Structural basis of the 9-fold symmetry of centrioles.

The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.

The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.

The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release.

Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far. Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Šresolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST. We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities.

Identification of compounds that bind the centriolar protein SAS-6 and inhibit its oligomerization.

Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerization of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organization of further organelle components. A dimerization interaction between SAS-6 N-terminal "head" domains was previously shown to be essential for protein oligomerization in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low-molecular-weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerization. We demonstrate using NMR spectroscopy that a ligand from this family binds at the head domain dimerization site of algae, nematode, and human SAS-6 variants, but also that another ligand specifically recognizes human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest that ligand specificity derives from favorable Van der Waals interactions with a hydrophobic cavity at the dimerization site.

A COVID moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.

The Plasmodium falciparum Hsp70-x chaperone assists the heat stress response of the malaria parasite.

Plasmodium falciparum is the most lethal of human-infective malaria parasites. A hallmark of P. falciparum malaria is extensive remodeling of host erythrocytes by the parasite, which facilitates the development of virulence properties such as host cell adhesion to the endothelial lining of the microvasculature. Host remodeling is mediated by a large complement of parasite proteins exported to the erythrocyte; among them is a single heat shock protein (Hsp)70-class protein chaperone, P. falciparum Hsp70-x (PfHsp70-x). PfHsp70-x was previously shown to assist the development of virulent cytoadherence characteristics. Here, we show that PfHsp70-x also supports parasite growth under elevated temperature conditions that simulate febrile episodes, especially at the beginning of the parasite life cycle when most of host cell remodeling takes place. Biochemical and biophysical analyses of PfHsp70-x, including crystallographic structures of its catalytic domain and the J-domain of its stimulatory Hsp40 cochaperone, suggest that PfHsp70-x is highly similar to human Hsp70 chaperones endogenous to the erythrocyte. Nevertheless, our results indicate that selective inhibition of PfHsp70-x function using small molecules may be possible and highlight specific sites of its catalytic domain as potentially of high interest. We discuss the likely roles of PfHsp70-x and human chaperones in P. falciparum biology and how specific inhibitors may assist us in disentangling their relative contributions.-Day, J., Passecker, A., Beck, H.-P., Vakonakis, I. The Plasmodium falciparum Hsp70-x chaperone assists the heat stress response of the malaria parasite.

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*.

The SARS-CoV-2 main protease (Mpro ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro . Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd =0.14±0.03 µM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.

Structural analysis of P. falciparum KAHRP and PfEMP1 complexes with host erythrocyte spectrin suggests a model for cytoadherent knob protrusions.

Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) and Knob-associated Histidine-rich Protein (KAHRP) are directly linked to malaria pathology. PfEMP1 and KAHRP cluster on protrusions (knobs) on the P. falciparum-infected erythrocyte surface and enable pathogenic cytoadherence of infected erythrocytes to the host microvasculature, leading to restricted blood flow, oxygen deprivation and damage of tissues. Here we characterize the interactions of PfEMP1 and KAHRP with host erythrocyte spectrin using biophysical, structural and computational approaches. These interactions assist knob formation and, thus, promote cytoadherence. We show that the folded core of the PfEMP1 cytosolic domain interacts broadly with erythrocyte spectrin but shows weak, residue-specific preference for domain 17 of α spectrin, which is proximal to the erythrocyte cytoskeletal junction. In contrast, a protein sequence repeat region in KAHRP preferentially associates with domains 10-14 of ß spectrin, proximal to the spectrin-ankyrin complex. Structural models of PfEMP1 and KAHRP with spectrin combined with previous microscopy and protein interaction data suggest a model for knob architecture.

The complex of Plasmodium falciparum falcipain-2 protease with an (E)-chalcone-based inhibitor highlights a novel, small, molecule-binding site.

BACKGROUND: Malaria kills over 400,000 people each year and nearly half the world's population live in at-risk areas. Progress against malaria has recently stalled, highlighting the need for developing novel therapeutics. The parasite haemoglobin degradation pathway, active in the blood stage of the disease where malaria symptoms and lethality manifest, is a well-established drug target. A key enzyme in this pathway is the papain-type protease falcipain-2. METHODS: The crystallographic structure of falcipain-2 at 3.45 Å resolution was resolved in complex with an (E)-chalcone small-molecule inhibitor. The falcipain-2-(E)-chalcone complex was analysed with reference to previous falcipain complexes and their similarity to human cathepsin proteases. RESULTS: The (E)-chalcone inhibitor binds falcipain-2 to the rear of the substrate-binding cleft. This is the first structure of a falcipain protease where the rear of the substrate cleft is bound by a small molecule. In this manner, the (E)-chalcone inhibitor mimics interactions observed in protein-based falcipain inhibitors, which can achieve high interaction specificity. CONCLUSIONS: This work informs the search for novel anti-malaria therapeutics that target falcipain-2 by showing the binding site and interactions of the medically privileged (E)-chalcone molecule. Furthermore, this study highlights the possibility of chemically combining the (E)-chalcone molecule with an existing active-site inhibitor of falcipain, which may yield a potent and selective compound for blocking haemoglobin degradation by the malaria parasite.

A spiral scaffold underlies cytoadherent knobs in Plasmodium falciparum-infected erythrocytes.

Much of the virulence of Plasmodium falciparum malaria is caused by cytoadherence of infected erythrocytes, which promotes parasite survival by preventing clearance in the spleen. Adherence is mediated by membrane protrusions known as knobs, whose formation depends on the parasite-derived, knob-associated histidine-rich protein (KAHRP). Knobs are required for cytoadherence under flow conditions, and they contain both KAHRP and the parasite-derived erythrocyte membrane protein PfEMP1. Using electron tomography, we have examined the 3-dimensional structure of knobs in detergent-insoluble skeletons of P falciparum 3D7 schizonts. We describe a highly organized knob skeleton composed of a spiral structure coated by an electron-dense layer underlying the knob membrane. This knob skeleton is connected by multiple links to the erythrocyte cytoskeleton. We used immuno-electron microscopy (EM) to locate KAHRP in these structures. The arrangement of membrane proteins in the knobs, visualized by high-resolution freeze-fracture scanning EM, is distinct from that in the surrounding erythrocyte membrane, with a structure at the apex that likely represents the adhesion site. Thus, erythrocyte knobs in P falciparum infection contain a highly organized skeleton structure underlying a specialized region of membrane. We propose that the spiral and dense coat organize the cytoadherence structures in the knob, and anchor them into the erythrocyte cytoskeleton. The high density of knobs and their extensive mechanical linkage suggest an explanation for the rigidification of the cytoskeleton in infected cells, and for the transmission to the cytoskeleton of shear forces experienced by adhering cells.