystate measurements show atypical regulation of actin dynamics; PbCPs facilitate rapid turnover of parasite actin I without affecting the apparent critical concentration. Possibly to rescue actin filament capping in life cycle stages where the CP {beta}-subunit is downregulated, homo- and heterodimeric PbCPs show redundant effects in vitro. However, our data suggest that homodimers may in addition influence actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a {beta}-subunit, as the asymmetric PbCP homodimer lacks the structural elements essential for canonical barbed end interactions, suggesting a novel CP binding mode. These findings facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium and the eligibility of PbCPs as drug targets against malaria. Significance statementMalaria parasites of the genus Plasmodium display a unique form of gliding motility, which depends on an unconventional actomyosin motor. Actin capping proteins (CPs) play a major role in regulating parasite motility. Here, we describe a unique Plasmodium berghei CP (PbCP) system, behaving contradictory to canonical heterodimeric CPs, more suited to regulate the fast dynamics of the parasite actin system. We present the crystal structure of a distinctive homodimeric form of PbCP and extensive biochemical data, describing the atypical behavior of each PbCP form. The PbCP homodimer displays capping even in the absence of canonical conserved structural elements, suggesting a novel actin-CP interaction mode. These distinct structural properties could provide opportunities for drug design against malaria.

molecular biology↗

bioRxiv2020

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 first high-resolution structure of the actomyosin motor complex from Plasmodium falciparum. Our structure includes the malaria parasite actin filament (PfAct1) complexed with the 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 an improved resolution, which gives new information about the nucleotide-binding site, including the orientation of the ATP/ADP sensor, Ser15, and the presence of a channel, which we propose as a possible phosphate exit path after ATP hydrolysis.Significance statement We present the first structure of the malaria parasite motor complex; actin 1 (PfAct1) and myosin A (PfMyoA) with its two light chains. We also report a high-resolution structure of filamentous PfAct1 that reveals new atomic details of the ATPase site, including a channel, which may provide an exit route for phosphate and explain why phosphate release is faster in PfAct1 compared to canonical actins. PfAct1 goes through no conformational changes upon PfMyoA binding. Our PfMyoA structure also superimposes with a recent crystal structure of PfMyoA alone, though there are small but important conformational changes at the interface. Our structures serve as an excellent starting point for drug design against malaria, which is one of the most devastating infectious diseases.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2019

Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organizing 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 important for folding of a select number 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 organizing 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 {degrees}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 {degrees}C. These findings contribute towards our understanding of the role of the Hop-mediated functional partnership between Hsp70 and Hsp90.

biochemistry↗

bioRxiv2018

Atomic view into Plasmodium actin polymerization, ATP hydrolysis, and phosphate release

Plasmodium actins form very short filaments and have a non-canonical link between ATP hydrolysis and polymerization. Long filaments are detrimental to the parasites, but the structural factors constraining Plasmodium microfilament lengths are currently unknown. Using high-resolution crystallography, we show that magnesium binding activates the Plasmodium actin I monomer before polymerization by a slight flattening, which is reversed upon phosphate release. 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 protonation, while Arg178 links the inner and outer domains. Hence, the A-loop is a switch between stable and non-stable filament conformations. Our data provide a comprehensive model for polymerization, phosphate release, and the inherent instability of parasite microfilaments.

biochemistry↗