Michèle C. Erat

Structural Basis of the 9-Fold Symmetry of Centrioles

Summary 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.

Identification and structural analysis of type I collagen sites in complex with fibronectin fragments.

Collagen and fibronectin are major components of vertebrate extracellular matrices. Their association and distribution control the development and properties of diverse tissues, but thus far no structural information has been available for the complex formed. Here, we report binding of a peptide, derived from the α1 chain of type I collagen, to the gelatin-binding domain of human fibronectin and present the crystal structure of this peptide in complex with the 8–9FnI domain pair. Both gelatin-binding domain subfragments, 6FnI1–2FnII7FnI and 8–9FnI, bind the same specific sequence on D-period 4 of collagen I α1, adjacent to the MMP-1 cleavage site. 8–9FnI also binds the equivalent sequence of the α2 chain. The collagen peptide adopts an antiparallel β-strand conformation, similar to structures of proteins from pathogenic bacteria bound to FnI domains. Analysis of the type I collagen sequence suggests multiple putative fibronectin-binding sites compatible with our structural model. We demonstrate, by kinetic unfolding experiments, that the triple-helical collagen state is destabilized by 8–9FnI. This finding suggests a role for fibronectin in collagen proteolysis and tissue remodeling.

Structural Analysis of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Intracellular Domain Reveals a Conserved Interaction Epitope

Background: PfEMP1 localization and connection to the red blood cell cytoskeleton is necessary for cytoadherence. Results: The PfEMP1 intracellular domain (ATS) is structurally conserved and interacts directly with a novel parasite protein through a flexible epitope. Conclusion: The ATS epitope mediate interactions that may be critical for cytoadherence. Significance: This is the first demonstration of ATS interacting with PHIST domains. Plasmodium falciparum-infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS center is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine-rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.

Solution Structure of Domain 6 from a Self-Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

Group II intron self‐splicing is essential for the correct expression of organellar genes in plants, fungi, and yeast, as well as of bacterial genes. Self‐excision of these autocatalytic introns from the primary RNA transcript is achieved in a two‐step mechanism that is apparently analogous to that of the eukaryotic spliceosome. The 2′‐OH of a conserved adenosine (the branch point) located within domain 6 (D6) acts as the nucleophile in the first step of splicing. Despite the biological importance of group II introns, little is known about their structural organization and usage of metal ions in catalysis. Here we report the first solution structure of a catalytically active D6 construct encompassing the branch point and the neighboring helical regions from the mitochondrial yeast intron ai5γ. The branch adenosine is the single unpaired nucleotide, and, in contrast to the spliceosomal branch site, resides within the helix, being partially stacked between two flanking GU wobble pairs. We identified a novel prominent Mg2+ binding site in the major groove of the branch site. Importantly, Mg2+ addition does not impair the stacking of the branch adenosine, rather it strengthens the interaction with the flanking uridines, as shown by NMR and fluorescence studies. This means that domain 6 presents the branch adenosine in a stacked fashion to the core of group II introns upon folding to the active conformation.

A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface

Uniquely among malaria parasites, Plasmodium falciparum‐infected erythrocytes (iRBCs) develop membrane protrusions, known as knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed sub‐telomeric (PHIST) domains has attracted attention, with members being implicated in host‐parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in knobs. PHIST variants that do not locate in knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST‐PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1‐cytoskeletal connection in knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.—Oberli, A., Slater, L. M., Cutts, E., Brand, F., Mundwiler‐Pachlatko, E., Rusch, S., Masik, M. F. G., Erat, M. C., Beck, H.‐P., Vakonakis, I. A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface. FASEB J. 28, 4420–4433 (2014). www.fasebj.org

Structural Analysis of the G-Box Domain of the Microcephaly Protein CPAP Suggests a Role in Centriole Architecture

Summary Centrioles are evolutionarily conserved eukaryotic organelles composed of a protein scaffold surrounded by sets of microtubules organized with a 9-fold radial symmetry. CPAP, a centriolar protein essential for microtubule recruitment, features a C-terminal domain of unknown structure, the G-box. A missense mutation in the G-box reduces affinity for the centriolar shuttling protein STIL and causes primary microcephaly. Here, we characterize the molecular architecture of CPAP and determine the G-box structure alone and in complex with a STIL fragment. The G-box comprises a single elongated β sheet capable of forming supramolecular assemblies. Structural and biophysical studies highlight the conserved nature of the CPAP-STIL complex. We propose that CPAP acts as a horizontal “strut” that joins the centriolar scaffold with microtubules, whereas G-box domains form perpendicular connections.

2:Methods to Detect and Characterize Metal Ion Binding Sites in RNA

Metal ions are inextricably associated with RNAs of any size and control their folding and activity to a large part. In order to understand RNA mechanisms, also the positioning, affinities and kinetics of metal ion binding must be known. Due to the spectroscopic silence and relatively fast exchange rates of the metal ions usually associated with RNAs, this task is extremely challenging and thus numerous methods have been developed and applied in the past. Here we provide an overview on the different metal ions and methods applied in RNA (bio)chemistry: The physical-chemical properties of important metal ions are presented and briefly discussed with respect to their application together with RNA. Each method ranging from spectroscopic over biochemical to computational approaches is briefly described also mentioning caveats that might occur during the experiment and/or interpretation of the results.

Soluble CD163 promotes recognition, phagocytosis and killing of Staphylococcus aureus via binding of specific fibronectin peptides

CD163 is a multi‐ligand scavenger receptor exclusively expressed by monocytes and macrophages, which is released after their activation during sepsis (sCD163). The biological relevance of sCD163, however, is not yet clear. We now demonstrate that sCD163 exhibits direct antimicrobial effects by recognizing a specific subfragment (6F11F22F27F1) of fibronectin (FN) bound to staphylococcal surface molecules. Moreover, contact with staphylococci promotes sCD163‐shedding from monocyte surface via induction of metalloproteinases ADAM10 and ADAM17. sCD163 subsequently binds to Staphylococcus aureus via FN peptides and strongly amplifies phagocytosis as well as killing by monocytes and to a lesser extend by neutrophils. This mechanism exhibits additional paracrine effects because staphylococci additionally opsonized by sCD163 induce higher activation and more efficient killing activity of non‐professional phagocytes like endothelial cells. Targeting pathogen‐bound FN by sCD163 would be a very sophisticated strategy to attack S. aureus as any attempt of the pathogen to avoid this defence mechanism will automatically bring about loss of adherence to the host protein FN, which is a pivotal patho‐mechanism of highly invasive staphylococcal strains. Thus, we report a novel function for sCD163 that is of particular importance for immune defence of the host against S. aureus infections.

Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry

Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species.

Divalent Metal Ions Tune the Self-Splicing Reaction of the Yeast Mitochondrial Group II Intron Sc.ai5γ

Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg2+ to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis–Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5′-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca2+, Mn2+, Ni2+, Zn2+, Cd2+, Pb2+, and [Co(NH3)6]3+ on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5γ. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg2+. For example, the presence of only 5% Ca2+ relative to Mg2+ results in a decrease in the maximal turnover rate kcat by 50%. Ca2+ thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg2+ in the folded state, the latter being indicative of at least one specific Ca2+ binding pocket interfering directly with catalysis. Similar results are obtained with Mn2+, Cd2+, and [Co(NH3)6]3+. Ni2+ is a much more powerful inhibitor and the presence of either Zn2+ or Pb2+ leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg2+ and raises the question of biological relevance at least in the case of Ca2+.