Biology subjects

Löw, C.

Publications and source records attributed to Löw, C..

4 recordsLinked to original sources

bioRxiv2021

PMRT1, a Plasmodium specific parasite plasma membrane transporter is essential for asexual and sexual blood stage development

Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures - the food vacuole, the apicoplast, and the parasite plasma membrane - and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1, PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have diverse intracellular localizations and putative functions. ImportancePlasmodium falciparum-infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes do not only facilitate movement of nutrients, metabolites and other molecules between these compartments, but are common therapeutic targets and can also confer antimalarial drug resistance. Orphan membrane transporter in P. falciparum without sequence homology to transporters in other evolutionary lineages and divergent to host transporters may constitute attractive targets for novel intervention approaches. Here, we localized six of these putative transporters at different subcellular compartments and probed into their importance during asexual parasite growth using reverse genetic approaches. In total, only two candidates turned out to be dispensable for the parasite, highlighting four candidates as putative targets for therapeutic interventions. This study reveals the importance of several orphan transporters to blood stage P. falciparum development.

molecular biology↗

bioRxiv2021

Deamidation drives molecular aging of the SARS-CoV-2 spike receptor-binding motif

The spike is the main protein component of the SARS-CoV-2 virion surface. The spike receptor binding motif mediates recognition of the hACE2 receptor, a critical infection step, and is the preferential target for spike-neutralizing antibodies. Post-translational modifications of the spike receptor binding motif can modulate viral infectivity and immune response. We studied the spike protein in search for asparagine deamidation, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, affecting both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike. Similar deamidation hotspots are frequently found at the spike receptor-binding motifs of related sarbecoviruses, at positions that are mutated in emerging variants and in escape mutants from neutralizing antibodies. Asparagine residues 481 and 501 from the receptor-binding motif deamidate with a half-time of 16.5 and 123 days at 37 {degrees}C, respectively. This process is significantly slowed down at 4 {degrees}C, pointing at a strong dependence of spike molecular aging on the environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold. A model for deamidation of the full SARS-CoV-2 virion illustrates that deamidation of the spike receptor-binding motif leads to the accumulation in the virion surface of a chemically diverse spike population in a timescale of days. Our findings provide a mechanism for molecular aging of the spike, with significant consequences for understanding virus infectivity and vaccine development.

biochemistry↗

bioRxiv2020

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 {micro}g/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the up ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

biophysics↗

bioRxiv2019

Structural role of essential light chains in the apicomplexan glideosome

AbstractApicomplexan parasites, such as Plasmodium falciparum and Toxoplasma gondii, traverse the host tissues and invade the host cells exhibiting a specific type of motility called gliding. The molecular mechanism of gliding lies in the actin-myosin motor localized to the intermembrane space between the plasma membrane and inner membrane complex (IMC) of the parasites. Myosin A (MyoA) is a part of the glideosome, a large multi-protein complex, which is anchored in the outer membrane of the IMC. MyoA is bound to the proximal essential light chain (ELC) and distal myosin light chain (MLC1), which further interact with the glideosome associated proteins GAP40, GAP45 and GAP50. Whereas structures of several individual glideosome components and small dimeric complexes have been solved, structural information concerning the interaction of larger glideosome subunits and their role in glideosome function still remains to be elucidated. Here, we present structures of a T. gondii trimeric glideosome sub complex composed of a myosin A light chain domain with bound MLC1 and TgELC1 or TgELC2. Regardless of the differences between the secondary structure content observed for free P. falciparum PfELC and T. gondii TgELC1 or TgELC2, the proteins interact with a conserved region of TgMyoA to form structurally conserved complexes. Upon interaction, the essential light chains undergo contraction and induce -helical structure in the myosin A C-terminus, stiffening the myosin lever arm. The complex formation is further stabilized through binding of a single calcium ion to T. gondii ELCs. Our work provides an important step towards the structural understanding of the entire glideosome and uncovering the role of its members in parasite motility and invasion. Author summaryApicomplexans, such as Toxoplasma gondii or the malaria agent Plasmodium falciparum, are small unicellular parasites that cause serious diseases in humans and other animals. These parasites move and infect the host cells by a unique type of motility called gliding. Gliding is empowered by an actin-myosin molecular motor located at the periphery of the parasites. Myosin interacts with additional proteins such as essential light chains to form the glideosome, a large protein assembly that anchors myosin in the inner membrane complex. Unfortunately, our understanding of the glideosome is insufficient because we lack the necessary structural information. Here we describe the first structures of trimeric glideosome sub complexes of T. gondii myosin A bound to two different light chain combinations, which show that T. gondii and P. falciparum form structurally conserved complexes. With an additional calcium-free complex structure, we demonstrate that calcium binding does not change the formation of the complexes, although it provides them with substantial stability. With additional data, we propose that the role of the essential light chains is to enhance myosin performance by inducing secondary structure in the C-terminus of myosin A. Our work represents an important step in unveiling the gliding mechanism of apicomplexan parasites.

biophysics↗